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Keywords:

  • ectomycorrhizal fungi;
  • zinc tolerance;
  • zinc toxicity;
  • genetic adaptation;
  • Suillus luteus;
  • Suillus bovinus;
  • Rhizopogon luteolus;
  • Paxillus involutus

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    Zn tolerance was investigated in populations of four ectomycorrhizal fungi: Suillus luteus, Suillus bovinus, Rhizopogon luteolus and Paxillus involutus. The fungi were collected in pioneer pine forests at 14 different locations, situated along a Zn pollution gradient. Genetic adaptation to Zn toxicity was previously presumed in a population of S. luteus.
  • • 
    Mycelial biomass production was assessed for 235 isolates exposed to increasing Zn2+ stress. EC50 concentrations were determined.
  • • 
    Adaptive Zn tolerance was found in the three species from the Suilloid clade and not in P. involutus. The Suilloid fungi collected within 5 km from a Zn smelter were highly Zn-tolerant, in contrast to isolates collected at least 15 km away from a pollution source. Mixed populations with tolerant and sensitive S. luteus isolates were found in a transition zone, between 5 and 15 km from the Zn smelters.
  • • 
    The severe Zn pollution in the surroundings of the Zn smelters has clearly triggered the evolution of an increased Zn tolerance in the pioneer Suilloid fungi. With increasing distances from the Zn smelters, the frequency of Zn-tolerant genotypes decreases.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Natural populations thriving in heavy metal contaminated ecosystems are often subjected to selective pressures for an increased resistance to toxic metals. Evolutionary adaptation to heavy metals is a well-documented process in several different groups of organisms including bacteria (Diels & Mergeay, 1990), animals (Levinton et al., 2003), marine algae (Nielsen et al., 2003), mosses (Shaw, 1988), etc. The phenomenon has been particularly studied in higher plants (Bradshaw & McNeilly, 1981; Baker et al., 1986; Schat & Verkleij, 1998). Zn-tolerant plant ecotypes are found on Zn-containing outcrops, on mine spoils, on soils heavily contaminated by Zn smelters or under galvanised metal constructions, such as fences and electricity pylons (Al-Hiyaly et al., 1990, 1993). The potential for the development of Zn tolerance has been demonstrated in monocotyledonous plants, in particular grasses (Al-Hiyaly et al., 1990). Evolution for metal tolerance in grasses can be very rapid because normal, nonadapted populations often contain a low frequency of Zn-tolerant individuals (Walley et al., 1974; Bradshaw & McNeilly, 1981; Al-Hiyaly et al., 1993). Relatively fewer dicots have been found to develop the Zn tolerance trait. These plants are often endemic to metalliferous soils and only a few species, for example Silene vulgaris, Thlaspi caerulescens and Arabidopsis halleri, have metallicolous and nonmetallicolous populations (Ernst, 1990; Assunção et al., 2003a). In trees with long reproductive cycles, the adaptive potential for metal tolerance seems to be low (Meharg & Cairney, 2000). Trees may resist extreme metal toxicity through large phenotypic plasticity and through their association with a small guild of well-adapted ectomycorrhizal (ECM) fungi (Wilkinson & Dickinson, 1995). Plant adaptation to selective pressures is often considered to be regulated by the plant genome, but it is evident that also mutualistic microorganisms can alleviate heavy metal toxicity in plants (Hall, 2002; Adriaensen et al., 2004).

Soil bacteria can adapt relatively quickly to toxic Zn environments (Díaz-Raviña & Bååth, 1996) and this might also be true for those soil-born microorganisms that live in mutualistic symbioses with plant roots (Wu & Lin, 1990; Meharg & Cairney, 2000; Lakzian et al., 2002). In a previous paper, we reported on the presence of a Zn-tolerant population of the ectomycorrhizal basidiomycete, Suillus luteus, in the immediate vicinity of the dismantled Zn smelter of Lommel-Maatheide in Belgium (Colpaert et al., 2000). The occurrence of several genets, some of which were quite large, as well as the yearly production of abundant basidiocarps of S. luteus on the Maatheide site suggest that the Zn-adapted genotypes are quite vigorous under the local toxic conditions. However, at that time, we did not know whether the Zn tolerance trait was present in other populations that were exposed to high Zn stress. Other surveys on Zn-contaminated sites in Europe could not demonstrate an increased Zn tolerance in S. luteus (Blaudez et al., 2000). Overall, there are still few reports that confirm that ECM fungi show evolutionary adaptation to heavy metal pollution (Hartley et al., 1997a; Meharg & Cairney, 2000).

Here, we further elaborate on the frequency and the spatial pattern of the Zn tolerance trait in ECM populations growing along a Zn gradient, which was caused in the previous century by the activities of several Zn smelters, all situated near the Belgian–Dutch border. We investigated fungal populations at 14 collection sites showing different degrees of Zn pollution. Because spores of basidiomycetes may be dispersed over long distances, we were curious to know how far the Zn tolerance trait could spread into populations that are hardly exposed to Zn contamination. Suillus luteus was the prime target species for the investigation, but because there were indications that adaptive Zn tolerance was also present in S. bovinus (Colpaert & Van Assche, 1987), this species was included in the study, as well as two other ECM fungi, Rhizopogon luteolus and Paxillus involutus. The two latter taxa were often present in the same fungal community as the Suillus species and both P. involutus and Rhizopogon sp. were previously reported from other Zn-polluted habitats (Denny & Wilkins, 1987; Turnau et al., 1996; Blaudez et al., 2000).

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Site descriptions

The geographical position of the five regional Zn smelters is shown on a map of the Belgian Limburg province (Fig. 1). The northern part of the region is most strongly polluted by the activities of four Zn smelters, whose activities started in the late nineteenth century: Balen (1888–), Lommel-Maatheide (1904–74), Overpelt (1888–) and Budel (NL). These four smelters produced Zn for a long period by pyrometallurgical processing of Zn ores. Until the mid-seventies, these factories emitted huge quantities of Zn and several other heavy metals into the atmosphere. Since the mid-seventies atmospheric Zn depositions were almost completely cut and the smelter of Lommel was closed. A fifth zinc factory in the eastern part of the province operated in Dilsen-Stokkem from 1928 to 1964. This factory had lower emissions because of its lower Zn production. Consequently the impact on the surroundings was smaller than in N-Limburg. The prevailing winds in the region are south-westerly winds so that the pollution is most seriously spread in a north-easterly direction (Fig. 1).

image

Figure 1. Map of the Limburg province in Belgium, showing the Zn smelters (solid triangle) of Lommel (1), Balen (2), Overpelt (3), Budel (4) and Dilsen-Stokkem (5). The ectomycorrhizal (ECM) fungal sampling sites (solid circle) are also plotted. The wind rose shows the direction of the long-year-average of the wind frequency in N-Limburg (Kleine Brogel, source KMI). The site abbreviations are explained in Table 1.

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All 14 sampling sites are situated in the Campine phytogeographic district, which is characterised by base-poor, sandy soils of low fertility. The 14 locations are also shown on the map in Fig. 1 and the characteristics of the studied forests are summarised in Table 1 (abbreviations of the sites are also reported in Table 1). On most sites, pines (Pinus sylvestris and P. nigra) were the dominant tree species, sometimes mixed with birches. The pine forests were usually younger than 30 yr except for the site in Z where deciduous trees were dominant and Scots pines were much older. Most forests were pioneer forests or primary plantations.

Table 1.  Study sites: distance between forest and nearest Zn smelter, number of isolates tested for Zn tolerance, age of the pine trees, and site characteristics
Site (abbreviation)Distance kmSuillus bovinus#Suillus luteus#Rhizopogon luteolus#Paxillusinvolutus#Age of pines yrLand use
  • 1

    , Species present, but not cultured. Site abbreviations are given in the first column.

Lommel Maatheide (Lm) 0.5 12 526Industrial area, mostly planted trees
Neerpelt (N) 1.2 9141+11–15Industrial area, spontaneous colonisation
Overpelt fabriek (Of) 1.3 5 72+1–25Sand dunes, spontaneous colonisation
Lommel sahara (Ls) 1.41427251–25Industrial area, reforested (plantation + spontaneous)
Lommel ring (Lr) 2.0  2 +8Second rotation forest
Overpelt zandgroeve (Oz) 3.8+ 4+10–22Second rotation forest
Eksel (E) 7.6 5181+1–15Road side, spontaneous colonisation
Maasmechelen (Mm)12.4+12 +1–25Sand quarry, spontaneous colonisation
Hechtelse heide (Hh)14.8 2224+1–25Sand dunes, spontaneous colonisation
Paal (P)16.6 91914(1–)24Industrial area, plantation + spontaneous colonisation
Houthalen remo (Hr)16.8  4 +8Industrial area, plantation
Winterslag (W)20.0  2 +5–10Road side, spontaneous colonisation
Meeuwen-Gruitrode (Mg)21.4 7 4 +(5–)30Mostly second rotation forest
Zolder (Z)22.6 7  6> 50Protected area, old mixed forest

Zinc pollution was assessed in two ways: we analysed Zn in soil pore water and we measured Zn in needles of the pine host plants. At each study site, four soil samples (to a depth of 20 cm) of about 2 kg each were collected beneath Suillus basidiocarps. Soils were transferred to large pots and incubated in a glasshouse under moist conditions. Pore water was sucked 4 wk later with Rhizon soil moisture samplers (Eijkelkamp Agrisearch Equipment, Giesbeek, NL) according to the procedure described in Knight et al. (1998). The pH of the pore water was measured and Zn was subsequently analysed with AAS.

Mixed samples of first year-old needles were collected in autumn from 10 different pine trees at each location. Needles were washed with plenty of tap water. They were dried (70°C, 120 h) and milled in a ball mill to a fine powder for combustion in a muffle furnace (600°C, 12 h). Ashes were dissolved in acid (final concentration 0.5 m HCl) and Zn was analysed with AAS. The analyses were performed in duplicate and certified reference plant material was included in the procedure.

Fungal material

Basidiomes of Suillus luteus (L. Fr.) Roussel, Suillus bovinus (L. Fr.) Roussel, Rhizopogon luteolus Fr. emend. Tul. & Tul. and Paxillus involutus (Batsch: Fr.) Fr. were collected in the forests selected for this study. S. luteus is typically found in pioneer pine forests and primary plantations, in particular on young or disturbed soils lacking litter layers. S. bovinus and R. luteolus fructify in both young and older pine forests and P. involutus is a generalist that also thrives in many other forest types (Arnolds et al., 1995).

Fungal cultures were established from basidiomes collected on a single sampling day for each site in 2000, 2001 or 2002. To avoid sampling of sporocarps from the same genet, a minimal distance of 10 m was respected between collected sporocarps. The majority of the cultures are kept in a culture collection on Fries medium without elevated Zn.

Metal tolerance analysis

All fungal isolates were screened from October 2001 to December 2002. To avoid carry over effects, the mycelia were subcultured at least three times on Fries medium before they were included in the screening tests. Zinc tolerance was tested on solid modified Fries medium (Colpaert et al., 2000). The final basic solution contained 28 mm glucose, 5.4 mm ammonium tartrate, 1.5 mm KH2PO4, 0.4 mm MgSO4·7H2O, 0.3 mm NaCl, 0.2 mm CaCl2·2H2O, 4 µm FeCl3·6H2O, 6 µm MnSO4·H2O, 0.8 µm CuSO4·5H2O, 56 µmmyo-inositol, 0.1 µm biotin, 0.5 µm pyridoxine, 0.3 µm riboflavin, 0.8 µm nicotinamide, 0.7 µmp-aminobenzoic acid, 0.3 µm thiamine, 0.2 µm Ca-pantothenate and 0.8% agar. Ten Zn treatments were established through addition of ZnSO4·7H2O to the nutrient medium. Zn2+ was added at concentrations of 0.15, 3, 6 up to 27 mm Zn increasing in steps of 3 mm. The pH of the final media was adjusted to 4.5.

Inocula (0.5 cm2 plugs) were prepared from 1-wk-old colonies. In order to obtain uniform inocula, a large number of plugs were preincubated on cellophane-covered agar plates with basic medium for 2 or 3 d. Single plugs showing emerging hyphae in all directions were then transferred to the centre of the test plates covered with cellophane. For each treatment, there were three replicates. Five untreated plugs of each isolate were immediately harvested to determine mean d. wt of the mycelia at the start of the treatment. Plates were incubated at 23°C in the dark. Mycelia were harvested during the exponential growth phase before exhaustion of the medium. In a preliminary experiment, a fast-growing isolate was used to determine the growth curve of each species on control medium (Hartley et al., 1997b). S. luteus and R. luteolus were harvested after 10 d of incubation, P. involutus and S. bovinus after 12 d. Mycelia were frozen at −80°C, and subsequently freeze-dried before weighing. The d. wt increment during the 10- or 12-d test period was determined. A tolerance index was calculated for each isolate as the percentage of biomass retained on the metal-enriched media compared with growth on the control Fries medium. The EC50 concentration (Zn concentration which inhibits growth by 50%) was determined for each isolate.

Statistical analysis

EC50 values of the isolates were regressed against the shortest distance from a Zn smelter or against soil pore water. Both a linear and a quadratic model were tested with the statistical software package SAS®.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The Zn gradient in the field

The Zn concentration in the needles of the pines decreased markedly with increasing distance from the Zn smelters (Fig. 2). In the forests with a low level of pollution, the Zn concentration in the pine needles varied from 20 to 40 µg g−1 d. wt. The Zn needle concentration increased above 100 µg Zn g−1 d. wt within the 2 km perimeter around the Zn smelters. The Zn concentration in the pore waters followed a similar pattern as Zn in needles. Both parameters were positively correlated (Fig. 3). The average Zn concentration in pore water varied from 0.6 µm in unpolluted soil up to 120 µm in the severely polluted Maatheide soil. The pH of the pore waters was within a rather narrow range of 3.4–4.2, except for the Maatheide soil, which had a much higher pH of 6.1, probably caused by the presence of heavy metal oxides.

image

Figure 2. Zn concentration in needles of the pine trees growing in the forests where the ectomycorrhizal (ECM) fungi were sampled. The sites are ordered according to their distance to a Zn smelter. The inset figure shows the decrease of Zn in the pine needles with increasing distance to the smelters. The dotted line represents the normal concentration of Zn in pine needles in the Campine district. Site abbreviations are explained in Table 1. Bars represent standard error of the mean (n = 10).

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image

Figure 3. Relationship between Zn in soil pore water and Zn in the pine needles at the different sampling sites in the Zn gradient. A linear regression line (y = 1.90x + 28.64, R2 = 0.88) is shown.

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Collection of material

S. luteus sporocarps were found in all potential habitats that were inspected for this study, often in large numbers. We obtained 146 successful isolations from 13 populations; the species was not found in the old Zolder forest (Table 1). Molecular fingerprints were made from sporocarps collected in a few, large S. luteus populations (Lm, Ls, N, P). These AFLP fingerprints indicated a low incidence (< 1%) of identical genotypes when sporocarps were sampled at a minimal distance of 10 m, probably because of relatively small genets in these pioneer conditions (L. Muller, unpublished). Sporocarps of S. bovinus are less dominant in the pioneer pine forests investigated, but the species is more widespread in the Campine district than S. luteus because it occurs also in older pine forests. For S. bovinus, we established 58 cultures from eight populations. R. luteolus has become a relatively rare species in Belgium and is on the red list of endangered species. Only 11 isolates were obtained from six sites. P. involutus has the broadest ecological amplitude of the fungi sampled and was present on all sites. Twenty isolations were made from four sites with contrasting degrees of Zn pollution. The number of isolates investigated at each site is reported in Table 1.

The Zn screenings

The careful preselection of uniform inocula resulted in a smooth, regular growth of the colonies and in small variance amongst replicates. Lag phases were greatly avoided. This is particularly important for a cumulative quantity such as biomass production. For the same reason, it is important to avoid nutrient exhaustion of the medium. In each of the four species tested, growth could continue on control medium for at least 16 d.

The average tolerance indices for each S. luteus population are shown in Table 2. Isolates from clean sites had an average EC50 of 4 mm Zn. Isolates with an EC50 above 6 mm Zn were considered as having an increased Zn tolerance. High-level zinc tolerance was predominantly found in S. luteus populations collected in the vicinity of the Zn smelters (Lm, Ls, Of, Oz, N). Three populations (E, Mm, Hh), situated at 7.6, 12.4 and 14.8 km from the nearest smelter, consisted of sensitive genotypes and genotypes with intermediate tolerance. In the most remote S. luteus populations only Zn-sensitive genotypes were discovered. The relationship between the EC50 values and the distance to the nearest Zn smelter is shown in Fig. 4. The distribution of the S. luteus isolates in Zn tolerance classes is shown in histograms (Fig. 5). A similar distribution pattern was observed for S. bovinus, although this species is overall less Zn-tolerant than S. luteus (Fig. 6). S. bovinus isolates with an EC50 above 3 mm Zn are considered as Zn-tolerant. All five isolates of R. luteolus that were found in the close surroundings of the Zn smelters were Zn-tolerant, whereas the 6 isolates growing in unpolluted sites were all very Zn-sensitive (Fig. 7). For Paxillus involutus both sensitive and moderately tolerant isolates were found in all four populations sampled (Fig. 8).

Table 2.  Zn tolerance indices of the Suillus luteus isolates, collected in 13 populations, situated along a Zn pollution gradient. Sites are ordered from a high to a low level of pollution
 Isolates #Zn concentration (mM)
0,15369121518212427
  • 1

    n.d. not determined.

Lommel Maatheide12100100 ± 3103 ± 485 ± 764 ± 1252 ± 1248 ± 1539 ± 1229 ± 1125 ± 10
Neerpelt14100100 ± 3 92 ± 579 ± 960 ± 1152 ± 1227 ± 1019 ± 8 8 ± 3 2 ± 1
Overpelt fabriek 7100112 ± 7100 ± 875 ± 1556 ± 1440 ± 1536 ± 1731 ± 1633 ± 1830 ± 15
Lommel sahara27100101 ± 3 94 ± 471 ± 763 ± 854 ± 846 ± 941 ± 940 ± 1031 ± 9
Lommel ring 2100104 ± 5 52 ± 3842 ± 3938 ± 3525 ± 2210 ± 11 3 ± 3 0 0
Overpelt zandgroeve 4100100 ± 6101 ± 890 ± 1175 ± 2051 ± 2020 ± 1212 ± 911 ± 11 8 ± 8
Eksel18100 86 ± 7 71 ± 944 ± 925 ± 818 ± 7 4 ± 3 1 ± 1 0 0
Maasmechelen12100 57 ± 8 21 ± 1017 ± 9 9 ± 6 4 ± 3n.d.1n.d.n.d.n.d.
Hechtelse heide22100 52 ± 8 33 ± 821 ± 714 ± 510 ± 5n.d.n.d.n.d.n.d.
Paal19100 15 ± 4  2 ± 1 2 ± 1 1 ± 1 1 ± 0n.d.n.d.n.d.n.d.
Houthalen remo 4100 73 ± 8  8 ± 2 4 ± 1 3 ± 1 0n.d.n.d.n.d.n.d.
Winterslag 2100 75 ± 7 15 ± 3 1 ± 1 0 0n.d.n.d.n.d.n.d.
Meeuwen-Gruitrode 4100 59 ± 10  9 ± 2 2 ± 1 1 ± 0 1 ± 0n.d.n.d.n.d.n.d.
image

Figure 4. Relationship between EC50 values of the Suillus luteus isolates and the distance to the nearest Zn smelter. The regression line for a quadratic model (y = ax2 + bx + c) is shown with a = 0.04 ± 0.02, b = −1.6 ± 0.3, c = 19.0 ± 0.9; P < 0.001; F2,145 = 79.58. Bars represent standard errors (n = 2–27).

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image

Figure 5. The distribution of zinc tolerance in the Suillus luteus populations along a Zn gradient. (a) Pooled isolates from the populations of Lm, Ls, Lr, N, Of and Oz, all within 5 km from a Zn smelter. (b) Populations of E, Hh and Mm, situated between 5 and 15 km from a Zn smelter. (c) Populations of P, Hr, Mg and W, all situated more than 15 km away from the Zn smelters in N-Limburg. The site abbreviations are explained in Table 1.

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image

Figure 6. The distribution of zinc tolerance in the Suillus bovinus populations along a Zn gradient. (a) Pooled isolates from the populations of Ls, N and Of, all within 5 km from a Zn smelter. (b) Populations of E and Hh, situated between 5 and 15 km from a Zn smelter. (c) Populations of P, Mg and Z, all situated more than 15 km away from the Zn smelters in N-Limburg. The site abbreviations are explained in Table 1.

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image

Figure 7. Zinc tolerance indices of the Rhizopogon luteolus isolates. Full lines are isolates from polluted sites: N (solid diamond, n = 1), Ls (solid triangle, n = 2), Of (solid square, n = 2); dotted lines are isolates from unpolluted sites: E (open diamond, n = 1), Hh (open triangle, n = 4), P (open square, n = 1). Bars represent standard error of the mean. The site abbreviations are explained in Table 1.

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image

Figure 8. The distribution of Zn tolerance in the Paxillus involutus populations. (a) Pooled isolates from the populations of Lm and Ls, both within 2 km from a Zn smelter. (b) Populations of P and Z, situated more than 15 km from the Zn smelters in N-Limburg. The site abbreviations are explained in Table 1.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The Zn concentrations in pine needles and soil pore waters illustrate that we are indeed studying ECM fungal communities situated along a gradient of Zn pollution. Both parameters were well correlated (Fig. 3). Zn in soil pore waters from the most polluted sites is high enough to cause toxicity in plants and microorganisms. The threshold for acute Zn toxicity in biosensor bacteria lies around 40 µm Zn in pore waters (Chaudri et al., 1999). In hydroponics, 40 µm Zn also causes reductions in root elongation in grasses not adapted to Zn stress (Al-Hiyaly et al., 1988) and 76 µm Zn reduces the growth as well as the N and P uptake capacity of Zn-sensitive S. bovinus associated with pine seedlings (Adriaensen et al., 2004).

The EC50 values for Zn toxicity obtained in this and other in vitro screening studies are very high. These EC50 concentrations as such should not be extrapolated to a field situation. We assume that the rich nutrient composition of most in vitro media used to cultivate and test microorganisms, including ECM fungi, is a major factor that determines the upward shift of the toxicity range. The isolates grow much faster in vitro than in symbiosis. The average relative growth rate of the S. bovinus isolates in the present study was 16% d−1; in an experiment with a host seedling it was only 3.4% d−1, even under optimal conditions (Adriaensen et al., 2004). However, the latter plant experiment also showed that the differential response obtained for a tolerant and a nontolerant isolate in vitro was maintained in symbiosis at much lower, more realistic Zn concentrations. Therefore, these in vitro tests can predict differences in growth when these fungi are exposed to elevated Zn in a contaminated soil.

Impacts of heavy metal pollution on plant communities and populations have received much more attention than effects on fungal communities or populations. Nevertheless, mycotrophic plants colonising extremely metal-polluted sites remain colonised by mycorrhizal fungi (Leyval et al., 1997). One might expect such sites to exert strong selection pressure on the symbiotic partners, a process that might result in genetic differentiation among fungal populations. To study such interpopulation differences, a large number of individuals must be studied, especially because in ECM fungi intraspecific variation as well as within population variation can be very large for many physiological responses (Cairney, 1999; Sawyer et al., 2003). If multiple genotypes are screened, frequency distributions can reveal interpopulation differences. In the present investigation, a strong differential response among the populations was observed in three of the four ECM species studied. The interpopulation differences in the response of S. luteus, S. bovinus and R. luteolus to Zn2+ are clearly related to the Zn status of their natural environment (Figs 4–7). This strong correlation between Zn tolerance and Zn pollution in three fungal species makes a strong case for a causal relationship. The Zn tolerance trait must be genetically determined because frequent subculturing (> 10 X) on basic medium does not cause a shift in the response towards elevated Zn. Physiological acclimation in Zn-exposed mycelia is likely but should disappear after subculturing on control medium.

Highest Zn tolerance was found in the populations of the Suilloid fungi, growing within a 5 km perimeter around the Zn smelters. Only in the recent plantation in Lr, it is not unequivocal whether tolerant isolates dominate (only two S. luteus isolates were tested, one of these being Zn-sensitive). The degree of Zn tolerance had a broad range within tolerant populations (Figs 5a and 6a), similar to what is found in metal-tolerant grass populations (Bradshaw & McNeilly, 1981; Al-Hiyaly et al., 1993). Mixed populations with both Zn-tolerant and nontolerant S. luteus isolates were found in E, Hh and Mm with, respectively, 62%, 32% and 17% of tolerant genotypes. The E and Hh populations are within the sphere of influence of the four northern Zn smelters. The Mm population is more than 30 km away from these Zn smelters but is relatively close to the former Dilsen smelter. We currently do not know whether there are Zn-tolerant Suillus populations in the immediate vicinity of the Dilsen factory. Populations situated more than 15 km from the Zn smelters are dominated by Zn-sensitive genotypes. This pattern in Zn tolerance suggests a gradual change from full Zn tolerance in the vicinity of the smelters to nontolerance in the remote sites. Pennanen et al. (1996) studied the effects of long-term heavy metal deposition on microbial community structure and the level of bacterial community metal tolerance in coniferous forest soils in the surroundings of the Rönnskär metal smelter in Sweden. In this area, an increased bacterial community metal tolerance was found up to 10–15 km from the smelter, a sphere of influence that is similar to the one we found here.

In the transition zone where we found Zn-tolerant and nontolerant Suillus genotypes we assume there is currently little selection pressure for Zn tolerance because plants and soils have almost normal Zn concentrations. Most fungal genets in E, Hh and Mm must have established less than 20 yr ago, in a period when atmospheric Zn deposition was negligible. Therefore, the presence of tolerant individuals might be caused by gene flow, bringing adapted genes for tolerance into normal Zn-sensitive populations. However, we cannot exclude the reverse hypothesis. If Zn deposition in the past was high enough in this zone to exert a temporary selection pressure then it is possible that Zn-sensitive genotypes are now migrating into areas previously occupied by tolerant genotypes. A low frequency of tolerant individuals might be present in normal Suillus populations. A similar phenomenon is known for grass species that can rapidly evolve metal-tolerant populations. Metal-tolerant mutants are present in normal unexposed grass populations at low frequencies, typically between 0.1 and 0.5% of the individuals (Walley et al., 1974; Bradshaw & McNeilly, 1981; Al-Hiyaly et al., 1993; Schat & Verkleij, 1998). A much larger number of fungal isolates should be screened to determine the frequency of Zn-tolerant mutants in normal unexposed populations.

The generation time of the Suilloids studied is relatively short. In primary plantations of pine, S. luteus and S. bovinus sporocarps show up 3–5 yr after planting and there are good indications that Suillus species invest relatively more energy in sexual reproduction than many other ectomycorrhizal fungi (Dahlberg & Finlay, 1999). High sporocarp productivity of R. luteolus has been reported from pine nurseries and its basidiospores have a high survival and inoculation potential (Molina et al., 1999). The short generation time and abundant reproduction may favour rapid selection for genotypes adapted to specific soil conditions.

The widespread distribution of Zn-tolerant S. luteus isolates even into areas with low contamination suggests high migration rates in these pioneer ECM fungi and a relatively low cost for the Zn tolerance. Some Rhizopogon and Suillus species are important colonisers of young conifer trees in pioneer conditions or in secondary successions after a major disturbance, such as stand-replacing fires (Baar et al., 1999; Jones et al., 2003). Spores seem to be the primary means by which these early colonist fungi colonise their hosts (Bruns et al., 2002). The fact that tolerant isolates are not rapidly outcompeted by the sensitive strains or by other pioneer fungi on nontoxic soils suggests that the cost for the tolerance might be relatively low. If Zn-tolerant fungi are less fit than normal fungi in unpolluted soil, then this should keep tolerance genes out of normal populations. If the differences in fitness are very small in clean soil, then tolerance genes can spread much farther away from the pollution source. On uncontaminated soils, metal-tolerant ecotypes are not necessarily inferior to their nontolerant counterparts (Schat & Verkleij, 1998; Rengel, 2000). Nevertheless, the elimination of a selection pressure, for example by cleanup, may lead to the disappearance of metal-tolerant populations as was observed for a Cd-adapted population of an oligochaete worm (Levinton et al., 2003). The speed of the disappearance is probably a function of the effective costs of tolerance, the mating system, gene flow and generation time.

Selection for increased Zn tolerance in Suillus sp. was not found in several other sites, affected by Zn deposition (Blaudez et al., 2000). Two explanations can be proposed for the discrepancy. Possibly selection pressure was not high enough because of low bio-availability of the metal. Determination of the available Zn fraction is a valuable tool to asses soil metal toxicity. Alternatively, local Suillus populations do not have the necessary mutant genes to develop Zn tolerance. Such a genetic constraint has been observed in grasses that normally can adapt to Zn pollution (Al-Hiyaly et al., 1993). However, this would also mean that these Zn-sensitive Suillus genotypes could survive in microsites with low toxicity. Here, we can mention that the Zn concentration in the pore water of the most polluted site (Lm) showed a large variation: from 80 to 190 µm Zn. Our investigation also indicates that P. involutus can survive on polluted sites even without specific genetic adaptation to the pollution, an observation supported by other reports (Denny & Wilkins, 1987; Blaudez et al., 2000). The presence of Zn-sensitive P. involutus genotypes in polluted habitats suggests that this species colonises soil patches with low bio-available Zn. Because organic matter is a good metal immobilising agent, Zn toxicity probably decreases when litter layers start to accumulate in the developing forest. It is likely that microsites with lower Zn concentrations can develop a few years after establishment of the tree seedlings.

The four ECM fungi studied showed a different pattern in Zn tolerance and sensitivity. On polluted sites S. luteus was on average more Zn-tolerant than S. bovinus and R. luteolus (Figs 5a, 6a and 7), and all three were more tolerant than P. involutus (Fig. 8a). However, on sites not under influence of the smelters, P. involutus populations seemed to be less sensitive towards Zn (Fig. 8b) than the Suillus populations (Figs 5c and 6c). EC50 values for P. involutus are in the same range as those observed in the 10 isolates tested by Denny & Wilkins (1987). Although we found adaptive Zn tolerance in three of the four ECM fungi studied, one should not conclude that genetic adaptation to high Zn stress is a very common phenomenon in ECM fungi, as it is also not the case in higher plants. Natural selection can more easily create an adapted population if the necessary variability is present in the original population. In higher plants, metal tolerance seems to be phylogenetically determined. Particular genera of higher plants (e.g. Silene, Agrostis, Festuca, Alyssum, Thlaspi) have a large potential for the development of metal-tolerant species and ecotypes (Ernst, 1990). Is it possible that the Suilloids have a similar genetic potential in the basidiomycetes? Many species in these plant and fungal genera are known as pioneer species. Early colonisers of metal-polluted spoils might have to cope with an extremely toxic environment with very few microsites where they can avoid metal toxicity. Less exposed microsites can develop as soon as vegetation can establish, followed by the immigration of organisms that are less adapted to extreme toxicity (Ernst, 1990). The production of litter and the biotransformations of heavy metals may play an important role in the local reduction of metal availability. Therefore, it is likely that selection pressure for higher tolerance decreases once the forest ecosystem becomes established when ‘late-stage’ fungi take over the dominance of the pioneer fungi. Ernst (1990) also indicates that the very first pioneer plants colonising mine spoils exhibit the highest metal tolerance and that these species create new niches for other organisms with a lower metal tolerance.

Plants with a high potential for metal adaptation often develop ecotypes that show multiple metal tolerances (Assunção et al., 2003b). In most cases multiple tolerance in a particular population is caused by the presence of toxic concentrations of specific metals that are present in the soil that supports this population. In the present investigation, we studied a Zn gradient caused by a number of Zn smelters. However, the Zn production in these factories was accompanied by a significant production, and thus emissions, of Cd, Cu and Pb. Bacteria collected at the Lm site show tolerance against Zn, Cu and Cd (Diels & Mergeay, 1990). At least some of the suilloids collected from the most polluted site also seem to have developed Cd tolerance, but only an exhaustive screening can give a better idea of possible multiple metal tolerances in these fungi.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank the Koninklijk Meteorologisch Instituut of Belgium (Brussels) for providing data on wind frequency and we are also grateful to all mycologists and foresters who helped us to localise the pioneer forests with S. luteus populations. We particularly thank Christel Faes for the statistical work and Klara Martens and Carine Put for the technical assistance with the screenings and the maintenance of the large fungal collection. The investigation was financially supported by the EU (MYCOREM project QLK3- 1999–00097) and by the Fund for Scientific Research – Flanders (Belgium) (project G0001.01). M.L and K.A. are supported by an IWT grant.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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