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A common characteristic of Cu distribution in soils is its accumulation in surface horizons, which reflects the bioaccumulation of the metal but also its deposition from anthropogenic sources (Tiller & Merry, 1981). Sources of soil copper contamination are diverse, including application of fertilisers, fungicide sprays, dumping of agricultural or municipal wastes, and emissions by industrial activities such as smelting and mining. High concentrations of Cu in soil have an adverse effect on vegetation (Lepp et al., 1997). Nevertheless, some tree species can establish and survive on Cu-polluted soils. Under natural conditions the majority of woody plants in temperate and boreal forests are associated with ectomycorrhizal (ECM) fungi and it seems that this mutualistic symbiosis persists on strongly metal (Cu)-contaminated sites which become slowly colonized by birches, pines and willows (Leyval et al., 1997; Vrålstad et al., 2000).
Copper, when present at elevated concentrations, is considered to be highly toxic for both plants and fungi (Fernandes & Henriques, 1991; Gadd, 1993). However, Cu sensitivity of ECM plants is poorly documented and there is no clear-cut evidence for a protective role of ECM fungi (Jones & Hutchinson, 1986; Dixon & Buschena, 1988). There are no studies in which ECM fungi have been demonstrated to alleviate growth depressions of tree seedlings due to toxic effects of Cu (Van Tichelen et al., 1999; Jentschke & Godbold, 2000). Only for the ericoid association between Calluna vulgaris and Hymenoscyphus ericae, has direct evidence for increased Cu resistance been found (Bradley et al., 1981).
The in vitro Cu sensitivity of a number of ECM fungi has been studied in several experiments. Considerable interspecific variation response to Cu was observed, but adaptive Cu tolerance has not been found (Blaudez et al., 2000; Colpaert et al., 2000). Relevant differences in metal sensitivity between ECM fungi and their hosts can, however, be best assessed in symbiosis experiments. The functioning of the mycorrhizal association as well as the development of the symbiotic partners should be studied together in a dose – response experiment.
In the present study, the Cu sensitivity of Pinus sylvestris seedlings and two ECM symbionts (Suillus bovinus and Thelephora terrestris) with high constitutive Cu resistance in vitro was compared. These fungi are common mycobionts under pines on metal contaminated sites (Colpaert & Van Assche, 1987). Phosphorus uptake, sorption of Cu on roots and extraradical mycelia, and Cu transfer to above-ground plant parts were assessed.
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Metal sensitivity or tolerance in trees is generally assessed in dilute nutrient solutions in hydroponics (Balsberg Påhlsson, 1989), whereas fungi are usually screened in vitro on nutrient rich culture media (Hartley et al., 1997). Comparisons of metal toxicity levels for both groups of organisms in absolute terms are consequently difficult and probably not very relevant. Nevertheless, in a metal-contaminated soil, ECM mycelia and roots are exposed to the same soil solution. In unpolluted soils, the water extractable Cu concentration is considerably < 1 µmol g−1 soil, but in the vicinity of copper processing factories, this soil copper fraction can increase > 100 µmol g−1 (Lepp et al., 1997). An important aspect of the present experiment is that Cu sensitivity of both symbiotic partners was assessed in the same culture system. Cu toxicity was determined in a dose – response experiment using different parameters: growth of both symbiotic partners, P and Cu status of the seedlings. Biomass of the fungi, in the roots as well as in the substrate, had to be quantified indirectly. Therefore, two methods were used for the quantification (Ekblad et al., 1998). Both the chitin and ergosterol assay gave similar results, which indicates that the proportion of active and inactive hyphae was hardly affected by the Cu treatments. The chitin : ergosterol ratio of our ECM fungi fell within the range observed in other ECM mycelia (Martin et al., 1990). Recently, Tarhanen et al. (1999) successfully used the ergosterol assay to assess the growth of the fungal partner in the lichen Bryoria fuscescens, exposed to elevated Cu and Ni. In the present experiment, the fungal biomass in roots was not significantly affected by the Cu treatments. Reductions in biomass of the extraradical S. bovinus mycelium indicated that this part of the thallus was more sensitive to Cu stress than the less exposed mantle mycelium on the roots (Tables 1, 2).
A beneficial effect of ECM fungi on host plants exposed to toxic levels of heavy metals has been observed in several experiments (Jentschke & Godbold, 2000). However, there remains a lot of controversy whether ECM fungi can confer added metal resistance to their host trees. In many cases alleviation of metal toxicity was largely based on growth promotion through a better nutrition, and hence was merely a matter of dilution of metals in the host plant tissues (Jentschke et al., 1999). Meharg & Cairney (2000) argue that ECM fungi merely fulfil their normal ecological function in polluted habitats and that most ECM fungi are as sensitive or even more sensitive than their hosts to metal contamination. The reduced transfer of metals to shoots of metal treated ECM plants, a phenomenon observed in many laboratory experiments (Jones & Hutchinson, 1986; Colpaert & Van Assche, 1992; Marschner et al., 1996; Jentschke et al., 1999) would only be active at moderate metal toxicity as long as the fungi survive. Meharg & Cairney (2000) further postulate that this reduced metal transfer aboveground in ECM hosts did not result in decreased metal toxicity. In many experiments, ECM plants did not outperform their NM counterparts at toxic metal levels when growth and nutrient status were considered.
The present experiment, however, shows that our T. terrestris and S. bovinus isolates are less sensitive to Cu than NM pine roots and it also demonstrated that both fungi could protect root growth and could decrease metal transfer to the host while maintaining P nutrition of the host. The presence of the fungi directly influenced Cu sensitivity of the host plants. A dilution effect in plant biomass was not present because the strictly controlled nutrient addition in the semihydroponics resulted in similar growth rates in ECM and NM plants (Colpaert & Verstuyft, 1999). In these circumstances mycorrhizal colonization may increase nutrient influx potential but not total nutrient assimilation in the host plant (Colpaert et al., 1999).
The most striking result of the experiment was seen in the comparison of the Cu sensitivity of NM pine roots and the fungal symbionts. Despite the well known fungitoxic nature of Cu, the ECM fungi clearly were less sensitive than the NM roots. Both the ergosterol and chitin assay confirmed that fungal growth was still substantial in the 32 and 47 mmol m−3 Cu treatment whereas the same Cu concentrations severely hampered or completely inhibited root growth of NM pine. Utriainen et al. (1997) found EC50 values for root elongation in NM birch clones, originating from metal (Zn, Cu, Ni)-polluted soil, to lie between 8 and 30 mmol m−3 Cu in hydroponics. Growth of our T. terrestris isolate was not affected at 47 mmol m−3 Cu. In the in vitro test, no growth inhibition was observed for this isolate at 790 mmol m−3 Cu, a concentration which is toxic to most ECM fungi in vitro (Hartley et al., 1997). Jones & Muehlchen (1994) report that growth of their T. terrestris isolate was hardly affected in liquid cultures at 3200 mmol m−3 Cu. These observations indicate that T. terrestris might possess a similar or even higher constitutive Cu resistance than Hymenoscyphus ericae. This ericoid mycobiont became inhibited in vitro at 1600 mmol m−3 Cu (Bradley et al., 1982), but it also conferred increased Cu resistance to its sensitive host plants (e.g. Calluna vulgaris). The apparently high Cu resistance of T. terrestris may explain the survival of this species on Cu polluted soils and it may also contribute to the success of this mycobiont in tree nurseries in which the application of Cu containing fungicides (e.g. copper oxychloride) is still common practice (Manninen et al., 1998).
Our study shows that at least some ECM fungi exhibit a higher constitutive Cu tolerance than the NM roots of their host plant. This finding supports the theory of Wilkinson & Dickinson (1995) who suggested that metal-resistant mycorrhizal fungi may allow trees to colonize extreme environments such as metalliferous soils. So far, there is little evidence that the tree species themselves have adapted genetically to metal contaminants. Both S. bovinus and T. terrestris clearly protected root growth of their host plant and reduced transfer of Cu to aboveground plant parts. Such a protective effect against elevated Cu has not previously been observed in studies with other fungal symbionts (Jones & Hutchinson, 1986).
Cu toxicity is considered to result in oxidative stress. This process is marked by an increase in plasmalemma permeability caused mainly by non-selective conductance increases and electrogenic pump inhibition. This process further leads to an imbalance and malfunctioning of transport processes localized in the plasmalemma (Demidchik et al., 1997). We assume that at the higher Cu concentrations, membrane damage was high in NM roots. First, this resulted in a sharp increase in Cu influx and translocation to shoots (32 mmol m−3), but eventually it led to a complete inhibition of root growth and Cu accumulation in roots (47 mmol m−3). P and Cu were no longer transported to the shoots, probably due to a strongly reduced transpiration stream. Jentschke & Godbold (2000) warned that metal transfer to shoots is a complex process that is affected by transpiration, which in turn might be influenced by metal toxicity and fungal symbionts. The reduced transfer of Cu in needles of NM plants at 47 mmol m−3 Cu (Fig. 4) is therefore misleading as it is probably due to a severely reduced transpiration stream. Despite the strong toxic effect of Cu on root development, shoot growth continued for some time in the NM plants although it was accompanied by a discoloration of the needles. Decreasing P concentrations in foliage (Table 3) explains the production of reddish foliage at the highest Cu concentration (Bergmann, 1992). It is also well known that root growth is more sensitive to Cu toxicity than shoot growth so that increases in shoot : root ratio are frequently found for plant seedlings exposed to elevated Cu (Jones & Hutchinson, 1986; Fernandes & Henriques, 1991; Bergmann, 1992).
After 36 d exposure to 16 and 32 mmol m−3 Cu, total amounts of Cu transferred to shoots were significantly lower with S. bovinus than with T. terrestris. The strong retention of Cu in the extraradical mycelia of S. bovinus might contribute to the low transfer of Cu to the shoots of its host. NM plants had shoot Cu concentrations above 20 µg g−1 d. wt, a critical level for Cu toxicity in pine needles (Balsberg Påhlsson, 1989). Metal sorption on fungal mycelia has often been postulated as a mechanism that restricts metal translocation to the host tissues (Jentschke & Godbold, 2000). Copper binding or sequestering in H. ericae was also proposed as a key phenomenon for the amelioration of Cu toxicity in the ericoid mycorrhizal symbiosis (Bradley et al., 1981). The Cu concentration in the extraradical mycelium of S. bovinus amounted to 0.13 mmol g−1 d. wt. ECM hyphae can adsorb large amounts of cations because of their very high CEC values (Browning & Hutchinson, 1991). Pb adsorption after exposure to 48 mmol m−3 Pb2+ was as high as 0.2–1.3 mmol g−1 d. wt (Marschner et al., 1998). The specific metal adsorbing capacity of mycelia is, however, highly species dependent and is largely affected by pH and other cations (Gadd, 1993). It was surprising that Cu could not be desorbed from the S. bovinus mycorrhizas suggesting that Cu is tightly sequestered in poorly soluble compounds or that it became largely enclosed in a hydrophobic fungal apoplast during mycorrhizal development. The production of oxalic acid by many fungi provides a means of immobilizing soluble metal ions as insoluble oxalates, decreasing availability and conferring resistance (Gadd, 1999; Ahonen-Jonnarth et al., 2000). Copper oxalate is very insoluble and has been observed on hyphae growing on Cu treated wood. A reduced availability of Cu can also be obtained when ageing hyphae become more hydrophobic. A considerable portion of the S. bovinus mycelia has hydrophobic features, in contrast to the highly hydrophilic T. terrestris mycelia (Unestam & Sun, 1995). It is likely that Cu2+ adsorbs on the newly formed, least hydrophobic, fungal cell walls of the S. bovinus mycorrhiza; during development of the mycorrhiza these cell walls become more hydrophobic and Cu becomes enclosed in the mantle. Hydrophobic cell walls may form a barrier between the fungal plasma membranes and the toxic soil solution. A similar immobilization mechanism is not possible with hydrophilic ECM fungi and explains why relatively more Cu ions can be desorbed from Thelephora mycorrhizas.
The excellent growth of T. terrestris in this experiment indicates that a high metal immobilization capacity is not an absolute prerequisite for a high constitutive Cu resistance of a mycorrhizal fungus at elevated Cu concentrations. The extraradical mycelia of T. terrestris contained far less Cu than those of S. bovinus. In addition, adsorbed copper could easily be desorbed by Pb2+ and protons. The high Cu resistance of Thelephora might be realized by an exclusion mechanism operating in the membranes (reduced uptake, a Cu efflux mechanism). Copper exclusion from cells has been demonstrated for several eukaryotic microorganisms (Meharg & Cairney, 2000).