Toxic interactions of metal ions (Cd +, Pb +, Zn +and Sb ) onin vitro biomass production of ectomycorrhizal fungi

Transcription

1 New Phytol. (1997), 137, Toxic interactions of metal ions (Cd +, Pb +, Zn +and Sb ) onin vitro biomass production of ectomycorrhizal fungi BY JEANETTE HARTLEY,, JOHN W. G. CAIRNEY, FRANCIS E. SANDERS AND ANDREW A. MEHARG * Institute of Terrestrial Ecology, Monks Wood, Huntingdon, Cambridgeshire, PE17 2LS, UK Department of Biology, University of Leeds, Leeds, LS2 9JT, UK Department of Biological Sciences, University of Western Sydney, Nepean, PO Box 1, Kingswood, NSW 2747, Australia (Received 24 April 1997; accepted 15 July 1997) SUMMARY A number of ectomycorrhizal (ECM) fungi, from sites uncontaminated by toxic metals, were investigated to determine their sensitivity to Cd +, Pb +,Zn +and Sb, measured as an inhibition of fungal biomass production. Isolates were grown in liquid media amended with the metals, individually (over a range of concentrations) and in combination (at single concentrations) to determine any significant interactions between the metals. Significant interspecific variation in sensitivity to Cd + and Zn + was recorded, while Pb + and Sb individually had little effect. The presence of Pb + and Sb in the media did however, ameliorate Cd + and Zn + toxicity in some circumstances. Interactions between Cd + and Zn + were investigated further over a range of concentrations. Zn + was found to significantly ameliorate the toxicity of Cd + to three of the four isolates tested. The influence of Zn + varied between ECM species and with the concentrations of metals tested. Key words: Ectomycorrhizal fungi, toxic metals, interactions, metal tolerance. INTRODUCTION Ectomycorrhizal (ECM) fungi play an important role in enhancing uptake of mineral nutrients for many plant species (Smith & Read, 1997). Essential and non-essential elements can be present in the environment at potentially toxic concentrations negatively affecting both hosts and symbionts. There is some evidence that ECM fungi can either evolve metal tolerance (Colpaert & Van Assche, 1992; 1993; Egerton-Warburton & Griffin, 1995) or that some ecotypes are constitutively tolerant (Denny & Wilkins, 1987). Studies have shown that significant intra- and interspecific variation exists in the metal sensitivity of ECM fungi (Brown & Wilkins, 1985; Colpaert & Van Assche, 1987; 1992; Jones & Hutchinson, 1988; Egerton-Warburton & Griffin, 1995; Hartley, Cairney & Meharg, 1997). For example, Lactarius rufus was shown to exhibit less sensitivity to Cd + than Laccaria bicolor and Lactarius hepaticus (Jongbloed & Borst-Pauwels, 199), whilst Suillus bovinus appeared less sensitive to Zn + * To whom correspondence should be addressed. a.meharg ite.ac.uk than a range of other ECM fungal species (Colpaert & Van Assche, 1987). Intraspecific studies have also revealed wide variation in metal sensitivity of isolates of Paxillus involutus (Batsch) Fr. (Denny & Wilkins, 1987) and Pisolithus tinctorius (Egerton-Warburton & Griffin, 1995). In the natural environment, contamination by single pollutants rarely occurs. Metal smelting, mining and manufacturing processes more often result in environments contaminated with a mixture of potentially toxic metals (Whitby & Hutchinson, 1974; Hunter, Johnson & Thompson, 1987; Alloway, 199). The interactions between these cocontaminants have been studied in some detail in plants where additive, antagonistic and synergistic interactions, with respect to toxicity, have been reported (Wu & Antonovics, 1975; Brar & Sekhon, 1976; Carlson & Bazzaz, 1977; Kabata-Pendias & Pendias, 1984; Burton, Morgan & Roig, 1986; Symeonidis & Karataglis, 1992). For ECM fungi, however, information on the interactions of potentially toxic metals is scarce. Colpaert & Van Assche (1992) investigating two S. bovinus isolates reported amelioration of Cd + toxicity by the ad-

2 552 J. Hartley and others dition of 765 mmol m Zn +. A similar type of interaction between Cd + and Zn + has also been reported in the basidiomycete Agrocybe aegerita (Brunnert & Zadrazil, 1985). Successful tree establishment on metal contaminated sites has both economic (forestry) and ecological benefits. It is believed that ECM fungi have an essential role in ameliorating metal toxicity to their hosts (Hartley et al., 1997). It was, therefore, the purpose of this study to investigate the toxic interactions of Cd +, Pb +, Zn + and Sb with regards to a number of ECM fungal isolates. This was carried out to determine their sensitivity to multiple metal contaminants. Zinc is an essential element required at low concentrations by all fungi (Carlile & Watkinson, 1994). By contrast, Cd +,Pb + and Sb are non-essential elements which can be toxic above threshold concentrations (Smith & Read, 1997). MATERIALS AND METHODS Culture of ECM fungi Five ECM fungal isolates were obtained from a number of uncontaminated sites. The isolates used were Suillus luteus (L. ex Fr.) S. F. Gray, Suillus granulatus (L. ex Fr.) O. Kuntze, Lactarius deliciosus Fr., suillus variegatus (Swartz ex Fr.) O. Kuntze and Paxillus involutus (Batsch) Fr. Cultures were maintained on modified Melin Norkrans (MMN) agar (Marx, 1969) amended with 71 mmol m glutamic acid. All experiments were conducted in solution culture using MMN media containing (mol m ): (NH ) HPO,3 79; KH PO,2 21; MgSO 7H O, 57; CaCl 6H O, 23; NaCl, 43; D-glucose, 55 5; and (mmol m ) thiamine, 3; glutamic acid, 71; distilled H O 1 l, adjusted to ph 5 5. Experiment 1 Determination of growth curves. For each of the five ECM fungal isolates, 24 Petri dishes were inoculated using the following method. Plugs (5 mm diameter) were taken from the edge of an actively growing colony. The tops of the plugs were sliced off to give a consistent weight of initial inoculum, and three discs of fungal mycelium were placed in a 9 mm Petri dish containing MMN solution (25 ml). Petri dishes were double wrapped in Parafilm and incubated at 22 C in the dark. To determine the mean weight of the discs used for inoculation, nine discs were taken from a colony of each isolate, and the mean d. wt calculated for three discs of each isolate. Inoculated Petri dishes were randomized within the incubator. Three replicates of each isolate were harvested on days 2, 4, 8, 12, 16, 2, 28 and 34. The d. wt of mycelium was determined at each harvest after filtering through pre-weighed 8 µm pore-size, membrane filters (Whatman), and drying at 8 C for 48 h. The final mean d. wt of sample minus initial inoculum was then plotted as a growth curve for each isolate. Dose response to Cd +, Zn +, Pb + and Sb. Using the method already described, the five fungal isolates were grown in liquid MMN amended with five concentrations of four metals (Cd +, Zn +,Pb +and Sb ). The concentrations used were: Cd +, 1, 5, 1, 1, 25 mmol m as Cd(NO ) ;Pb +, 125, 5, 2 5, 25, 125 mmol m as Pb(NO ) ; Zn +, 15, 1 5, 7 5, 75, 375 mmol m as Zn(NO ) ; Sb, 125, 1, 5, 5, 2 mmol m as SbKHO CCH(OH)CHCOHCO H (antimony potassium tartrate). From the biomass growth curves a standard point 75% along the exponential phase was calculated as the time to harvest each isolate, to ensure harvests would be conducted at a similar point in the growth for each isolate. The times estimated were: L. deliciosus, 12 d.; S. luteus, 13 d.; S. variegatus, 13 d.; S. granulatus, 19 d.; and P. involutus, 27d. (see Fig. 1). Mycelia were harvested on their pre-determined harvest dates as described above. The mean dose response data for each isolate were plotted as a proportion of the biomass in the absence of metal. The experiment was designed so that the three replicates of each treatment were harvested on three separate days. Replicates were randomized within blocks, a block being harvested on each of the separate days. This experimental design was used in all subsequent experiments. Curves were fitted to the dose response values obtained for each day, using the curve-fitting program Tablecurve 2D (Jandel Corporation, Germany). Effective concentrations inhibiting growth by 5% (EC ) values, for each replicate dose response curve were calculated. Mean EC and standard error (SE) values were then determined by averaging the EC obtained for each day. Where an EC value could not be calculated, as occurred in most cases with Sb and Pb +, the minimum concentration which caused the maximum inhibition was determined for use in subsequent experiments. Experiment 2 Effects of interactions between Cd +, Zn +, Pb + and Sb on biomass production. Based on their demonstrated sensitivities to the four metals, L. deliciosus, S. granulatus and S. variegatus were then used to investigate the interactive effects of the four metals at the EC values (or minimum values causing maximum inhibition) reported in Table 1. All combinations of metals were investigated in combinations of one, two, three and four metals using the methods

3 Metal toxicity and in vitro production of ectomycorrhizal fungi Fungal d. wt (mg) (a) 1 (b) Fungal d. wt (mg) (c) (d) Fungal d. wt (mg) (e) Time (d) Figure 1. Growth curves of (a) L. deliciosus,(b) P. involutus,(c) S. variegatus,(d) S. granulatus and (e) S. luteus. Each point is the mean of three replicates, bars represent the SE of the mean, curves were fitted using a sigmoid equation (Tablecurve 2D, Jandel Corporation). outlined above. The harvest times used were those determined in expt 1, and each treatment was replicated three times. Growth was measured as the mean fungal d. wt minus initial inoculum. The results were then expressed as a percentage of each individual metal treatment and displayed as histograms. Analysis of variance, using Minitab v. 1, was performed on the data to determine significant differences between treatments. Experiment 3 Cd + Zn + interactions. Results from the dose response experiment indicated that Cd + and Zn + were the most toxic metals to the fungal isolates, at the concentration ranges tested. The interactions between the two metals, over a range of concentrations, were therefore, investigated. Treatments used were all possible combinations of the following Cd + and Zn + concentrations. Cd +:, 1, 5, 1, 1, 25 mmol m as Cd(NO ) and Zn +:, 1, 1, 1, 1, 5 mmol m as Zn(NO ). The isolates investigated were S. luteus, S. variegatus, S. granulatus and P. involutus as they exhibited a range of sensitivity to the two metals (Table 1). L. delicious was not used in this experiment because of its poor biomass production, as demonstrated in expts 1 and 2. Isolates were grown and harvested on predetermined harvest dates as outlined in expt 1. Results were statistically analysed using ANOVA and presented graphically using contour plots (Sigmaplot, Jandel Corporation, Germany). Data for EC

4 554 J. Hartley and others Table 1. EC data for Cd +, Pb +, Zn + and Sb for five ECM fungal isolates EC (mmol m ) Cd + Pb + Zn + Sb Lactarius deliciosus 7931 a a 6616 Paxillus involutus 2 32 b a,b 5 Suillus variegatus 86 c b 5 S. granulatus d b 5 S. luteus 4215 ac b 5 EC data were calculated from curves fitted to individual replicate data using Tablecurve 2D, curve fitting procedure. Where EC values could be calculated, they were compared using analysis of variance (Minitab v. 1). Each value is the mean of three determinations SE of the mean. Data are presented graphically in Figure 2. Where an EC value could not be calculated, the minimum concentration which caused the maximum inhibition was determined for use in subsequent experiments. For Cd + and Zn + treatments, EC values followed by the same letter do not differ at P 5. were also calculated from Cd + dose response curves, plotted at each concentration of Zn + for each replicate. Curves were fitted with a non-linear curvefitting procedure, utilising the Marquart algorithm for least-squares estimation in Tablecurve 2D (Jandel Corporation). Analysis of variance was performed on the EC data to determine whether Zn + had a significant effect on Cd + EC values. RESULTS Growth curves The growth curve of each isolate displayed in Figure 1 was sigmoid in shape, typical of micro-organisms grown in batch culture. The exception was L. deliciosus which showed exponential growth for the first 12 d but then entered stationary phase. The final d. wt of L. deliciosus was significantly smaller than that of the other ECM isolates. This might result from unsuitable media constituents. L. deliciosus grew better on media containing more complex carbohydrates (e.g. potato dextrose agar) (J. Hartley, unpublished) and amino acids (Smith & Read, 1997). However, as medium may affect metal speciation and solubility, MMN medium was used in all experiments for consistency. Dose response experiment Summary EC data for all five ECM isolates are given in Table 1. The dose responses to Cd + for all five isolates followed exponential decay curves, but were presented in Figure 2a on a logarithmic axis for clarity. Sensitivity of the isolates to Cd + varied significantly (Table 1) by four orders of magnitude, the most sensitive being S. variegatus (EC 8 6 mmol m ), and the least S. granulatus (EC mmol m ). L. deliciosus was the only isolate whose growth was inhibited significantly by Pb + (EC 45 6 mmol m ), at the concentrations tested (Table 1, Fig. 2 c). Inhibition of the remaining isolates by Pb + ranged from 38% inhibition of growth of S. variegatus to 12 7% inhibition of S. luteus, both at 125 mmol m Pb + (Fig. 2c). In the case of Sb, L. deliciosus was again the only isolate which demonstrated dose-dependant inhibition, where biomass decreased with increasing metal concentration (EC 6616 mmol m ). The remainder of the results were variable, with slight inhibition occurring over a range of concentrations (Fig. 2 b). For example, growth of S. variegatus and S. luteus was inhibited by 24% at 24% at 5 mmol m and 5 mmol m Sb, respectively. P. involutus, however, showed no inhibition of growth up to 2 mmol m Sb (Fig. 2b). All five isolates showed similar sensitivity to Zn + with EC data within a relatively narrow range (1 45 to mmol m ). Up to 1 mmol m, Zn +appeared to have little effect on the ECM isolates tested, however, above this concentration there was a decrease in biomass production (Fig. 2 d). L. deliciosus was the only isolate to show significantly more sensitivity to Zn + than three of the remaining isolates (Table 1, Fig. 2d). Effects of interactions between Cd +, Zn +, Pb + and Sb on biomass production The results from expt 2 indicated wide interspecific differences in response of ECM fungi to metals, singly and in combination (Fig. 3a d). L. deliciosus was the only isolate sensitive to all four metals at the concentrations tested in expt 1 (Table 1). In expt 2, Sb interacted with Cd + to reduce its toxicity to L. deliciosus, with biomass production increased by 1% compared with the single Cd + treatment

5 Metal toxicity and in vitro production of ectomycorrhizal fungi 555 Fungal d. wt (mg)/d. wt at mmol m 3 Cd (a) Cd concentration (mmol m 3 ) Fungal d. wt (mg)/d. wt at mmol m 3 Pb (b) Sb concentration (mmol m 3 ) Fungal d. wt (mg)/d. wt at mmol m 3 Pb (c) Pb concentration (mmol m 3 ) Fungal d. wt (mg)/d. wt at mmol m 3 Zn Zn concentration (mmol m 3 ) Figure 2. Growth of L. deliciosus ( ), P. involutus ( ), S. variegatus ( ), S. granulatus ( ), and S. luteus ( ) in the presence of a range of concentrations of (a) Cd +,(b)sb,(c)pb +and (d) Zn +. Each point is the mean of three replicates, bars represent the SE of the mean, curves were fitted using linear and non-linear curve-fitting procedures (Tablecurve 2D, Jandel Corporation). (d) (Fig. 3a, Table 2). The metals Sb, Cd +and Zn + also interacted, with growth in their presence, 5% greater than the Cd + treatment alone (Fig. 3a). The individual Cd + and Zn + treatments each reduced growth by 5% of the control treatment, as expected from the results of expt 1 (Table 1). Nevertheless, when the two elements were combined they still only reduced growth by 5% of the control treatment (Fig. 3d). The combination of Cd + Pb + Zn + was shown to be less toxic to L. deliciosus than the individual or paired treatments, with growth c. 5% greater than that of the Cd +, Cd + Pb + and Cd + Zn + treatments (Fig. 3a). Despite the ability of Zn + and Sb to reduce Cd + toxicity, individually and in combination (P 1), the addition of Pb + to the solution caused the four metals to be more toxic to biomass production of L. deliciosus (Fig. 3 a, Table 2). In the case of S. variegatus, there was no additive toxicity between Cd + and Zn + (Table 2), with growth in the presence of both metals, approx. equal to that of the two individually (Fig. 3 a). The significant interactions between Sb Cd + (P 13), Sb Zn + (P 31) and Sb Cd + Zn + (P 2) were also similar to those of L. deliciosus (Table 2). The investigation of S. granulatus revealed that both Pb + and Sb ameliorated the toxicity of Cd + to the fungus, with growth in the two treatments (Cd + Pb +, Cd + Sb ) almost equal to the control (Fig. 3a). However, this amelioration by Sb did not occur in the presence of Cd + Zn + (Cd + Sb Zn +), where there was a significant interaction between the three metals (P 6) resulting in an 85% inhibition of growth, compared to the single Cd + treatment (Table 2, Fig. 3a). This effect was ameliorated to some extent by the addition of Pb + (Cd + Zn + Sb Pb +), although there was still a highly significant interaction (P 1) between the four metals (Table 2). S. granulatus grew significantly better in the presence of Pb + Zn +, than with Zn + by itself (P 4) implying that Pb + also had an ameliorative effect on the toxicity of Zn +.

6 556 J. Hartley and others Fungal d. wt (% of Cd treatment) Cd Cd+Pb Cd+Sb Cd+Zn Cd+Pb+Sb Cd+Pb+Zn Cd+Sb+Zn Cd+Pb+Sb+Zn Cd Cd+Pb Cd+Sb Cd+Zn Cd+Pb+Sb Cd+Pb+Zn Pb+Sb+Zn Cd+Pb+Sb+Zn Cd Cd+Pb Cd+Sb Cd+Zn Cd+Pb+Sb (a) Cd+Pb+Zn Cd+Sb+Zn Cd+Pb+Sb+Zn Fungal d. wt (% of Sb treatment) Sb Sb+Cd Sb+Pb Sb+Zn Sb+Cd+Pb Sb+Cd+Zn Sb+Pb+Zn Sb+Cd+Pb+Zn Sb Sb+Cd Sb+Pb Sb+Zn Sb+Cd+Pb Sb+Cd+Zn Sb+Pb+Zn Sb+Cd+Pb+Zn Sb Sb+Cd Sb+Pb Sb+Zn Sb+Cd+Pb (b) Sb+Cd+Zn Sb+Pb+Zn Sb+Cd+Pb+Zn Fungal d. wt (% of Pb treatment) Pb Pb+Cd Pb+Sb Pb+Zn Pb+Cd+Sb Pb+Cd+Zn Pb+Sb+Zn Pb+Cd+Sb+Zn Pb Pb+Cd Pb+Sb Pb+Zn Pb+Cd+Sb Pb+Cd+Zn Pb+Sb+Zn Pb+Cd+Sb+Zn Pb Pb+Cd Pb+Sb Pb+Zn Pb+Cd+Sb (c) Pb+Cd+Zn Pb+Sb+Zn Pb+Cd+Sb+Zn Treatments Figure 3. Interactive effects of (a) Cd +,(b)sb,(c)pb +and (d) Zn +at EC values, on the growth of L. deliciosus ( ), S. variegatus ( ) and S. granulatus ( ). Each column is the mean of three replicates, bars represent the SE of the mean. Fungal d. wt (% of Zn treatment) Zn Zn+Cd Zn+Pb Zn+Sb Zn+Cd+Pb Zn+Cd+Sb Zn+Pb+Sb Zn+Cd+Pb+Sb Zn Zn+Cd Zn+Pb Zn+Sb Zn+Cd+Pb Zn+Cd+Sb Zn+Pb+Sb Zn+Cd+Pb+Sb Zn Zn+Cd Zn+Pb Zn+Sb Zn+Cd+Pb (d) Zn+Cd+Sb Zn+Pb+Sb Zn+Cd+Pb+Sb The ANOVA conducted on the three species together revealed interspecific differences in response to the metal treatments (Table 2). There were significant interactions between species Pb + (P 1) and species Zn + (P 1), showing that sensitivity to the two metals varied between ECM species (Table 2). Whilst interspecific variation in response to metal interactions was demonstrated, it was clear that for all three species Zn + decreased toxicity to Cd +. By contrast, the influence of Pb + and Sb varied significantly between the species studied. Antimony reduced the toxicity of Cd + in the presence of L. deliciosus and S. granulatus (Fig. 3b), while Pb + only reduced Cd + toxicity in the presence of S. granulatus. Furthermore, Pb + neither ameliorated nor increased the toxicity of the other metals to S. variegatus or L. deliciosus (Fig. 3c). Cd + Zn + interactions The results from expt 3 revealed the ability of Zn + to ameliorate the toxicity of Cd + to three of the four isolates tested. Nevertheless, the degree of interaction varied interspecifically and with the concentrations of metals involved. Significant amelioration of Cd + toxicity to P. involutus by Zn + (P 1) occurred at all Zn + concentrations tested ( 5 mmol m ) (Fig. 4a, Table 3). Growth was considerably inhibited at Cd + concentrations 5 mmol m, however, fungal biomass was still increased by the presence of Zn +. For example, at 1 mmol m Cd +, fungal biomass increased from 3 9 mg, in the absence of Zn +, to 19 5 mg in the presence of 5 mmol m Zn + (Fig. 4 a). This positive effect on fungal biomass production was indicated by the contour pattern above 5 mmol m Cd + (Fig. 4a). Amelioration, was also illustrated by Figure 5a where increasing Zn + concentration significantly increased the Cd + EC (P 1) (Table 4). The initial increase in EC at low levels of Zn + reflected the stimulation of growth of P. involutus at low Zn + concentrations (Fig. 5c). S. variegatus was the most sensitive of the isolates to Cd + in expt 1, with an EC of 8 6 mmol m Cd + (Table 1). The contour plot of the Cd + Zn + interactions (Fig. 4b) was therefore plotted with a limited x axis range of 5 mmol m Cd +, as growth above this concentration range was extremely low. Significant (P 1) amelioration of Cd + toxicity by Zn + was

7 Metal toxicity and in vitro production of ectomycorrhizal fungi 557 Table 2. Interactions between Cd +, Pb +, Zn + and Sb on the growth of three ECM fungal isolates, as determined by ANOVA Source Lactarius deliciosus (n 48) Suillus variegatus (n 48) S. granulatus (n 48) All species (n 144) Species n.d. n.d. n.d. 1*** Cd 1*** 1*** 1*** 1*** Pb 11* 1*** 1* 4** Sb 59 1* Zn * 1*** 1*** Species Cd n.d. n.d. n.d. 122 Species Pb n.d. n.d. n.d. 1*** Species Sb n.d. n.d. n.d. 55 Species Zn n.d. n.d. n.d. 1*** Cd Pb 2** * 544 Cd Sb 89 13* 3** 27* Cd Zn 14 2** 1*** 567 Pb Sb Pb Zn 27* 1** 4** 1** Sb Zn 44* 31* 1** 2** Cd Pb Sb 5** Cd Pb Zn Cd Sb Zn 1** 2* 6** 1*** Pb Sb Zn * 888 Cd Pb Sb Zn *** 18* Four models were used for analysis, three for each species individually and a model for all three species in combination. All data were log transformed before analysis of variance and residuals were checked for normality using the Anderson Darling Test (Minitab v. 1). Data are presented graphically in Figure 3. * P 5, ** P 1, *** P 1; n.d., not determined. determined for S. variegatus (Table 3). For example, biomass at 1 mmol m Cd + in the absence of Zn + was 8 4 mg, whilst at the same Cd + concentration, with 1 mmol m Zn +, biomass production was 21 mg (Fig. 4b). Figure 5a illustrated that whilst the EC data were lower than those of P. involutus, the pattern of response was very similar. However, the relative increase in EC compared with that at mmol m Zn + was greater for S. variegatus than that for P. involutus (Fig. 5b), with the EC of S. variegatus increasing 113-fold between and 5 mmol m Zn + compared with a 17-fold increase for P. involutus. A significant increase in Cd + EC (P 1) for S. variegatus, with increasing Zn + concentration, also occurred (Fig. 5a), where Cd + EC increased from 6 mmol m at mmol m Zn + to 6 2 mmol m at 5 mmol m Zn + (Table 4). However, Zn + did not appear to have an ameliorative effect at low Zn + (as with P. involutus) but only at concentrations of Zn + 1 mmol m. In expt 1, S. granulatus was demonstrated to be the isolate least sensitive to Cd +, and one of the least sensitive to Zn + (Table 1). The contour plot (Fig. 4c) also revealed this lack of sensitivity to Cd +. However, Zn + was only beneficial to the growth of S. granulatus at low Zn + concentrations, unlike the remainder of the ECM isolates (Fig. 4 c). Above 1 mmol m Zn + the effect was reversed, with Zn + causing an increased inhibition of fungal growth (Fig. 4c). Therefore, in contrast to the other ECM isolates, the significant effect (P 13) of Zn + on the Cd + EC was negative (Table 4). This contrasting response was illustrated in Figure 5a, where after an initial increase in EC at low Zn + concentrations, there was a sharp decrease in the presence of increasing Zn +. Growth of S. luteus in the presence of 1 mmol m Cd + was increased by the addition of 1 mmol m Zn + (Fig. 4d). For example, at 1 mmol m Cd +, biomass production increased from 22 mg in the absence of Zn +, to 48 mg in the presence of 1 mmol m Zn + (Fig. 4d). There was a significant interaction (P 1) between Cd + and Zn + in the presence of S. luteus (Table 3), and Zn + was shown to increase significantly (P 2) the Cd + EC (Table 4). This was confirmed in Figure 5a where the increase in EC was clearly illustrated up to 1 mmol m Zn +. Whilst this increase appeared relatively small compared with the other ECM isolates, Figure 5 b demonstrated that in relation to the EC at mmol m Zn + the increase was similar to that of P. involutus. The statistical analysis of data from all four species together revealed significant effects and interactions between Cd + and Zn + for the ECM isolates studied

8 558 J. Hartley and others 5 (a) 5 (b) Zn concentration (mmol m 3 ) (c) (d) Cd concentration (mmol m 3 ) Figure 4. Interactive effects of Cd + and Zn + over a range of concentrations on the growth of (a) P. involutus, (b) S. variegatus, (c) S. granulatus and (d) S. luteus. Contour plots were achieved by interpolation (using Sigmaplot v3., Jandel Corporation) of the fungal biomass values at 36 Zn + Cd + co-ordinates. Contour lines represent fungal biomass (mg) at fixed intervals. Table 3. Interactions between Cd + and Zn + over a range of concentrations on the growth of four ECM fungal isolates, as determined by ANOVA (Minitab v. 1) Source Paxillus involutus (n 18) Suillus variegatus (n 18) S. granulatus (n 18) S. luteus (n 18) All species (n 432) Species n.d. n.d. n.d. n.d. 1*** Cd 1*** 1*** 1*** 1*** 1*** Zn 1*** 1*** 1*** 1*** 1*** Cd species n.d. n.d. n.d. n.d. 1*** Zn species n.d. n.d. n.d. n.d. 9** Cd Zn 1*** 1*** 6** 1** 1*** Cd Zn species n.d. n.d. n.d. n.d. 1*** Four models were used for analysis, three for each species individually and a model for all three species in combination. Data are presented graphically in Figure 4. ** P 1, *** P 1; n.d., not determined. (Table 3). From the results discussed above, both Cd + and Zn + have been shown to have significant effects on biomass production of ECM fungi. However, ANOVA showed significant interspecific variation in response of the ECM isolates to Cd + (P 1), Zn + (P 9) and Cd + Zn + interaction (P 1) (Table 3). The values of Cd + EC also varied interspecifically (P 1), as did the effect of Zn + on Cd + EC values (P 1) (Table 4).

9 Metal toxicity and in vitro production of ectomycorrhizal fungi 559 Cd EC 5 (mmol m 3 ) (a) 1 Cd EC 5 /(Cd EC 5 at mmol m 3 Zn) (b) 8 (c) Fungal d. wt (mg) Zn concentration (mmol m 3 ) Figure 5. (a) Cd +EC values of four ECM fungi over a range of Zn + concentrations. (b) Relative Cd + EC values (compared to EC at mmol m Zn +) of ECM fungi over a range of Zn + concentrations. (c) Response to Zn + of ECM fungi in the absence of Cd +. S. variegatus ( ), P. involutus ( ), S. luteus ( ) and S. granulatus ( ). Table 4. The effects and interactions of Zn + concentration on Cd + EC values of three ECM fungi, analysed using individual models and a combined model by ANOVA Factor Paxillus involutus (n 6) Suillus variegatus (n 6) S. granulatus (n 6) S. luteus (n 6) All species (n 24) Species n.d. n.d. n.d. n.d. 1*** Zn 1*** 1*** 13* 2** 1*** Species Zn n.d. n.d. n.d. n.d. 1*** All data were log transformed (except where stated) before ANOVA and residuals were checked for normality using the Anderson Darling Test (Minitab v. 1). Data are presented graphically in Figure 5. Data are square root transformed; * P 5, ** P 1, *** P 1; n.d. not determined.

10 56 J. Hartley and others DISCUSSION This study demonstrated that the response of ECM fungi to toxic metals varied widely, and that the response depended on the ECM fungal species, the metal concentration in the growth media and the presence of other toxic metals in the growth media. Sensitivity of the four ECM fungi to Cd + was shown to vary significantly with EC values differing by over three orders of magnitude (Table 1). Interspecific variation had also been demonstrated by a number of other authors for axenically cultured ECM. For example, the Cd + EC of P. involutus was 3 mmol m Cd + (Darlington & Rauser, 1988), whilst the EC of Amanita muscaria was between mmol m Cd + (McCreight & Schroeder, 1982). Sensitivity to Zn + was less variable between the four ECM isolates (Table 1, Fig. 2 d), which suggested a similar physiological response to Zn + exposure existed for the four species. However, other workers found wide variation in sensitivity of ECM fungi to Zn +, with EC values ranging from mmol m in agar media and mmol m in liquid media (Brown & Wilkins, 1985; Denny & Wilkins, 1987; Tam, 1995). In the present study, L. deliciosus was the only isolate which was sensitive to Pb +, with an EC of 45 6 mmol m. Growth of the remainder of the isolates was not significantly inhibited by Pb +, but not significantly so (Fig. 2c). This was in contrast with some studies where Pb + EC values ranged from 1 to 255 mmol m for a number of species (McCreight & Schroeder, 1982). The same pattern was true for Sb where only L. deliciosus demonstrated a significant response to the metal. However, whilst sensitivity to Pb + and Sb was low, later experiments showed that their presence in the growth media could alter Cd + toxicity (Fig. 3). The lack of growth inhibition of ECM fungi by Pb + and Sb could have been a result of low solubility of the two metals in solution, especially given the high phosphate status of the growth media (Hartley et al., 1997). However, the alteration of Cd + toxicity (both amelioration and enhanced toxicity) in the presence of the two metals suggested that they were in a form available to ECM fungi. Our study showed wide variation in sensitivity to metals was present in species from uncontaminated populations. A number of studies had compared the response of ECM isolates from contaminated and uncontaminated sites in an attempt to relate metal sensitivity to the contamination of the soil of origin. The results of the investigations were, however, contradictory. Ten isolates of P. involutus, from a range of Zn + contaminated and uncontaminated soils, were shown to have no relationship between their sensitivity and indices of contamination of the soil of origin (Denny & Wilkins, 1987). By contrast, however, the sensitivity of a range of P. tinctorius isolates to Al +, was related to the Al + status of the soil of origin (Egerton-Warburton & Griffin, 1995). Interaction between metals influences their relative toxicity to ECM fungi. Inhibition of growth caused by multiple metals can not, therefore, be predicted from their individual toxicity (Figs 3, 4). Metal interactions vary between ECM fungal species (Fig. 3, Table 2); however, the result of most interactions was reduced toxicity compared to the toxicity of individual metals. Antimony ameliorated the toxicity of Cd + for all three ECM fungi and the mixture of Cd + Pb + Zn + Sb was only slightly more toxic to the three fungi tested than the individual Cd + treatment (Fig. 3, Table 2). This would suggest that soils contaminated by a mixture of metals might not be as toxic to ECM fungi as predicted from toxicity assessment of individual metals. At certain concentrations, the presence of Zn + significantly reduced the toxicity of Cd + to some ECM fungi (Table 3). The influence of Zn + varied between ECM species and appeared to be dependent on the initial sensitivity of the isolate (Fig. 4). For example, S. variegatus was shown to be highly sensitive to Cd + in the absence of Zn + (EC 4 mmol m ) yet in the presence of 5 mmol m Zn + the Cd + EC increased significantly to 6 5 mmol m (Table 4, Fig. 5). By contrast, whilst S. granulatus was significantly less sensitive to Cd + in the absence of Zn + (EC 22 mmol m ), in the presence of 5 mmol m Zn + the Cd + EC decreased to 4 6 mmol m. This amelioration by Zn + was also recorded in the basidiomycete A. aegerita where increasing the Zn + concentration in agar media suppressed Cd + translocation into the mycelium (Brunnert & Zadrazil, 1985). Furthermore, Colpaert & Van Assche (1992) recorded amelioration by Zn + of Cd + toxicity to the ECM fungus S. bovinus, where 765 mmol m Zn + reduced the toxicity of 8 9 and 89 mmol m Cd +. Competitive interactions between metals have also been documented in plants. Cataldo, Garland & Wildung (1983) reported that root absorption of Cd + was competitively inhibited by Zn +, Cu +and Mn +, whilst in Holcus lanatus L. Cd + was shown to interact with both Zn + and Pb + (Symeonidis & Karataglis, 1992). There are a number of possible explanations for the interactions between Cd + and Zn + reported here. The plasma membrane is the main selective barrier to influx efflux of cations and anions and is the principal site of active transport (Meharg & Macnair, 1992). Ions being transported from the external solution to the cytoplasm bind to transport sites in the plasma membrane. It is, therefore, possible that competition between ions of the same valency or size may occur, assuming that the number of binding sites is small in relation to the con-

11 Metal toxicity and in vitro production of ectomycorrhizal fungi 561 centration of competing ions (Marschner, 1995). Studies in both fungi and plants have demonstrated that at low concentrations metals are taken up by high affinity transporters, whilst at high concentrations a general divalent cation transporter appears to operate (Clarkson & Lu ttge, 1989; Gadd, 1993). It is possible, therefore, that interactions between Cd + and Zn + would vary dependent on the concentration of metals involved. At high Zn + concentrations amelioration of Cd + toxicity, for three out of four of the ECM species in this study, was highly significant (Fig. 4). If a general transporter is operating for both metals, then the high Zn + concentrations, in comparison to Cd + could result in preferential uptake of Zn + through competitive inhibition of Cd + uptake. Also, as Zn + is an essential element for fungal nutrition, divalent metal transporters might have a higher affinity for Zn + than for the non-essential Cd +. A second hypothesis could be that a physiological response in ECM fungi is induced by the presence of Zn +, but not Cd +. This response might result in decreased sensitivity to Zn +, and coincidentally confer decreased Cd + sensitivity to the ECM fungus. A range of mechanisms have been proposed by which ECM fungi protect themselves and their hosts against metal toxicity. These fall into four main categories: reduced influx; extracellular binding; intracellular compartmentation and complexation with polyphosphate granules; and intracellular chelation (Hartley et al., 1997). It appears from the results of expt 1 that ECM fungi do not all possess the same physiological responses, or at least have differing expression of physiological responses for metal exposure, as some species are much less sensitive to metals than others. It is possible that reduced influx could be induced by the presence of high levels of Zn + through feedback regulation of the Zn + transporters (Marschner, 1995), and if a common transporter is in operation this would also decrease the concentration of Cd + entering the fungus and so decrease Cd + toxicity. Liquid media was chosen as the growth substrate as opposed to agar media, as there was some evidence that complexation of metals within the substrate matrix could occur in agar media (Gadd, 1983; Hartley et al., 1997). Nevertheless, in both liquid and agar media, phosphate at high concentrations might form precipitates with toxic metals, thus reducing their availability to the fungus. For example, concentrations of phosphate 2 1 mol m were found to reduce significantly the toxicity of Cd + to Rhizobium spp. (Angle, McGrath & Chaudri, 1992). The concentration of phosphate used in liquid MMN in this study was 6 mol m, this would therefore, be expected to have some effect on the availability and toxicity of the metals studied. Liquid media would still, however, be a valid substrate for comparison between ECM species as relative differences in sensitivity could still be assessed (Hartley et al., 1997). The addition of NO at various concentrations with the metal treatments, added as NO salts, could also have affected the results of this study. It was possible that NO stimulated the growth of the ECM fungal isolates. However, the concentration of NO - N added as metal salts were 2 mol m, compared with concentrations of NH+ -N in the media of 7 5 mol m. Ammonium is also the preferred N source of ECM fungi (Smith & Read, 1997) and as fungal NO transporters are repressed in the presence of NH+ (Schloemer & Garrett, 1974), it is unlikely that the relatively small additions of NO would have a significant effect on fungal growth. CONCLUSIONS In summary, whilst single metal studies are essential for unravelling the complex relationships between ECM fungi and potentially toxic metals, it is likely that these studies would have little practical relevance for predicting the effects of multiple metal exposure on ECM fungi in contaminated soils. Potentially toxic metals are very rarely present singly in the environment, but occur in mixtures through mineral ore deposits and as a result of anthropogenic pollution. Further work is required to examine the interactive effects of metals on ECM fungi in symbiosis with host plants. The relationship between metal sensitivity of ECM fungi in pure culture and symbiosis is unclear for single metals (Jones & Hutchinson, 1988; Hartley et al., 1997) and uninvestigated for multiple metals. Further research is, therefore, also needed in this area. REFERENCES Alloway BJ Heavy metals in soils. London: Blackie and Sons. 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