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Intraspecific variability in growth response to cadmium of the wood-rotting fungus Piptoporus betulinus

Petr Baldrian ¹
Jií Gabriel

     Laboratory of Biochemistry of the Wood-rotting Fungi, Institute of Microbiology AS CR, Vídeská 1083, 14220, Prague 4, Czech Republic

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

The intraspecific variability in growth response to cadmium (Cd) on agar media and in liquid culture was studied among fourteen strains of a wood-rotting fungus Piptoporus betulinus. The variability of Cd tolerance was found to be very high. The ED₅₀ ranged from 6.8 µM Cd in the most sensitive strain, up to 255.1 µM in the most resistant one. On agar media the addition of Cd to nutrient media resulted in reduction of relative growth rate and increased lag time. While the reduction of growth rate was already apparent at 10 µM Cd, the lag time was significantly increased in higher Cd concentrations. Five strains of P. betulinus failed to grow at 250 µM Cd and none grew at 500 µM metal. Biomass production in liquid culture was less sensitive to addition of Cd than the growth rate on solid media. At 100 µM Cd the radial growth rate of the mycelium was reduced to 27%, whereas the dry mass of mycelium was 77% of the respective control value. A group of four Cd-sensitive strains was found, showing low metal tolerance both on solid media and in liquid cultures. Although the isolates originated from sites with different Cd-pollution level, no correlation between level of Cd-pollution and resistance (ED₅₀) was found. The growth rate of fourteen tested strains displayed lower variability than biomass production, showing that radial growth rate is more species-specific and therefore more valuable for interspecific comparisons of growth response.

Key words: biomass production, heavy metals, Piptoporus betulinus, radial growth rate

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Human activities result in the increased release of heavy metals in the environment. Heavy metals in atmosphere, soils, water, and sediments pose a serious problem: they can enter and pass through the food chains, and—in contrast to organic xenobiotics—cannot be degraded by microorganisms. The problems with metal contamination are particularly pronounced in localities where industrial exploitation has led to accumulation of extreme concentrations of these substances, like the surroundings of smelters, tanneries, waste treatment plants, or mining sites. The concentrations of heavy metals in such places are high, and their influence on ecosystems obvious (Nordgren et al 1983 , Giller et al 1998 )

Like other microorganisms, fungi, including basidiomycetes, face the problem of metal contamination. Until now, mycological research concerning metal stress was mainly focused on the accumulation of metals in fungal fruiting bodies (Allen and Steinnes 1978 , Gast et al 1988 ), the study of heavy metal impact on mycorrhizal associations (Jones and Hutchinson 1986 , Colpaert and van Assche 1993 , Hartley et al 1997b, 1999 ) and on fungal communities in soil (Nordgren et al 1983 , Del Val et al 1999 ). The investigation of physiological consequences of metal contamination in fungi showed that there are several mechanisms of active or passive defense, which can help fungi tolerate metal toxicity. These systems include extracellular chelation of metals by extracellular compounds, binding of metals to components of the cell wall, intracellular detoxification by binding of metals to phytochelatins and metallothionein-like proteins, or sequestration of metals in vacuoles or within cytoplasmic inclusions (Gadd 1993 , Tomsett 1993 ). Although these defense mechanisms exist, fungi cannot completely evade metal toxicity. The toxicity of metals is mostly due to their binding to biomolecules, including enzymes, and the induction of oxidative stress (Vallee and Ulmer 1972 , Stohs and Bagchi 1995 ). The metals thus interfere with many different physiological processes in fungi in a concentration-dependent manner.

From the viewpoint of metal toxicity, the growth response has been mostly studied as a complex physiological process, which is in direct relation to the ability of fungi to colonize the substrate, to spread in the ecosystem, and to exploit its resources. The studies of growth response to metals were mainly focused on interspecific comparisons, both in lower fungi (Arnebrant et al 1987 , Weissenhorn et al 1993 , Plaza et al 1998 , Del Val et al 1999 ) and basidiomycetes, among them mostly ectomycorrhizal species (McCreight and Schroeder 1982 , Willenborg et al 1990 , Hartley et al 1997a ).

Growing interest in wood-rotting fungi led recently to the first studies of their metal tolerance (Sanglimsuwan et al 1993 , Baldrian and Gabriel 1997 , Mandal et al 1998 ). Although wood-rotting fungi usually have no direct contact with soil—which is usually the main source of metals for saprophytic species—they have to cope with the metal input from the atmosphere. The understanding of their growth response in metal-contaminated environments is particularly important because of their potential use in bioremediation technologies in contaminated soil (Paszczynski and Crawford 1995 ). The ability of fungi to grow in contaminated environments is necessary for substrate colonization and long-term survival and therefore crucial for their use in in situ bioremediation technologies (Baldrian et al 2000 ). It was found that wood-rotting fungi differ significantly in their growth response to heavy metals (Baldrian and Gabriel 1997 , Mandal et al 1998 ). However, there is no information about intraspecific variability of metal sensitivities, so the results obtained with one strain can therefore hardly be extended to the whole species. Since wood-rotting fungi can grow well and produce fruiting bodies even in an environment with a high level of metal pollution (Gabriel et al 1997 ), the question arises whether more metal-resistant strains can be found on such sites in nature. Some results indicate that resistant fungal strains might be selected at contaminated sites (Gildon and Tinker 1981 , Arnebrant et al 1987 , Weissenhorn et al 1993 ).

In our previous work (Baldrian and Gabriel 1997 ) we found the wood-rotting basidiomycete Piptoporus betulinus (Bull. : Fr.) P. Karst. sensitive to low concentrations of Cd, although the species in nature grows even in highly contaminated areas. In this study we concentrated on the analysis of growth response of P. betulinus to Cd, with the aim to describe the intraspecific variability of growth response to a heavy metal within a species. The next aim was to look for the occurrence of metal-resistant isolates of wood rotting fungi on metal-contaminated sites. Cd was chosen since it is a nonessential, highly toxic metal element and one of the most important contaminants of the atmosphere.

As there is no direct contact with soil, the main source of metals in mycelium of wood-rotting fungi is atmospheric deposition (Gabriel et al 1997 , Baldrian et al 1999 ). The transport of heavy metals from soil through stem to fungal fruiting bodies is negligible due to transport barriers at soil/root, root/vascular tissue and wood/mycelium interfaces. Therefore, the isolates were collected in sites differing in Cd pollution of the atmosphere. The common occurrence of P. betulinus enabled us to isolate sufficient number of strains from different sites (Table I ). In addition to freshly isolated strains, a strain from the culture collection used in our previous work was involved in the study.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation and maintenance of cultures – Fungi were aseptically isolated on petri dishes with MEG medium (malt extract 20 g L^-1, glucose 20 g L^-1, agar 25 g L^-1, pH adjusted to 6.0 with NaOH prior to autoclaving) containing 200 mg L^-1 streptomycin and 10 mg L^-1 benomyl. The fruiting body was cut and a piece of flesh was placed on MEG agar using sterile techniques. After two weeks, strains were reinoculated on petri dishes with fresh GC medium (corn steep 7 g L^-1, glucose 20 g L^-1, agar 25 g L^-1, pH 6.0). After 7 d at 28 C, colonized petri dishes were transferred to 4 C and used as stock cultures. Every two months, fungi were re-inoculated to fresh medium. The strain P. betulinus CCBAS585 was maintained on GC medium by the same procedure.

Growth on solid media – For growth experiments MM medium was used containing the following (per L): MgSO₄·7H₂O 0.50 g, KH₂PO₄ 0.60 g, K₂HPO₄ 0.40 g, (NH₄)₂SO₄ 0.50 g, glucose 20 g, agar 20 g, with pH adjusted with NaOH to 6.0 prior to autoclaving. Cd was added to the medium as Cd(NO₃)₂ before autoclaving to final concentrations (w/v) 10 µM, 50 µM, 100 µM, 250 µM, and 500 µM. Five replicates of each concentration and control (without Cd) were used. The plates were inoculated with 7-mm agar plugs from fungal colonies, pre-grown on MM agar for 8 d at 28 C. The fungus was grown at 28 C in the dark. Maximum colony diameters were measured at 24–72 h-intervals. All chemicals used for media preparation were of analytical grade, malt extract was supplied by Sigma, corn steep was from ZD Boleráz (Slovak Republic).

Biomass production – Fifteen mL of liquid MM medium (without agar), containing Cd(NO₃)₂ in concentrations (w/v) 10 µM, 100 µM, and control (without Cd) were placed in 100-mL Erlenmeyer flasks. Each flask was inoculated with two 7-mm agar plugs from the edge of an actively growing colony, precultivated on MM agar for 8 d at 28 C. The liquid stationary cultivation proceeded at 28 C in the dark. Three replicates of each concentration were used. After 12 d, mycelia were harvested, washed with distilled water and dried at 105 C to constant mass. After cooling to room temperature, mycelia were weighed. The mass of agar plugs used as inoculum was subtracted. The results were expressed in µg per mL of the cultivation medium.

Calculation of growth characteristics – From colony diameters and dry biomass, measured in growth experiments, strain specific growth characteristics were calculated. Radial growth rate (K_r) was defined as K_r = (R₁ - R₀)/(t₁ - t₀) (Trinci 1971 ), where R₀ and R₁ are the colony radii at time t₀ and t₁, respectively. The K_r was calculated by linear regression of colony radius versus time during the phase of linear growth. Lag time (T_l) was determined from the linear extrapolate of linear growth phase to the initial colony diameter. Radial growth rate, lag time, and biomass dry mass were also expressed as percentages of values of the metal-free controls. Effective dose inhibiting radial growth rate by 50% (ED₅₀) values were calculated from dose-response curves (regression of growth rate vs Cd concentration). Statistical analyses (linear regression, Students t-test, and the frequency analysis combined with ²-test for distribution fitting) were performed by the use of statistical software Microcal^TM Origin^TM 5.0 Professional (Microcal Software).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Effect of Cd on fungal growth and biomass production – The impact of metal on different stages of the growth curve was apparent even at the lower metal concentrations used. Figure 1 shows that the presence of Cd (50, 100, or 250 µM) in growth media caused reduction of radial growth rate and increased the length of the lag phase. The reduction of K_r was present already in the lowest Cd concentration used (10 µM) (Table II ). Although there was a high variability among the strains tested and some strains had no K_r reduction at 10 µM Cd, in average the K_r was reduced to 73% of control. At 50 and 100 µM Cd, the average radial growth rate was reduced to 47 and 27% of the growth rate of control, respectively. Five of fourteen strains failed to grow at 250 µM Cd and none of the tested strains grew at 500 µM.

View larger version (23K): [in this window] [in a new window]    FIG. 1. Growth of selected strains of Piptoporus betulinus on solid agar media supplemented with 0 µM, 50 µM, 100 µM, and 250 µM of cadmium nitrate. The data plots represent means of five replicates

The variance of growth rates and lag times among isolates increased with increasing metal concentration. In higher Cd concentrations, there was also higher variability in growth rates among replicates of the same strain. While in the control the standard error (SE) of K_r within one strain was less than 3%, at 100 and 250 µM Cd, the SE was 3–10% of the mean. A similar phenomenon was observed with T_l.

The morphology of mycelial colonies was not affected by Cd at concentrations up to 50 µM. Here, a relatively sparse mycelium forming circular colonies was observed. At 100 µM, and even more at 250 µM Cd, the mycelium was much more dense, sometimes beginning to expand from one side of the inoculation plug with a circular colony forming later. No differences in pigmentation were observed between metal-treated and control mycelia, except that the mycelium at higher Cd concentrations was slightly darker.

In the biomass production experiment, strains of P. betulinus were grown in liquid MM media without agitation until the end of the stationary phase of growth (12 d). A mycelial mat similar to an agar colony developed on the surface of the medium during the cultivation. In contrast to inhibition of K_r, no inhibition of biomass production was found at 10 µM Cd (Table II ). Also at 100 µM Cd, the decrease of dry mass of mycelium was not found in all tested strains. The mean dry mass of mycelium was decreased to 77% of control. If we compare the reduction of radial growth rate and of mycelial dry mass at 100 µM Cd, it is apparent that the radial growth rate is much more sensitive to Cd than biomass production. In liquid culture, the mycelium formed a mat, which in the control and at 10 µM Cd extended wide, whereas at 100 µM Cd the "colony" was smaller in size, but more dense.

Intraspecific variability of Cd response in P. betulinus – Values of K_r, T_l, biomass production, and ED₅₀ for all fourteen strains tested in this work are summarized in Table II . The strains differed not only in their sensitivity towards Cd, but also in their growth rate on metal-free media. The K_r of the controls varied between 0.239 and 0.426 mm h^-1 and the biomass production ranged from 4.50 to 19.50 µg mL^-1. Distribution of both variables within the species fits best with the normal distribution, but the range of estimated dry mass values were much broader with the maximum being almost four times higher than the minimum. There was also a broad range of T_l values among the strains tested, indicating that there was a difference in the time it took to adapt to fresh culture medium before growing at a linear rate.

When Cd was present in the growth media, the differences between strains were more pronounced. For example, the K_r of the most sensitive strain OR was already reduced to 25% of the control at 10 µM Cd. However, this was an exception, as the second most sensitive strain—CCBAS—was able to grow at 52% of the K_r of control and the mean for all strains was 76% of control growth. The strain SS grew at the same rate in 10 µM Cd as in medium without Cd. The inhibition of growth rates was more pronounced at 50 µM Cd, where the mean growth rate of tested strains was lower than 50% of control (Table II ). All strains were able to grow at 100 µM Cd, although the radial growth rate of strains KR, DP and OY was more than 20 times lower than in media without metal. Although most strains were able to grow at 250 µM Cd, none of them could grow at 500 µM Cd. It is particularly interesting in the cases of strains KR and LT, which at 250 µM Cd grew at 40 and 52% of the control growth rates, respectively. Furthermore, the latter of these two strains had its estimated ED₅₀ at higher than 250 µM. A very broad range of ED₅₀ values was found among the strains. The highest value of the effective dose that limited the growth of the mycelium by 50% was more than 30 times higher than the value of the most sensitive strain. The distribution of ED₅₀ within the species follows a log-normal distribution with most strains having a value close to median.

Biomass production among strains was much more variable than K_r. For three strains (DE, P, and SS) dry mass after cultivation in 100 µM Cd was comparable to or greater than in metal-free media. There was a group of four Cd-sensitive strains (OR, CCBAS, OY, and CM) showing the lowest ED₅₀ values and also the greatest inhibition of biomass production at 100 µM Cd. In contrast to most other strains their dry masses were also substantially decreased after cultivation in 10 µM Cd.

The Cd-resistance of strains was found to be independent of the pollution level at the site of origin. The most resistant strain based on the ED₅₀ calculation was LT from a highly polluted site, but only one of four sensitive strains (OR) originated from a site with low pollution levels, whereas the three others were found on sites with relatively high levels of Cd deposition.

Interactions between growth variables – To elucidate the interrelations between different growth variables on Cd resistance/sensitivity of the fungus, the interactions between them were analyzed by regression analysis. The results are summarized in Table III . The growth performance of P. betulinus strains was compared at 0 and 100 µM Cd, where all strains were able to grow. There was a significant negative correlation between the K_r values and the length of the lag phase at 100 µM Cd in both the presence and the absence of metals. The faster-growing strains exhibited shorter lag phases. Interestingly, the radial growth rates at 100 µM and in the control were not found to be dependent. This is also confirmed by the independence of ED₅₀ and the K_r at 0 µM Cd. It means that the fast growing strains are not the most tolerant. Absolute and relative K_r at 100 µM were found to have a strong correlation with the ED₅₀. This is natural, since both values reflect fungal tolerance to Cd. The length of the lag phase on Cd decreased with the increasing tolerance of the fungus both absolutely and relatively. No correlation was confirmed between T_l at 0 and 100 µM Cd and between the growth variables in solid state cultivation and the biomass production in the liquid culture except in the four metal-sensitive strains. The inhibition of dry mass was lower in strains exhibiting higher dry mass yields in control, although direct correlation between dry mass in control and on Cd was not observed.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The effect of Cd on the growth curve of P. betulinus consisted of two components: the extension of the initial lag phase and the reduction of radial growth rate. A linear relationship between the rate of radial growth and the logarithm of Cd concentration as proposed by Trinci and Gull (1970) was observed. Whereas the reduction of growth rate is a typical response of fungi to toxicants (Gadd et al 1993 ), the lengthening of the lag phase is not always present. Jones and Hutchinson (1988) found an increase in lag time among different ectomycorrhizal basidiomycetes cultivated on Zn- and Cd-amended media. On the other hand, Darlington and Rauser (1988) did not find any dependence of T_l on Cd concentration in Paxillus involutus (Batch : Fr.) Fr. It seems likely that the lag time is increased significantly only in nutrient low media or when inoculation proceeds from a nutrient low medium (Mandal et al 1998 ). Because nutrient limitation is common under natural conditions, the increase of T_l seems to be an important factor, affecting substrate colonization. The very long lag times found in our experiments could be the cause of the inability of P. betulinus to grow at more than 250 ppm Cd, since the adaptation to growth in metal-containing medium has to occur prior to the death of the mycelium. No correlation between lag times and growth rates of different strains in controls and in metal-containing media shows that fast growth rate and quick adaptation on the fresh medium do not ensure fast growth and adaptation in the presence of Cd.

Biomass production of the fungus was less affected than the radial growth rate by Cd. The mycelia grown at higher Cd concentrations both in liquid culture and on agar plates were more dense, which can probably be attributed to changes in hyphal branching. This is in agreement with the results of Darlington and Rauser (1988) , who found that in Paxillus involutus Cd decreases the rate of elongation of fungal hyphae, but significantly decreases the distance between branch points and increases the number of laterals per branch point. Moreover, in liquid culture, where P. betulinus forms spherical pellets, pellets in absence of metals are "hairy" with loose, longer hyphae, whereas in the presence of Cd the surface of a pellet is smooth, formed by a dense layer of hyphal tips. Also in other wood-rotting fungi such as Schizophyllum commune Fr : Fr. and Daedalea quercina (L.) Pers. Cd increases the density of mycelium by increased branching (Lilly et al 1992 , Gabriel et al 1996 ). It was demonstrated that the K_r of a fungal colony is related to the specific growth rate obtained in submerged culture by the equation K_r = w x µ, where w is the width of peripheral growth zone and µ is the specific growth rate (Trinci 1971 ). As the hyphal branching only occurs in the peripheral growth zone, Cd probably decreases the width of the peripheral growth zone (Darlington and Rauser 1988 ), as is also the case with other inhibitors (Trinci 1985 ). Although the inhibition of radial growth rate and biomass production were found to be independent, strains with the lowest ED₅₀ values (i.e., the highest sensitivity to metal estimated on solid medium) also showed the highest inhibition of dry mass in liquid culture.

In recent decades, much attention has been paid to the effect of metals on the growth of fungi, mostly soil fungi (Arnebrant et al 1987 , Plaza et al 1998 ), and mycorrhizal species (reviewed in Hartley et al 1997a ). Different methods used by different authors for the measurement of growth and the use of different media make any comparisons between published works practically impossible. In some comparative studies it was proposed that fungal species differ in their sensitivities to metals (Baldrian and Gabriel 1997 , Hartley et al 1997a , Plaza et al 1998 ). However, our experiments show that there is high intraspecific variability in metal response. The highest value of ED₅₀ estimated in our experiments was 40x higher than the lowest one and similar results were obtained with biomass production. Therefore, it is necessary to use more strains from each species in interspecific comparisons.

Intraspecific variability of growth and metal response was studied in ectomycorrhizal basidiomycetes with aluminum (Leski et al 1995 , Rudawska and Leski 1998 ). Fungal strains studied exhibited tolerance to very high Al concentrations, with minor intraspecific variability, which is in opposite to our results obtained with Cd.

In some cases, metal-tolerant fungal strains were isolated from contaminated sites (Gildon and Tinker 1981 , Arnebrant et al 1987 ) and tolerance of several fungi was increased by repeated cultivation on increasing metal concentrations (Ashida 1965 , Garcia-Toledo et al 1985 ). In addition, the fact that the content of metals in fungal fruiting bodies reflects the metal concentrations in their environment (Gast et al 1988 , Gabriel et al 1997 ) raised the question of whether fungal strains growing on polluted sites are more tolerant than fungi from nonpolluted areas. This was particularly studied in ectomycorrhizal fungi. However, although some authors found that the strains isolated from contaminated soils were more tolerant than strains from unpolluted regions (Gildon and Tinker 1981 , Weissenhorn et al 1994 , Leski et al 1995 ), there are also studies that do not confirm this (Brown and Wilkins 1985 , Denny and Wilkins 1987 , Jones and Hutchinson 1988 , Howe et al 1997 , Rudawska and Leski 1998 ). In all cases, reported differences between strains from polluted and unpolluted sites were low. One reason may be that fungi are tolerant to higher concentrations of metals than are present in contaminated areas. In P. betulinus, higher resistance to Cd was not common for strains originating from metal-contaminated sites. Thus, the presence of Cd may not act as a selective pressure for Cd resistance. In fruiting bodies of P. betulinus collected in the Czech Republic, the concentration of Cd did not exceed 5 µg g^-1 dry mass (Baldrian et al 1999 ). In experiments with metal accumulation, this concentration of metal in fungal mycelium corresponded to 1 µM Cd in the growth medium (Gabriel et al 1996 ). It must be also taken into account that the contamination in polluted sites is usually not caused by a single metal and that the selection is probably driven either by the most toxic element or by more metals acting synergistically. Three of the four most Cd-sensitive strains in this work were collected in areas with a high level of Cd contamination. Similarly, Howe et al (1997) isolated a strain of Scleroderma citrinum (Pers.) from copper-polluted soil that was unable to grow even at a 10-times lower copper concentration than all other fungal strains tested.

Although under natural conditions fungi are usually not limited in growth by metals in the environment, there are locations with extreme metal contents, where the effect on fungal growth is obvious (Nordgren et al 1983 ). It has to be added that metal impact on fungal physiology is not only limited to alteration of growth. In particular, the activity of extracellular enzymes including ligninolytic complex enzymes (Baldrian et al 1996, 2000 ) and germination of spores (Leyval et al 1994 ) were found to be inhibited at metal concentrations substantially lower than those affecting fungal growth. Therefore, more research in the field of fungal ecology in metal-polluted habitats should be helpful.

	ACKNOWLEDGMENTS

 
We thank I. Stoychev and J. Velík for help with collection and isolation of fungal strains.

This work was supported by grant 204/99/1528 from the Grant Agency of the Czech Republic.

	FOOTNOTES

 
¹ Corresponding author, baldrian{at}biomed.cas.cz

Accepted for publication September 25, 2001.

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