Annals of Microbiology, 50, 157-166 (2000) Genetic polymorphism and metal sensitivity of Oidiodendron maius strains isolated from polluted soil* I. LACOURT1, S. D’ANGELO1, M. GIRLANDA1, K. TURNAU3, P. BONFANTE 1,2, S. PEROTTO2,4** 1 Dipartimento di Biologia Vegetale, and 2 Centro di Studio sulla Micologia del Terreno, Viale Mattioli 25, 10125 Torino, Italy. 3 Department of Plant Taxonomy and Phytogeography, Institute of Botany of the Jagiellonian University, ul. Lubicz 46, PL-31-512 Cracow, Poland. 4 Istituto di Meteorologia e Oceonografia, I.U.N., Via De Gasperi 5, 80133 Napoli, Italy. Abstract - Mycorrhizal isolates of Oidiodendron maius, recovered from Vaccinium myrtillus that recolonized experimental plots polluted with different mixtures of toxic metals in the Niepolomice forest (Poland), were investigated for their genetic polymorphism and their ability to grow on metal ions. RAPD fingerprinting revealed the presence of distinct fungal genets. The same genet could be found in plots polluted with different metal mixtures. Growth on culture media containing zinc, cadmium or aluminium ions showed that isolates collected on the polluted site were in general less sensitive to metals than strains of O. maius from non-polluted soils. However, their response on the different metal ions could not be correlated with the plot of origin or with the RAPD cluster. O. maius isolates with high and stable metal tolerance could be useful for bioremediation or to investigate the mechanisms of tolerance. Key words: Oidiodendron maius, ericoid fungi, polluted soils, polymorphism, heavy metal. INTRODUCTION Increase of heavy metal pollution has stimulated the development of bioremediation strategies using tolerant organisms able to grow in the presence of heavy metals and to extract them from soils (Ow, 1996). Microorganisms possess a natural ability to resist and adapt to toxic metals, although plants are often preferred in bioremediation because they can be easily harvested once they have fixed met- * Research was funded by Progetto Finalizzato Biotecnologie, CNR (Subproject 2), by MIPA Project N. 451 and by Italian MURST. I.L. was supported by a fellowshinp of the Fondazione per le Biotecnologie. ** Corresponding author. Phone: +39-0116502927; Fax: +39-0116707459; e-mail: perotto@bioveg.unito.it 157 als. Mycorrhizal fungi could be a significant component of bioremediation systems against heavy metal pollution (Donnelly and Fletcher, 1994), since most plants are naturally associated with these symbiotic fungi and often display better heavy metal tolerance than non mycorrhizal plants (Galli et al., 1994). Ericaceous plants are able to grow on acidic nutrient poor soils, and their fine hair roots are colonized by soilborne fungi that play an important role in mineral cycling and plant nutrition (Read, 1991) They can also grow on soils containing high amounts of toxic metals (Marrs and Bannister, 1978; Oxbrow and Moffatt, 1979). Bradley et al., (1981) demonstrated that increased tolerance of Calluna vulgaris to zinc and copper was due to their mycorrhizal association. Ericaceous mycorrhizal plants (Vaccinium myrtillus) were among the first to grow on a site experimentally polluted with heavy metals (Turnau, 1991). In that experiment, started in 1980, dusts collected from industrial filters and containing different combinations of toxic metals were applied separately on 240 m2 plots in the Niepolomice forest in Poland (Greszta et al., 1987). In 1995, two ericoid mycorrhizal fungi were isolated and identified as Oidiodendron maius. They could grow in vitro on zinc concentrations than prevented growth of isolates from non-polluted soils (Martino et al., 2000). Thirteen more isolates were collected two years later in the same site. Randomly amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic diversity of all isolates and to determine if specific clones were associated with the different dust treatments. Moreover, growth of the isolates was tested on media containing different concentrations of zinc, cadmium or aluminium, the most toxic components in the industrial dusts applied to the forest plots. MATERIALS AND METHODS Fungal isolates and culture conditions. The fungal isolates used in this study are listed in Table 1. Isolate C-1 of O. maius was derived from a non-polluted soil in Italy (Perotto et al., 1996) and was used for comparison. Fifteen isolates were obtained from Vaccinium myrtillus plants growing in the experimental site of Niepolomice forest (Poland), whereas C-2 was isolated from a natural site a few kilometers away. The percentage of zinc, cadmium and aluminium in the dusts were respectively (Greszta et al., 1987): 22.06% ZnO, 0.63% CdO and 8.13% Al2O3 (zinc plot); 3.02% CdO, 1.75% ZnO and 21.83% Al2O3 (cadmium plot); 21.42% Al2O3 and 0.01% ZnO (aluminium plot). Fungal isolates were maintained on 2% malt, 1% agar medium at 24 °C in the dark. To test growth in the presence of metal ions, different concentrations of zinc, cadmium or aluminium were added to the malt agar medium as salts. The pH values ranged from 4.2 to 4.4 in the control medium and in the media containing ZnSO4 (2.1, 2.7 and 4 mM) and CdSO4 (0.6 and 1.35 mM). A culture medium of pH 3.9 was obtained when Al(OH)3 and AlCl3 were mixed to yield total Al3+ concentrations of 14.4 and 140 mM, although some precipitates were observed in the culture medium. Radial growth of the fungal colonies was measured from a central mycelial plug in four replicate plates for each metal treatment, four weeks after inoculation. Growth relative to the mean value obtained on the control medium was calculated to eliminate growth rate variability among isolates. Statistical 158 TABLE 1 – List of the fungal isolates used in this study and their RAPD groups Isolates* Site of origin Reference N.** RAPD group C-1 non polluted site CLM1356.98 not tested C-2 non polluted site CLM1383.98 A Zn-1 zinc plot CLM1381.98 B Cd-1 cadmium plot CLM1382.98 B Cd-2 cadmium plot CLM1385.98 C Cd-3 cadmium plot CLM1386.98 D Cd-4 cadmium plot CLM1387.98 E Cd-5 cadmium plot CLM1388.98 E Cd-6 cadmium plot CLM1389.98 E Cd-7 cadmium plot CLM1390.98 F Cd-8 cadmium plot CLM1391.98 F Cd-9 cadmium plot CLM1392.98 F Al-1 aluminium plot CLM1393.98 F Al-2 aluminium plot CLM1394.98 F Al-3 aluminium plot CLM1395.98 F Al-4 aluminium plot CLM1396.98 F Al-5 aluminium plot CLM1397.98 F * The names used to indicate the experimental plots and the fungal isolates that were derived from them refer to the most toxic metal present in the applied mixture (see Materials and Methods). ** All isolates are held in the M.U.T. (Mycotheca Universitatis Taurinensis) at the Plant Biology Department, University of Torino, Italy. analysis (ANOVA and Tukey test) was carried out with the SYSTAT package, release 5.2, to determine significant growth differences (P<0.05). Axenic synthesis of ericoid mycorrhiza. Isolates were inoculated onto seedlings of Calluna vulgaris according to Pearson and Read (1973) to test their symbiotic abilities under axenic conditions. Root samples taken four to six months from inoculation were observed by light microscopy. Analysis of genetic variability. DNA was extracted as described in Longato and Bonfante (1997). The internal transcribed spacer (ITS) was amplified by PCR using the universal ribosomal primers ITS1 and ITS4 (White et al., 1990). Amplified DNA fragments were digested with 5 units of AluI or cloned into the pGEMT vector (Promega, USA) and sequenced at the University of Laval (Québec, Canada). Alignment with Oidiodendron sequences available in databases was performed with the CLUSTAL option in PC Gene package (IntelliGenetics). RAPD markers were generated with the decamer primers OPA1, OPA2, OPA4, 159 OPA13, OPA14, OPB7, OPB18 (Operon Technologies Inc., Alameda, California, USA). The PCR reaction mix was prepared according to Wyss and Bonfante (1993) with 5 and 100 ng of template DNA. PCR and restriction products were separated by electrophoresis on 1% to 1.5% agarose gels run in 0.5X TBE buffer and stained in ethidium bromide. A Jaccard pairwise similarity matrix was calculated based on the RAPD banding patterns, according to the formula: S= Nab/Nab+Na+Nb where Na and Nb are the number of bands specific to the individuals a and b, and Nab the bands found in both with the seven primers tested. A dendrogram was then obtained by the UPGMA method using the SYN-TAX 5.0 package (Podani, 1994). RESULTS Identification of isolates Isolates C-2, Zn-1 and Cd-1 from Poland and the isolate C-1 from Piemont (Italy) were previously identified as O. maius on the basis of their morphology (Martino et al., 2000). Morphological identification according to taxonomic keys (Barron, 1962) of the 13 remaining isolates revealed that they all belonged to O. maius. The expected Alu1 restriction pattern of O. maius (Hambleton et al., 1998) was found in the ITS of all isolates (Fig. 1). All isolates also formed typical coils in the epidermal cells of Calluna hair roots, demonstrating their mycorrhizal ability (not shown). Intraspecific polymorphism of O. maius isolates RAPD markers were analysed to determine intraspecific variability of the isolates collected in the various polluted plots. Isolate C-2, collected a few kilometers away from the experimental area was used as an external reference (Fig. 2 A, B). Six groups showing identical RAPD patterns with all primers tested (genets) were identified. The dendrogram built according to the similarity values for 63 poly- FIG. 1 – PCR-RFLP of the ITS region with Alu 1. Lane m, DNA marker size (1 kb ladder, Life technologiesTM, Italy). Lanes 1 to 14 correspond to isolates Cd-2, Cd3, Cd-4, Cd-5, Cd-6, Cd-7, Cd-8, Cd-9, Al-1, Al-2, Al-3, Al-4, Al-5, Cd-1. Lane 15, authenticated reference strain of Oidiodendron maius (MUCL 14539). 160 FIG. 2 – RAPD patterns obtained for O. maius isolates collected from polluted soils using primers OPA-14 (Fig. 2A) and OPA-2 (Fig. 2B). Lanes m, m’, DNA marker size, respectively 50 kb ladder, and l digested by Hind III + Eco R1 (Life tecnologies). Lanes 1 to 16 correspond to isolates C-1, Zn-1, Cd-1, Cd-2, Cd-3, Cd-4, Cd-6, Cd-8, Cd-9, Cd-5, Cd-7, Al-1, Al-2, Al-3, Al-4, Al-5. 161 FIG. 3 – Dendrogram obtained from UPGMA analysis of RAPD markers. Six RAPD group were distinguished among the O. maius isolates. Group A: isolate C-2. Group B: isolates Cd-1, Zn-1. Group C: isolate Cd-2. Group D: isolate Cd-3. Group E: isolates Cd-6, Cd-8, Cd-9. Group F: isolates Cd-4, Cd-5, Cd-7, Al-1, Al-2, Al-3, Al-4, Al-5. morphic bands (Fig. 3) shows that one isolate collected in 1997 (group C) clustered with the isolates collected in 1995 (group B). All other isolates from polluted soils fell into three groups that clustered together (groups D, E, F). A third branch (group A) corresponded to the external reference. The same genet could be found in plots polluted with different types of dust, like in group B (zinc and cadmium plots) or in group F (cadmium and aluminium plots). On the other hand, isolates from plots polluted with the same type of dust could belong to different groups (cadmium plot). Sensitivity of O. maius isolates At the metal concentrations tested, a general decrease of radial growth was observed in all isolates. In the case of zinc and aluminium, discriminating concentrations could be found that completely inhibited growth of only part of the isolates. For zinc, although growth variation among fungal isolates was observed at all concentrations, the most significant differences were found at 4 mM (Fig. 4). Half of the isolates, including both controls strains, were unable to grow whereas six isolates (Zn-1, Cd-1, Cd-2, Cd-4, Cd-9 and Al-2), collected from plots polluted with different kinds of dust and belonging to different RAPD groups, were significantly less sensitive (Fig. 4). Only three isolates (Cd-6, Cd-9 162 FIG. 4 – Fungal growth on culture medium with 4mM Zn2+, the highest concentration tested, four weeks after inoculation. Each column is the mean of four replicates. Bars indicate standard errors, and isolates showing statistically significant differences by ANOVA (P<0.05) are indicated by different letters. FIG. 5 – Fungal growth on culture medium added with either 0.6 mM Cd2+ or 14.4 mM Al3+, four weeks after inoculation. Each column is the mean of four replicates, and bars indicate standard errors. 163 and Cd-4) were able to grow, although to a limited extent, at the highest concentration tested for aluminium. At the lowest concentration (Fig. 5), C-2 and Cd-2 showed a significant inhibition compared to the other isolates. No discriminating metal concentration could be found for cadmium, as either growth or complete inhibition of all isolates was observed. Only the growth of control isolate C-1 was significantly reduced at 0.6 mM Cd2+ (Fig. 5). Except in the case of the isolate Zn-1, significantly less sensitive to zinc than all the other isolates, no significant correlations were identified of the results of radial growth on metals with either the RAPD groups or the plots of origin. DISCUSSION Few fungal species have been identified as forming ericoid mycorrhizas: Hymenoscyphus ericae was reported as the dominant European species (Straker, 1996), but sampling in heathlands and temperate forests also indicates the ubiquitous occurrence of Oidiodendron maius, largely dominant on other Oidiodendron species and concomitant with other ericoid fungal strains (Douglas et al., 1989; Perotto et al., 1996; Hambleton and Currah, 1997). Our results indicate that O. maius dominates in the Niepolomice forest and show that more than one genotype was able to adapt to the stress caused by the presence of toxic metal ions. Different genets of O. maius were identified in the cadmium plot, and the genetic relatedness found with some isolates from other plots (e.g. group B and group F) indicates fungal spreading across the polluted area either via hyphal growth or by propagules. Comparison of genetic polymorphisms and fungal growth on individual metal ions outlined a complex situation. The sensitivity of isolates belonging to the same genet to a given toxic metal could be strikingly different. This was clearly revealed on the zinc-containing medium for the isolates from the aluminium plot (group F) and for isolates derived from the cadmium plot and belonging to groups E and F. These data suggest that the results of RAPD fingerprinting, although very sensitive to reveal genetic relatedness among individuals, cannot be used to imply similarities in physiological behaviour. This absence of evident links between genetic and functional diversity of ericoid fungi has also been found in the case of mycorrhiza formation by ericoid fungi (Read, 2000). From the study of Cd-1 and Zn-1, the first isolates collected in the Niepolomice site, Martino et al. (2000) suggested that tolerance towards one metal could increase tolerance to another. In this study, the analysis of a larger range of fungal isolates confirms this hypothesis in some cases. For example, isolates from the aluminium plot grew well on zinc containing media, even though the latter was absent from the dust used to contaminate the plot. This situation, however, cannot be generalised, as several isolates showed different sensitivity to the metal ions tested, thus suggesting that the mechanisms involved in tolerance may vary from one metal to another. At the Niepolomice site, the content of toxic metals in the soil closely mirrored the metal content of the dusts that had been applied to the plots (Greszta et al., 1987). However, several factors such as soil composition, pH, humidity, and temperature modulate the availability of metals to microrganisms (Gadd, 1993) and interactions between multiple metals modify their relative toxicity (Hartley et 164 al., 1997). It is therefore difficult to evaluate the real availability of individual metals in the experimental plots and their selective pressure. For these reasons, in vitro tests of fungal growth allowed quantification of fungal responses to one metal stress under defined conditions. In few cases, high standard errors values were found (Fig. 4) although four repetions have been done, outlining the capacity of adaptation of these fungi to metal stress. However hypotheses on a possible adaptive response of O. maius to the different dust treatments would remain speculative. The two control isolates were always among the most sensitive isolates, even though C-2 was less sensitive to cadmium than isolates collected from the cadmium plot itself and C-1 grew quite well at the lower aluminium concentration. Occurrence of spontaneous resistance to toxic metals is not rare in fungi and may explain the behaviour of these reference isolates on specific metals. However, tolerant isolates are more frequently found in polluted areas (Gadd, 1993) and our results would strengthen this observation. Both O. maius isolates Zn-1 and Cd-1, collected in 1995 have been sub-cultured in our laboratory for several years on malt agar medium without loosing their tolerant phenotype. By contrast, it is recommanded to continuously expose mutants to high metal concentrations to maintain resistance (Tomsett, 1993). Therefore, the insensitivity to metal measured on the isolates collected in Niepolomice forest could be based on other mechanisms than the ones resulting from an in vitro selection of mutants, thus offering new elements to understand better the mechanisms of metal resistance. In conclusion, O. maius isolates presenting high, stable and diverse tolerance to toxic metals have been obtained and can be useful for different purposes. 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