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The effects of nickel and copper on the axenic growth of ectomycorrhizal fungi
Abstract
Isolates of several ectomycorrhizal fungi, some from a Cu- and Ni-contaminated smelter site near Sudbury, Ont., and some from an uncontaminated site, were grown on solid media containing 25 or 100 mg nickel/L, or 25 or 100 mg copper/L. The influence of the metals on lag time prior to the commencement of growth, on the maximum increase in colony diameter, and on the final colony dry weight was determined. Isolates from the metal-contaminated site did not outperform those from the uncontaminated site on any of the metal-amended media. In a second experiment, isolates of Laccaria proximo, Lactarius hibbardae, Lactarius rufus, and Scleroderma flavidum were grown in liquid media containing 2 or 5 mg Ni/L or 4 mg Cu/L. These fungi have previously been shown to vary in their effect on the metal tolerance of Betula papyrifera, at these concentrations. The axenic growth of the fungi in the presence of Ni showed no relationship to their effect on the birch seedlings. In general, the fungi that were least tolerant to Cu in liquid culture, Lactarius rufus and Laccaria proximo, were the same ones that proved most detrimental to the birch grown in the presence of Cu. These results indicate that axenic screening tests will not necessarily select fungi that will increase host metal tolerance.
... Sembratowicz and Rusinek-Prystupa showed that fungi derived from areas contaminated with heavy metals were more resistant to higher concentration of lead and cadmium ions in the substrate compared with mycorrhizal fungi that came from uncontaminated areas [16]. It is worth to notice that in this kind of research it should be extremely careful to draw conclusions because the lab eliminates many factors that may play a role in natural biocenoses and in vitro tests as evidenced by Jones and Hutchinson [17] can be downright confusing. Their research showed that in vitro studies on ectomyccorhizal fungi are little connected with the real behavior of these fungi in the natural symbiosis. ...
... Mushroom Scleroderma flavidum was unable to grow in vitro as a vegetative mycelium with even low concentrations of nickel, although it managed to produce fruiting bodies in a very high concentration of nickel in the soil [17]. The contradictions between the results of different authors may be only apparent, because the ability to adapt can be a feature of the species and even strains of the mycorrhizal fungus. ...
Plants together with water and minerals actively take from the soil heavy metals such as cadmium and lead. The negative role of ions of these metals on plant growth and development depends not only on their concentration in the soil, but also on a number of factors that may affect the transport of minerals from the soil to the roots. The harmful effects of xenobiotics getting from the soil to the plants are limited by the organic compounds contained in the soil, soil structure and pH. Particularly noteworthy are biotic factors, such as bacteria and fungi which greatly limit the translocation of heavy metals. Stream of new scientific reports show that the symbiotic combination of fungi with plant roots so called mycorrhizae is a factor that may be important in reducing the impact of soil contamination by heavy metals. Mycorrhiza by filtering solutions of water and mineral salts stop a considerable amount of heavy metals in the internal mycelium or on its surface. It was proved that plants with properly formed mycorrhiza grow better in hard to renew lands, such as salty, sterile soils contaminated with industrial waste. Questions to which answer was sought in this study are: 1) whether mycorrhizal fungi for many years growing in the contaminated areas have managed to adapt to these adverse conditions and 2) do the same species derived from clean areas are less resistant to contamination by heavy metals? Stated problems tried to be solved based on the fruiting bodies of fungi collected from ectomycorrhizal fungi picked from the areas contaminated by industrial emissions and areas free of contamination. The interaction of cadmium and lead ions on the growth of mycelium was examined by plate method and binding of heavy metals in fruiting structures of fungi were done by colorimetric method with use of methylene blue. It has been shown that the fungal resistance, even of the same species, to high concentration of heavy metals varies depending on the origin of symbiont. Isolated fungi from contaminated areas are better adapted to high concentrations of xenobiotics. Ability to bind cadmium and lead to fruiting bodies of fungi varies.
... For example, Blaudez et al. [64] showed that there is no difference in metal tolerance between ectomycorrhizal fungi from a polluted soil and from a non-polluted soil. Jones and Hutchinson [65] had similar results regarding Ni and Cu bioremediation mechanisms. However, Egerton-Warburton and Griffin [59] claimed that strains isolated from metal contaminated soils where more Al-tolerant than strains from less polluted sites. ...
We investigated a microbe-based approach to be used for the biorecovery of valuable metals from e-waste. E-waste is a heterogeneous matrix at the microbial scale. Therefore, this study aims at taking advantage of bacterial-fungal (BF) interactions in order to mobilize and immobilize a selected metal present in e-waste. We used cadmium (Cd) and a selection of Cd-tolerant microorganisms from our culture collection or isolated from a naturally cadmium-contaminated soil. Several experiments were designed in order to use the synergistic bioremediation capabilities of BF couples to mobilize and immobilize Cd from a culture medium. Initial results showed that the selected synergistic BF couples are more tolerant to Cd concentrations than the organisms alone. However, setting the conditions leading to effective immobilization of this toxic metal still need further work. Using microbial consortia rather than single species represents an innovative alternative to traditional bioremediation approaches for the development of new biotechnological approaches in urban mining.
... They can also withstand contamination with heavy metals (Cumming and Weinstein, 1990;Denny and Wilkins, 1987;Jones and Hutchinson, 1988;Jongbloed and Borst-Pauwels, 1990; Morselt et al., 1986;Wilkins and Hodson, 1989), as well as marginal and doughty soils (Coleman et al., 1989;Richter and Bruhn, 1989). They can tolerate soils with suboptimal pH's for plant growth (Jongbloed and Borst-Pauwels, 1990;Mcafee and Fortin, 1987), and soils with high incidence of soil borne plant pathogens (Chakravarty et al, 1990;Duchesne et al., 1989;Grahm, 1988;Marx, 1969;Moser and Haselwandter, 1983;Sylvia and Sinclair, 1983;Smith, 1988;Tinker, 1984). ...
Plant-microbe community dynamics influence the natural succession of plant species where pioneer vegetation facilitates the establishment of a distantly related, later successional plant species. This has been observed in the case of restoration of the American chestnut (Castanea dentata) on abandoned mine land where Virginia pine (Pinus virginiana) facilitated the establishment of chestnut seedlings. This was apparently due to the natural mycorrhizal networks of pine, which aided the survival and growth of chestnut seedlings. In this study, we assessed the survival and propensity of introduced mycorrhizal fungi on Virginia pine to colonize pure American and backcrossed American chestnut. Seedlings were planted in Perry State Forest located in southeastern Ohio. This area was mined for coal in the 1950s and had very little reclamation done aside from experimental tree plantings. The selected site, with little topsoil or organic matter, was characterized by high concentrations of Al, high soil temperatures, and a pH of 3.6. Virginia pine seedlings were inoculated using ectomycorrhizal (ECM) cultures of Amanita rubescens, Laccaria laccata, and Pisolithus tinctorius via liquid media. After three months, roots were tested for the presence of mycorrhizae. They were then transplanted and grown for two years in the greenhouse. After verifying mycorrhizal colonization, 600 pines were out planted in May of 2005. Chestnut seedlings (100 one-year-old seedlings) inoculated with P. tinctorius by the Ohio state tree nursery had been planted by other researchers at the same time. After eight growing seasons, pines and chestnuts were measured and sampled for ECM colonization. Growth measurements showed that pines and hybrid chestnuts had significantly more aboveground biomass compared to pure American chestnut (P = 0.01). Eleven fungal species were detected using DNA sequencing. With the exception of Amanita, the inoculum that were out planted with both chestnut and Virginia pine were replaced after 8 field seasons by fungi native to the site. More fungal species were sampled from the Virginia pines than from chestnut roots, which contributed to the significant differences in ECM fungal community composition between the two species (P = 0.005).
... For example, inoculation of wattle (Acacia spirorbis Labill.) and blue gum (Eucalyptus globulus Labill.) with a metal-sensitive isolate of Pisolithus albus (Cooke and Massee) stimulated shoot growth on an ultramafic soil high in Co, Cr, Fe, Mn and Ni more than several metal-tolerant isolates of the same fungal species ). Jones and Hutchinson found that fungal isolates with very low tolerance to Ni in pure culture (Jones and Hutchinson 1988a) produced the most biomass and provided the most protection for plants against Ni toxicity (Jones and Hutchinson 1988b). These results confirm the importance of testing a wide range of combinations of plant genotypes and fungal isolates in symbiosis in realistic substrates when determining the efficacy of growth enhancement by ECM fungi on metal-contaminated sites. ...
In natural environments, tree roots are almost always in intimate, symbiotic association with particular species of fungi through the formation of mycorrhizae. Most mycorrhizal fungi provide soil resources, particularly nitrogen (N), phosphorus and/or water to the tree, and can increase the abiotic and biotic stress resistance of their hosts. The fungi benefit by receiving fixed carbon from the tree. The association is of particular benefit on harsh or degraded sites. This review surveys recent literature on ectomycorrhizal (ECM) associations of temperate and boreal forest trees as it relates to N-nutrition and restoration of forests on sites where native mycorrhizal communities have been altered or depleted. Part I emphasizes the ECM fungal partners. Changes in ECM communities through primary and secondary succession are reviewed and related to the influence of N availability. The effect of N-related functional traits of ECM fungi on their distribution is discussed. Part II focuses on the ECM plant partners. The influence of ECM fungi on plant N uptake, and effects of N deposition and fertilization are presented. The benefit of ECM inoculation under different disturbance regimes and the benefit of greater ECM diversity are reviewed. Variations among and within tree and ECM fungal species in the forms of N taken up and utilized are highlighted. Conclusions include recommendations for including ECM fungi in forest restoration projects.
... Small mammals are an integral part of a complicated ecosystem triad involving ectomycorrhizal (ECM) fungi and trees. Ectomycorrhizal fungi form mutualistic symbiotic associations with the roots of vascular plants and aid the plants in acquisition of nutrients and water Thompson et al., 1994;Eggerton-Warburton et al., 2007), as well as protection from heavy metals (Jones et al., 1988;Wilkinson et al., 1995) and pathogens (Brazanti et al., 1991). In return, the plant symbiont provides the fungus with organic carbon in the form of photosynthates. ...
... Further studies examining the same sites over a longer period of time and throughout the year could shed more light on the way that these fungal communities change over time. Thompson et al., 1994; Eggerton-Warburton et al., 2007), as well as protection from heavy metals (Jones et al., 1988; Wilkinson et al., 1995) and pathogens (Brazanti et al., 1991). In return, the plant symbiont provides the fungus with organic carbon in the form of photosynthates. ...
Background/Question/Methods
Ectomycorrhizal fungi grow symbiotically on the roots of trees and provide them with nutrients and protection in exchange for carbon from photosynthesis. Many of these fungi form underground fruiting bodies; it is thought that ectomycorrhizal fungi gain long distance dispersal when small mammals eat these truffles and deposit their spores elsewhere in their feces. This study examined the role of small mammals in transporting spores from mature forests into disturbed areas. Twelve sites were chosen in the Interior Cedar Hemlock biogeoclimatic zone, based on the time (from 7 to over 100 years) since their last disturbance (either clearcutting or forest fire). In each case, small mammals (chipmunks (Tamias amoenus), flying squirrels (Glaucomys sabrinus), red squirrel (Tamiasciurus hudsonicus), red-backed vole (Clethrionomys grapperi), and deer mice (Peromyscus maniculatus)) were trapped and their feces were collected in both disturbed and adjacent mature areas, in both the early summer and fall of 2008. Truffles were collected on the same plots. Fungal DNA was extracted, amplified, and processed for t-RFLP from both truffles and feces for identification and comparison purposes. Fecal pellets were also observed microscopically for the presence, identification, and quantification of truffle spores.
Results/Conclusions
Chipmunks were the most frequently trapped spore-carriers on the sites followed by red-backed voles then flying squirrels. The percentage of fecal samples that were positive for fungal spores was 95.5% for flying squirrel (n=17), 83% for red squirrels (n=8), 78% for red-back voles (n=20), 71.5% for chipmunks (n=136), and negligible for deer mice (n=418). Thus, of the small mammals sampled in this biogeoclimatic zone, chipmunks appear to be the most important dispersers of fungal spores. Deer mice have been known to be opportunistic mycophagists in many areas; this study demonstrated that although they are abundant in the Interior Cedar Hemlock zone, they are not important dispersers of fungal spores in this area. The dominant mycorrhizal fungi forming truffles on these sites were Hysterangium separabile, Rhizopogon vesiculosus, and R. vinicolor; preliminary results indicate that Rhizopogon spp. spores were dominant in chipmunk feces. Implications of these results on forest management will be discussed.
... In addition, ECM associations can alleviate heavy-metal toxicity in trees and enable trees to survive in heavy-metal contaminated soils (Wilkinson and Dickinson, 1995;Meharg and Cairney, 2000). Heavy-metal tolerance varies among ECM fungal species and isolates, as revealed by mycelial culture experiments (Jones and Hutchinson, 1988;Hartley et al., 1997;Blaudez et al., 2000;Colpaert et al., 2000;Goncalves et al., 2007), which suggests that heavy metals could be a significant determinant of the structure of ECM fungal communities in the field. ...
The ecological responses of ectomycorrhizal fungi to heavy-metal contamination could affect the establishment and survival of trees under stress conditions due to industrial mining operations. In this study, we investigated species composition and diversity of ectomycorrhizal fungi associated with Masson pine (Pinus massoniana) and white oak (Quercus fabri) growing in Xiangtan manganese mining area in China. Canonical correspondence analysis revealed that soil Mn, Cu, Cd and several other soil cofactors related to manganese mining activities simultaneously structured the ectomycorrhizal community; in addition, a strong host effect was observed. The pine ectomycorrhizal community was dominated by four taxa (Atheliaceae, Thelephoraceae, Russulaceae, and Cenococcum), while the oak community was dominated by Thelephoraceae and Cenococcum. The relative abundance of Atheliaceae was positively correlated with soil manganese concentrations while that of Russulaceae decreased with increasing soil manganese. These ecological responses of ectomycorrhizal fungi may reflect the different physiological sensitivity of ectomycorrhizal fungal species to manganese.
A cadmium resistant fungal strain belonging to the genera penicillium was obtained by carrying out successive enrichments from soil samples collected near a metal processing industry in Mumbai. The culture demonstrated resistance to 1.4 mg/ml cadmium. Optimum pH and temperature growth conditions for the isolate were determined. Study of growth pattern of this culture revealed a low specific growth rate, with an extended lag period in the presence of cadmium. Screening for resistance to other heavy metals showed significant tolerance to zinc, lead, nickel and copper. The isolate was found to remove 67%, 84%, and 95 % of cadmium from solution after 48, 72 and 96 hours respectively. This study highlights the important role that penicillium cultures exhibiting high metal tolerance, could play in bioremediation of polluted environments.
The minimum inhibitory concentration (MIC) value of Aspergillus awamori for Cd(II) was 700 mg/l. A complete inhibition of biomass production was observed at 400 mg/l concentration of Cd(II). A significant deformation in Cd(II)-stressed conidiophores and conidia was observed by Scanning electron microscopy (SEM) investigation. Quantification of Cd(II) was performed by EDX microanalysis. Transmission electron microscopy investigation (TEM) confirmed the involvement of extracellular adsorption, intracellular penetration through the cell wall and vacuolation. Cadmium(II) stress induced noticeable changes in the activities of polyphenol oxidase (PPO), glutathione reductase (GR) and peroxidase (POD), and in the concentrations of total antioxidants, soluble protein and thiols. High performance liquid chromatography analysis (HPLC) revealed that Cd(II) stress stimulated the production of oxalic acid. Maximum Cd(II) uptake capacity was achieved at pH 5.0, initial metal ion concentration 500 mg/l and biomass dosage 1 g/l. Maximum Cd(II) uptake capacity was reached after 6 h for live biomass and after 2 h for dead biomass. Fourier transform infrared spectroscopy (FTIR) results gave an indication on chelation between oxygen-, nitrogen-, phosphorus- and especially sulphur-containing ligands of biomass with metal ions. X-ray diffraction analysis (XRD) revealed the presence of CdSO4
.H2O by live and dead biomass. EDX confirmed the occurrence of sulphur, oxygen and Cd(II) on the cell wall.
The toxicity of a sublethal cadmium concentration to ectomycorrhizal Pinus sylvestris L. seedlings was studied. Nine mycorrhizal strains, collected from a Zn- and Cd-polluted soil or from an unpolluted area, were compared for their ability to increase Cd tolerance in their host plant. Plants were cultivated in a semi-hydroponic system that allowed visual observation of the root systems. The degree of soil colonization as well as the density of the extramatrical mycelium was determined. The water use of the host plants was carefully monitored during the treatment period. Cadmium concentrations were determined in all plant parts. Although Cd addition resulted in decreases in transpiration in the non-mycorrhizal plants, disturbances in water relations could be more or less pronounced in the mycorrhizal pines. The treatment strongly influenced the growth of the fungi in the substrate: several mycobionts hardly survived in the polluted substrate. Fungi producing a large mycelial biomass showed the greatest effect in overcoming toxicity. Some s trains were even able to increase their turnover growth when treated with cadmium. In a dense extramatrical mycelium the potential for Cd retention increases, while the individual hyphae are exposed to a lower Cd concentration than in a sparse mycelium. A protective effect against cadmium toxicity in the host was observed with all mycobionts, since Cd uptake was highest in the non-mycorrhizal seedlings. The treatment had no effect on the growth of the seedlings or on the concentrations of the other cations, probably due to the relatively low toxicity and the relatively short observation period. The results are discussed in relation to the in vitro Cd tolerance of the fungi.
S ummary
The role of ectomycorrhizas in the metal tolerance of birch seedlings from Sudbury, Ontario was investigated. Betula papyrifera Marsh, is one of the few tree species able to survive in the copper‐ and nickel‐polluted area near Sudbury. Two types of birch seedlings were tested; those inoculated with Laccaria proxima (Boud.) Pat., Lactarius hibbardae Peck, Lactarius rufus (Scop, ex Fr.) Fries, or Scleroderma flavidum E. & E. isolates originating from the Sudbury area, and those which were not inoculated. Once the seedlings were infected, they were grown in sand culture containing 34 or 85 μM Ni, 32 or 63 Cu, or a control solution. At the low nickel concentration, the mycorrhizal plants, especially those infected with S. flavidum , grew significantly better than did non‐mycorrhizal plants. At the higher nickel concentration, S. flavidum ‐infected seedlings weighed 86% of controls without nickel, while the other seedlings weighed only 52 to 61 % of control. The good growth of S. flavidum ‐infected birch seedlings may have been due to retention of nickel in the mycorrhizas. These seedlings had the highest root nickel contents and the lowest stem nickel contents. At the high concentration of copper, growth of the mycorrhizal seedlings was significantly reduced compared with the non‐mycorrhizal seedlings. This reduction in growth of infected seedlings did not relate to an increase in the uptake or translocation of copper. Under the low copper treatment, infection with mycorrhizal fungi did not affect seedling growth. The potential role of iron and phosphorus in affecting host metal tolerance was investigated. Changes in the tissue concentrations of these elements did not relate to the effect of the fungi on metal tolerance.
Growth of Amanita muscaria, Cenococcum graniforme, Laccaria laccata, Pisolithus tinctorius, Rhizopogon roseolus, Suillus brevipes, S. grevellei, S. luteus, and Thelephora terrestris on Hagem Nutrient Agar as modified by Modess at 20°C for 28 days was inhibited by cadmium, lead, and nickel. All fungi were arrested by 350 μg cadmium per ml (ppm) or less. Lead arrested five species at 200 ppm or less; Cenococcum graniforme, L. laccata, and S. luteus were arrested at 2,000 ppm lead. Nickel arrested growth of six fungi at 20 ppm or less. Amanita muscaria, S. luteus, and L. laccata were arrested at 40, 175 and 225 ppm nickel, respectively. Metal concentrations that did not arrest delayed growth for 7–21 days after which the growth rate was comparable to the control.
S ummary
Seedlings of Betula spp. were grown in conjunction with isolates of Amanita muscaria Hooker and Paxillus involutus Fr. under aseptic conditions. Mycorrhizas were established in sterilized peat and vermiculite to which nutrient media had been added. Zinc was supplied at various levels and growth of the host was assessed after eight weeks. The mycorrhizas increased the tolerance to Zn of both tolerant and non‐tolerant Betula . This was coupled with a reduction in the translocation of zinc to the shoots of Betula , but an accumulation of zinc in the mycorrhizas.
Thesis (Ph. D.)--University of Toronto, 1983. Includes bibliography.
Copper-binding proteins were extracted from a copper-resistant strain of Saccharomyces cerevisiae which was obtained by repeated subculturing in a copper-containing medium. They were separated into three types through purification steps such as salt fractionation, gel filtration and preparative polyacrylamide gel electrophoresis. They resembled each other in amino acid composition. Acidic amino acids, lysine, serine, glycine and half-cystine constituted a large part of the protein, with a small amount of hydrophobic amino acids. Aromatic amino acids and methionine were almost absent. The molecular weight of the components was estimated to be about 10,000 by Sephadex gel filtration and electrophoresis on polyacrylamide gel (slope method). Absorption spectra of the components exhibited a broad band at 275 nm, but none in the visible region, thus resembling that of copper-thionein. Moreover, the absorption band at 275 nm changed markedly on addition of Ag+, Hg2+, CN− or H 2 O 2 , which are well known as thiol reagents. These components were abo produced in the parent cells, if they could grow in a copper-containing medium. Based the results of experiments using various culture conditions and some other yeast species, a possible role of the components is discussed.
Within 2 km of a zinc (Zn) smelter in Palmerton, Pennsylvania, near the Lehigh Water Gap, up to 13.5% Zn by weight has been measured in the O2 horizon of the soil, and up to 8% Zn in the A1 horizon. The total numbers of bacteria, actinomycetes, and fungi (measured by dilution plate counts) were greatly reduced in the most severely Zn-contaminated soils compared with control soils. The reduction of microbial populations may be a partial cause of the decreased rate of litter decomposition at Lehigh Gap. Growth of most bacteria from control sites was reduced by 100 to 200 muM Zn, most actinomycetes by 100 muM Zn, and most fungi by 100 to 1000 muM Zn in thin-Pablum extract agar (TPab). All the tested actinomycetes and non-spore-forming bacteria isolated from Zn-contaminated Lehigh Gap soils were Zn-tolerant, growing normally in media containing 600-2000 muM Zn. Most fungi, regardless of source, were capable of at least 50% of normal growth at 700 muM Zn. Zinc-tolerant bacteria, actinomycetes, and fungi were readily isolated from low-Zn soils, suggesting that selection for Zn tolerance may proceed rapidly. Acidophilic Mortierella species have been selectively eliminated near the smelter, apparently because of elevated soil pH. Peryronellaea glomerata (Corda) Goidanich and Coniothyrium spp. were found only in the high-Zn soils.
A defined solid medium has been used to examine the responses of Aureobasidium pullulans, Saccharomyces cerevisiae and Sporobolomyces roseus to cadmium, copper and zinc. Experiments where aliquot volumes of metal salt solutions were added to wells in the centre of agar plates revealed marked differences between these organisms. The yeast-like fungus A. pullulans was the least sensitive but S. cerevisiae was more sensitive to cadmium than zinc: the reverse was true for S. roseus. Quantitative data, which complemented the qualitative results, were obtained by measurement of metal concentrations in the plates.
Some aspects of the fungal flora in nickel con-taminated and non-contaminated soils near Sudbury Effects of nickel contamination on nitro-gen cycling in boreal forests in northern Ontario Ecological aspects of heavy metal responses in microorganisms
- A Toronto
- De
- J B Catanzaro
CARTER, A. 1978. Some aspects of the fungal flora in nickel con-taminated and non-contaminated soils near Sudbury, Ontario, Canada. M.Sc. thesis, University of Toronto, Toronto. DE CATANZARO, J. B. 1983. Effects of nickel contamination on nitro-gen cycling in boreal forests in northern Ontario. Ph.D. thesis, University of Toronto, Toronto. DUXBURY, T. 1985. Ecological aspects of heavy metal responses in microorganisms. Adv. Microb. Ecol. 8: 185-235.
Fungal flora of the soil as conditioned by varying concentrations of heavy metals
- L M Hartman
HARTMAN, L. M. 1976. Fungal flora of the soil as conditioned by varying concentrations of heavy metals. Ph.D. thesis, University of Montana, Missoula.