Screening for lignocellulolytic enzymes and metal tolerance in isolates of wood-rot fungi from Chile/ Determinacion de enzimas lignoceluloliticas y tolerancia a metales en cepas de hongos pudridores de madera de Chile/ Determinacao de enzimas lignoceluloliticas e tolerancia a metais em isolados de fungos da decomposicao da madeira de Chile.
Wood-rot fungi have been shown to be powerful agents in many biotechnological processes. However, there may be great inter- and intra-specific variability in their performance. Consequently, it is not only important to screen a broad range of species but also different isolates of the same species in order to obtain strains with specific biotechnological profiles. In this study, the presence of wood-rot fungi was monitored in Southern-Central Chile and the biotechnological potential of the isolates was analyzed by determining lignocellulolytic enzymes and tolerance to metal ions (Cu and Cd) in solid medium. Seventy-one strains were isolated from cultures of a total of 144 basidiomes collected from wood substrates and 59 species of 18 different genera were identified, of which four are first records for Chile: Antrodia xantha, Gloeophyllum abietinum, G. protractum and Stereum rameale. Cellulase and xylanase activity were detected in all strains and 20 strains showed significant ligninolytic activity. The great majority of the strains showed tolerance to 3mM Cu in solid medium, but were inhibited by 1mM Cd. In contrast, some strains of the white-rot fungi Ganoderma australe, Stereum hirsutum and Trametes versicolor presented high lignocellulolytic potential combined with metal tolerance. Possible applications of these strains in biodegradation or bioremediation processes are discussed.
El enorme potencial de los hongos pudridores de madera en muehos procesos biotecnologieos ha sido demostrado. Sin embargo, estos pueden presentar grandes variaciones inter- e intraespecificas en su desempeno. Asi, cuando se desea obtener especies fungicas para usos biotecnologicos especificos, es necesario analizar no solo una amplia variedad de especies, sino tambien diferentes cepas de una misma especie. En este estudio fue investigada la presencia de hongos pudridores de madera en una region del centro-sur de Chile, y su potencial biotecnologico fue evaluado a traves de la deteccion de enzimas lignoceluloliticas y de la tolerancia a iones metalicos como Cu y Cd, en medio de cultivo solido. De los 144 basidiomas recolectados a partir de sustratos lenosos fueron obtenidos 71 cultivos puros y de estos, 59 especies de 18 generos diferentes fueron identificadas, de las cuales cuatro son informadas por primera vez en Chile: Antrodia xantha, Gloeophyllum abietinum, G. protractum y Stereum rameale. Se detecto actividad de celulasa y xilanasa en todas las cepas fungicas y solo 20 mostraron una significativa actividad ligninolitica. La mayoria de las cepas fue tolerante a 3mM Cu, pero fueron inhibidas por 1mM Cd. Algunas cepas de los hongos de pudricion blanca Ganoderma australe, Stereum hirsutum y Trametes versicolor presentaron un eficiente potencial lignocelulolitico combinado con una alta tolerancia a metales. Se discuten posibles aplicaciones de estas cepas en procesos de biodegradacion o bioremediacion.
O enorme potencial dos fungos da decomposicao da madeira em uma variedade de processos biotecnologicos tem sido demonstrado. Contudo, tais fungos podem apresentar uma grande variabilidade inter- e intraespecifica no seu desempenho. Assim, quando se deseja obter especies fungicas com fins biotecnologicos especificos, e necessario analisar nao somente uma ampla variedade de especies, senao tambem diferentes isolados de uma mesma especie. No presente estudo, a presenca de fungos da decomposicao da madeira foi pesquisada numa regiao do centro-sul do Chile, e seu potencial biotecnologico foi avaliado mediante a deteccao das enzimas lignoceluloliticas e da tolerancia a ions metalicos tais como Cu e Cd, em meio de cultura solido. Dos 144 basidiocarpos recoletados a partir de sustratos lenhosos, 71 cultivos puros foram obtidos e desses, 59 especies de 18 generos diferentes foram identificadas, das quais quatro sao descritas pela primeira vez no Chile: Antrodia xantha, Gloeophyllum abietinum, G. protractum e Stereum rameale. As atividades de celulase e xilanase foram detectadas em todos os isolados fungicos e somente 20 mostraram uma significativa atividade ligninolitica. A maioria dos isolados foi tolerante a 3ruM Cu, mas foi inibida por 1mM de Cd. Alguns isolados dos fungos da decomposicao branca Ganoderma australe, Stereum hirsutum e Trametes versicolor apresentaram um eficiente potencial lignocelulolitico junto com uma elevada tolerancia a metais. Discutese a respeito das possiveis aplicacoes desses isolados em processos de biodegradacao ou biorremediacao.
KEYWORDS / Brown-rot Fungi / Ligninocellulolytic Activity / Metal Tolerance / White-rot fungi /
Received: 05/04/2011. Modified: 11/14/2011. Accepted: 11/15/2011.
Whereas in the past wood-rot fungi have been mainly considered as an economic threat for causing substantial losses of wood and wood products, they have since been rediscovered as powerful agents in biotechnological processes. The biochemical mechanisms responsible for their lignocellulolytic capacities are the main focus of current studies which aim at their application in solving technological and environmental problems, particularly in the paper industry (Ferraz et al., 2008), and the bioremediation of contaminated soils and industrial wastes (RodriguezCouto and Toco-Herrera, 2006; Gao et al., 2010; Magan et al., 2010). When the fungi attack the wood, a range of degradative extracellular, enzymatic and nonenzymatic activities are produced which chemically and morphologically alter the substrate, resulting in three main types of rot: white, brown and soft (Blanchette, 1995). Depending on the type, wood-rot fungi secrete a battery of enzymes and low-molecular weight agents that cause depolymerization of cellulose and hemicelluloses as well as fragmentation of lignin (Kirk and Cullen, 1998). This enzymatic potential, together with tolerance to metals that some wood-rot fungi can develop, has been used in research related to metal biosorption, biopulping, biobleaching and bioremediation of soils, industrial effluents and preserved wood that have been discarded as waste (Pointing, 2001; Baldrian, 2003; Illman and Yang, 2004; Ferraz et al., 2008; Gao et al., 2010; Dashtban et al., 2010).
Chile possesses large areas of forest plantations and is among the main exporting nations of wood products, especially pulp and paper. The international market increasingly demands the use of environmentally friendly techniques, especially in processes like cellulose production. Many studies on the degradative properties of wood-rotting fungi have been performed using model organistas such as the white-rot fungi Phanerochaete spp., Trametes spp. or Pleurotus spp. However, it is also important to study a broad range of fungal species and strains from the same geographical area, where these could be applied in the future, given that locally distributed taxa may show enhanced performance, having adapted to local climate and substrate types (Atagana, 2004; D'Annibale et al., 2006). Moreover, legal restrictions on the use of allochthonous microorganisms in natural environments may impede their biotechnological application in some countries (Matheus et al., 2000). In this context, it must be emphasized that Chilean mycobiota, especially their lignocellulolytic capacities, have not been studied in depth despite their economic importance, and few studies on biotechnological potential of native fungi exist (Donoso et al., 2008; Mendonca et al., 2008; Tortella et al., 2008; Rubilar et al., 2011; Acevedo et al., 2010, 2011). For this reason, the aim of the present study was to isolate and identify wood-rot fungi (Basidiomycota) from SouthernCentral Chile and determine their lignocellulolytic capacity. To this end, pure mycelial cultures of the basidiomes collected in the field were subjected to different qualitative enzyme assays in solid medium to detect hydrolytic and ligninolytic enzymes (Gramms et al., 1998; Pointing, 1999; Kjoller et al., 2000). These assays also enable corroboration of the type of wood rot caused by some species of xylophagous fungi, mainly the traditional Bavendamm test (with tannic or gallic acid), used for many years to distinguish between white- and brown-rot fungi based on the presence or absence of phenoloxidase production, respectively (Kaarik, 1965). In addition, the effect of Cu and Cd ions on mycelial growth rates in solid medium was analyzed in order to select strains with biotechnological potential to be used in the bioremediation of substrates and/or environments contaminated with metals.
Materials and Methods
Basidiomes from wood-rot fungi were collected between latitudes 36[degrees] and 38[degrees]S in Southern Central Chile, in different locations in the Provinces of Concepcion, Bio-Bio and Nuble (Figure 1). The climate of the region is Mediterranean with a dry, hot summer that can last up to four months, contrasted by a rainy and cool winter (Hoffmann, 1998). Most fungal species tested were collected in native forests dominated by southern beech (Nothofagus spp.) and sclerophyllous trees of the Myrtales and Laurales orders (Table I). Other species were collected from commercial plantations, on living or dead wood of Pinus radiata D. Don, Eucalyptus globulus Labill and E. nitens Maiden. Samples were collected from substrates including standing or uprooted stems and/or wood structures at different stages of decomposition, between autumn and spring 2004 and 2005.
Isolation and identification of wood-rot fungi
Pure mycelial cultures were obtained under aseptic conditions, placing small fragments or spores of the basidiomes collected on malt extract agar medium prepared with 2% (w/v) malt extract (Fluka) and 2% (w/v) agar (Merck). Petri dishes were incubated at 24 [+ or -]1[degrees]C in the dark and after variable incubation periods the axenic cultures were transferred to test tubes with 2% MEA and kept constantly at 3[degrees]C in the Laboratory of Fungi Biotechnology at the Universidad de Concepcion, Campus Los Angeles, Chile. Furthermore, the respective basidiomes were oven-dried at 40-50[degrees]C for 48h and deposited as voucher material in the Fungi Collection of the Herbarium of the Universidad de Concepcion (CONCF). Classification of genera and species was performed according to the techniques suggested by Ryvarden (1987) and Rajchenberg (2006). For microscopy, hand-cut sections of specimens were mounted in 3-5% KOH and stained with Melzer's reagent and 1% phloxine. Sources of identification keys and species descriptions were the publications by Wright and Deschamps (1972), Horak (1979), Breitenbach and Kranzlin (1986), Ryvarden (1987, 1991), Bernicchia (2005) and Rajchenberg (2006). Some of the basidiomes were compared to material deposited in the herbarium at the Patagonian Andes Forest Research and Extension Center (CIEFAP), Esquel, Argentina.
[FIGURE 1 OMITTED]
Detection of lignocellulolytic enzymes
The qualitative assays for detection of lignocellulolytic enzymatic activity in mycelial fungi culture were performed in solid medium. The substrates were added to 0.1% MEA (pH 4.5) after sterilization, in different concentrations (w/v): 2% carboxymethylcellulose (CMC), 2% xylan from birchwood, 0.01% guaiacol, 0.04% Remazol Brilliant Blue R (R-BBR), 0.02% Poly R-478 and 0.5% tannic acid, following Pointing's methodology (1999). Two 7mm-diameter disks obtained from the active growth zone of each fungal strain culture were placed equidistantly at opposing edges of a Petri dish. The dishes were incubated in the dark at 24 +l[degrees]C for 10 days, a period in which the enzymatic activity could be seen by the formation of coloration or discoloration halos in the media containing the different enzymatic indicators. The brown-rot fungi Wolfiporia cocos ATCC 62778 and Gloeophyllum trabeum ATCC 11539 and white-rot fungi Trametes versicolor DEBIQ and Ganoderma australe A464, cultivated under conditions similar to those of the native strains, were used as references in the lignocellulolytic enzyme detection assays.
Effect of Cu and Cd on growth rate
In order to evaluate the effect of metal ions on the growth of fungal strains, Cu or Cd was added to the 2% MEA (pH 4.5) medium as solid salts (CuS[O.sub.4].5[H.sub.2]O or CdS[O.sub.4].8[H.sub.2]O) after sterilization when the medium had reached an appropriate temperature, to final concentrations of 3mM and 1mM, respectively. Petri dishes (9cm diameter) containing MEA medium with or without metal ions were inoculated in the center with a 7mm diameter disk obtained from the stock cultures and incubated in the dark at 24 [+ or -]1[degrees]C for a maximum of two months. Growth was determined by measuring the radius of the mycelial colonies at various time intervals. Growth rates for each fungal strain were calculated from the respective growth curves and expressed in mm/day (Guillen and Machuca, 2008). The assay was performed with three replicates for each isolated strain with its respective control (MEA medium without metal).
Results and Discussion
Many studies related to wood-rot fungi have been conducted with different biotechnological goals, but the vast majority of these studies have been performed with a limited number of well-known fungal species and strains, mainly white-rot. While this concept of model organistas makes sense for basic research, studies of specific properties for future use in defined geographical areas may require the use of local taxa adapted to site-specific climates, substrates and other variables. In Chile, a country with abundant fungal diversity, extensive areas of native forests and forest plantations (Conaf-ConamaBirf, 1999a, b; Mujica et al., 1980), few studies on native mycobiota exist. Within this context, the aim of the present work was to study the enzymatic activity and heavy metal tolerance of endemic taxa like Anthracophyllum discolor and Bondarzewia guaitecasensis (Wright and Deschamps, 1972; Lazo, 2001), as well as to test different strains of cosmopolitan species like Trametes versicolor of Stereum hirsutum for intraspecific variability of properties that are of particular interest for the biodegradation or bioremediation of contaminated substrates.
A total of 144 collections of basidiomes were taken from a variety of woody substrates at different sites in Southern-Central Chile. Of these, 71 strains of pure cultures were obtained and 59 species of 18 different genera could be identified at the species (55) or genus (4) level (Table I). Most taxa (78.9%) had aphyllophoroid basidiomes; the rest (19.7%) had agaricoid morphology. Stereum hirsutum, Trametes versicolor, Ganoderma australe and Lenzites betulina were the most frequently encountered species and provided several strains (Table I). Other species which could be isolated more than once were Bjerkandera adusta, Flammulina velutipes, Gymnopilus spectabilis and Pleurotus ostreatus. The remaining taxa were isolated only once (Table I).
Among Chilean lignicolous mycobiota, two large groups can be distinguished: endemic fungi that colonize similar endemic tree species and adventives, usually widely distributed species that have been established with introduced trees. It has been suggested that a balance between both fungal groups may develop over time (Butin and Peredo, 1986). The present findings extend the list of known Chilean wood-rot fungi by four newly recorded species: Antrodia xantha, Gloeophylluto abietinum, Gloeophyllum protractum and Stereum rameale, all species of wide geographic distribution (Krieglsteiner and Kaiser, 2000). Among these, A. xantha, which has also been reported in Argentina (Wright and Alberto, 2006), is a brown-rot species of reference in assays about the effectiveness of wood preservatives, due to its tolerance to Cu (AWPA, 2004).
The enzymatic potential of 59 strains of identified genera and species was determined qualitatively in the MEA medium using different indicators (Table II). All strains hydrolyzed carboxymethyl-cellulose (CMC) to a greater or lesser extent, proving their ability to produce enzymes from cellulolytic complex. The majority of the native strains (71%) showed a strong positive reaction and the remainder performed only a partial CMC hydrolysis. Among the strains of B. adusta, G. australe, L. betulina, T. versicolor, and particularly S. hirsutum, differentiated responses in the hydrolysis of CMC were observed (Table II). With birchwood xylan as the indicator substrate for hemicellulolytic enzymes, all strains presented a positive reaction (Table II), with no differences in the color intensity of the reaction zone, but varying in the size of the hydrolysis halos. The reference strains showed similar positive cellulolytic and hemicellulolytic reactions, except W. cocos, which reacted only moderately with CMC. The hydrolysis of CMC can be attributed not only to the production of endoglucanase-type cellulases, but also to [beta]-glycosidases, whereas hydrolysis of xylan is usually performed by endoxylanases and [beta]-xylosidases (Pointing, 1999), providing good evidence that all the fungal strains analyzed can produce various types of cellulolytic and hemicellulolytic enzymes. The biotechnological applications of these strains or their fungal extracts with hydrolytic activity are of great interest in the textile, detergent and food industries, as well as for bleaching processes in the paper industry (Kuhad et al., 1997; Bhat, 2000).
To detect ligninolytic enzymes different enzymatic substrates were used, related to the production of polyphenoloxidases, laccases, peroxidases, lignin-peroxidases (LiP) or Mn-peroxidases (MnP). Most strains demonstrated ability to oxidize at least one of the substrates assayed and only eight strains did not oxidize any of them (Table II). Of these, six are species belonging to the brown-rot fungi group, showing coherence with the results obtained with the reference brown-rot fungi W. cocos and G. trabeum. Remarkably, two of these strains ate of species belonging to the white-rot fungi group (F. velutipes 98, S. rameale 71). In addition, the strain of P. panuoides, a brown-rot fungus, reacted positively with the RBB-R dye, an indicator of ligninolytic activity. A high degree of strain-specific variability in enzymatic activity was observed in G. australe and S. hirsutum: only strains 64 and 142 of G. australe and strain 19 of S. hirsutum oxidized all ligninolytic substrates; the rest presented varying reactions to the different indicator substrates. Nevertheless, among other species like B. adusta, L. betulina and T. versicolor, a much more homogenous behavior was observed. All four strains of B. adusta behaved similarly, discoloring the polymeric dyes RBB-R and Poly-R 478; only one was able to oxidize tannic acid and none of them oxidized guaiacol. Most of the strains of L. betulina reacted positively to all the substrates, and only three of them did not oxidize guaiacol. Most of the strains of Z versicolor reacted positively with all the ligninolytic substrates, except strain 7, which did not oxidize guaiacol or tannic acid (Table II). Among native strains, the majority discolored RBB-R (78%) and oxidized tannic acid (73%), and only a smaller proportion reacted with Poly R-478 (58%) and guaiacol (44%).
A considerable intra- and interspecific variety of responses was obtained among the 59 native fungal strains and only eight strains were unable to react with any of the substrates used. These results were expected since seven of the strains belong to species classified as brown-rot fungi. Interestingly, S. rameale 71, a white-rot fungus, was also among the strains that showed no reaction. This may be related to the selected substrate indicators, because when [alpha]-naphthol, p-cresol and pyrogallol were used as substrates, S. rameale reacted positively, oxidizing these compounds (Stalpers, 1978). On the other hand, some white-rot species (B. adusta, F. velutipes 95 and 98, G. australe 28 and 86, S. rameale 71 and T. versicolor 7) showed inconsistent results with the Bavendamm test as they reacted negatively. These results confirm the importance of using more than one enzymatic substrate in studies regarding the selection of ligninolytic enzyme-producing fungi. Confirming the type of wood rot (white or brown) through enzymatic assays in solid medium is considered of great importance for the taxonomy of xylophagous fungi (Stalpers, 1978). In the reviewed literature there is no prior information with respect to the wood-rot type or lignocellulolytic enzymes produced by B. guaitecasensis. The assay-based classification of this species as a white-rot fungus (Table II) is consistent with the same status described for B. montana (Quel.) Singer, from the Northern Hemisphere (Breitenbach and Kranzlin, 1986; Krieglsteiner and Kaiser, 2000).
It is worthy of note that P. panuoides 83, of the brown-rot fungi group, decolorizes the RBB-R dye, suggesting that in addition to the expected hydrolytic enzyme production, this strain is also able to produce oxidative enzymes. Although few studies exist on the detection of ligninolytic enzymatic activity in brown-rot fungi, some describe the production of laccases by this type of fungi and the detection of the genes responsible for coding these enzymes (Pelaez et al., 1995; D'Souza et al., 1996; Lee et al., 2004). The discoloration and/or degradation of RBB-R and Poly R-478 have been correlated with the production of peroxidases (LiP and MnP) and laccases, and also with organopollutant degradation. This suggests that the fungi that react positively with these dyes present a wider range of ligninolytic enzymes, making them potential candidates for the treatment of effluents from the textile industry, biopulping, kraft pulp bleaching or the bioremediation of organopollutants, among others (Freitag and Morrell, 1992; Okino et al., 2000; Pointing et al., 2000; Minussi et al., 2001; Hakala et al., 2004; Hernandez-Luna et al., 2008).
Additionally, the ability of the native strains to grow in solid medium containing Cu or Cd ions was evaluated through the radial growth rate of the colonies. The metal tolerance of fungi is of importance for some biotechnological applications, such as the bioremediation of contaminated substrates. Soil contamination with polycyclic aromatic hydrocarbons (PAHs) is frequently accompanied by contamination with metal ions, which makes it essential that the fungi selected for bioremediation programs, in addition to being able to degrade PAHs, also show tolerance to metals (Baldrian et al., 2000; Riis et al., 2002; Baldrian, 2003). Some metals can also interfere significantly with the activity of extracellular enzymes and colonization capacity of fungi (Baldrian, 2003).
Radial growth rates in MEA medium varied between 0.39 and 11.6mm/day among all species and strains (Table II). Twelve strains, mainly belonging to white-rot fungi, presented the fastest growth rates (>7mm/day), among which two strains of B. adusta and most of the T. versicolor strains stood out, followed by G. australe 28, L. betulina 22 and G. abietinum 132. The remaining strains showed a moderate growth rate and only S. lacrymans 141 grew very slowly under the assay conditions. Generally, growth rates were comparable and in some cases superior to those of the reference strains. The addition of 3mM Cu or 1mM Cd to the MEA medium reduced the growth rate in all strains tested compared to the control (without metal), with the exception of S. lacrymans 141, a dry and brown-rot fungus. This strain, despite its slow growth rate, displayed unusual behavior in that it was not inhibited by either of the metal ions, whereas all the strains of white-rot fungi F. velutipes and L. betulina exhibited a similar sensitivity to both. The majority (43) of the native strains grew in the presence of Cu, showing varying degrees of tolerance to the metal, reflected in rates that fluctuated between 0.07 and 3.36mm/day (Table II). The remaining strains showed a high sensitivity to the metal. Most strains of the white-rot fungi G. australe, T. versicolor and S. hirsutum were tolerant to 3mM Cu and only one strain of each species exhibited sensitivity. Among the native strains, the white-rot fungus G. australe 100 and brown-rot fungus Gloeophyllum sp. 8 displayed the lowest growth inhibition in the presence of Cu (23 and 28%, respectively), compared with their respective controls. On the other hand, the majority of the native strains were unable to grow in the presence of 1mM Cd and only 17 strains were tolerant to the metal, with growth rates varying between 0.11 and 1.27mm/day (Table II). Growth inhibitions caused by Cd were always elevated and higher than those ob served in the assays with Cu. However, some strains such as G. australe 64 and T. versicolor 12 showed tolerance to Cd but not to Cu. A group of 15 native strains belonging to different white-rot (B. adusta, C. dusenii, G. australe, S hirsutum and T. versicolor) and brown-rot (A. xantha, P. panuoides and S. lacrymans) species showed tolerance to both metal ions.
After mercury (Hg), Cd is considered one of the most toxic metals for some species of wood-rot fungi (Baldrian and Gabriel, 1997; Tham et al., 1999). This explains why Cu exerted a lower inhibiting effect on the growth of most of the native strains than Cd, even though the later was used at a lower concentration. The growth of colonies in the presence of metals showed a great intra-specific variability between strains of G. australe, S. hirsutum and T. versicolor, all white-rot fungi (Table II). Some strains of G. australe were tolerant to Cu, but not to Cd; others were tolerant to both metals and only one was tolerant to Cd, but not to Cu. In the case of S. hirsutum and T. versicolor, most strains were tolerant to Cu, but not to Cd. Only among the strains of F. velutipes and L. betulina, also white-rot fungi, the sensitivity to Cu and Cd was similar. The results do not allow to generalize the behavior of the different brown-rot and white-rot fungi species analyzed in this study with respect to Cu and Cd tolerance, since in the case of brown-rot fungi, only one strain of each species was assayed. However, among the brown-rot species, A. xantha and P. panuoides presented relatively low growth inhibition in the presence of both metals. Furthermore, G. abietinum and Gloeophyllum sp. showed tolerance to Cu but not to Cd. Among the reference strains, the brown-rot fungus W. cocos displayed the greatest tolerance to Cu, but in the presence of Cd its growth was significantly inhibited (Table II).
In order to avoid erroneous generalizations regarding the metal tolerance of brown-rot and white-rot fungi, relevant studies should always include the maximum possible number of strains of a given species (Collet, 1992; Woodward and De Groot, 1999; Clausen et al., 2000). According to various studies, brown-rot fungi presenta higher Cu tolerance than white-rot fungi, which could be related to an increased production and accumulation of oxalic acid in the brown-rot fungi cultures (Murphy and Levy, 1983; Green and Clausen, 2003; Baldrian, 2003). Organic acids, together with other extracellular metal-chelating agents such as siderophores, have been considered among detoxification mechanisms that allow various organisms to tolerate high concentrations of some metal ions (Gadd, 1993, 2004). Most studies have been conducted, however, comparing only single strains of a range of species.
As the result of the collection of wood-rot fungal basidiomes from different sites in Southern-Central Chile, four species are reported for the first time in Chile: Antrodia xantha, Gloeophyllum abietinum, G. protractum and Stereum rameale. In addition, all the fungal strains analyzed through qualitative assays produced cellulases and xylanases, according to the observed hydrolysis of CMC and birchwood xylan, respectively, and only some strains produced ligninolytic activity. With regard to metal sensitivity of the strains the great majority showed tolerance to 3mM Cu in solid medium, but were inhibited by 1mM Cd. According to the lignocellulolytic enzymatic potential and/or the degree of tolerance to metal ions presented by some native strains identified in this study, these could be selected for future biotechnological applications. A. xantha, G. abietinum, Gloeophyllum sp. and P. panuoides, for example, will be used in future studies on tolerance to wood copper preservatives and preserved wood waste biodegradation, whereas some strains of G. australe, S. hirsutum and T. versicolor will be used in studies on soil bioremediation or industrial effluents contaminated with organopollutants and heavy metals.
The authors thank the valuable technical assistance of D. Chavez and E. Hermosilla, and G. Pereira for their help related to the description of the collection sites.
Acevedo F, Pizzul L, Castillo M, Gonzalez ME, Cea M, Gianfreda L, Diez MC (2010) Degradation of polycyclic aromatic hydrocarbons by free and nanoclay-immobilized manganese peroxidase from Anthracophyllum discolor. Chemosphere 80: 271-278.
Acevedo F, Pizzul L, Castillo M, Cuevas R, Diez MC (2011) Degradation of polycyclic aromatic hydrocarbons by the Chilean white-rot fungus Anthracophyllum discolor. J. Hazard. Mat. 185: 212-219.
Atagana HJ (2004) Biodegradation of phenol, o-cresol, m-cresol and p-cresol by indigenous soil fungi in soil contaminated with cresolate. World J. Microbiol. Biotechnol. 20: 851-858.
AWPA (2004) El0-01 Standard methods of testing wood preservatives by laboratory soilblock cultures. In Book of Standards. American WoodPreservers'Association. Granbury, TX, USA. pp 1-9.
Baldrian P (2003) Interactions of heavy metals with white-rot fungi. Enz. Microb. Technol. 32: 78-91.
Baldrian P, Gabriel J (1997) Effect of heavy metals on the growth of selected wood-rotting basidiomycetes. Folia Microbiol. 42: 521-523.
Baldrian P, der Wiesche C, Gabriel J, Nerud F, Zadrazil F (2000) Influence of cadmium and mercury on the activities of ligninolytic enzymes and degradation of polycyclic aromatic hydrocarbons by Pleurotus ostreatus in soil. Appl. Env. Microbiol. 66: 2471-2478.
Bernicchia A (2005) Fungi Europaei. Polyporaceae s.l. 2nd ed. Candusso. Alassio, Italy. 808 pp.
Bhat MK (2000) Cellulase and related enzymes in biotechnology. Biotechnol. Adv. 18: 355-383.
Blanchette R (1995) Degradation of the lignocellulose complex in wood. Can. J. Bot. 73: S999-S1010.
Breitenbach J, Kranzlin F (1986) Fungi of Switzerland. Vol. 2. Non-gilled fungi. Mykologia. Lucerne, Switzerland. 412 pp.
Butin H, Peredo H (1986) Hongos parasitos en coniferas de America del Sur con especial referencia a Chile. Cramer. Berlin-Stuttgart, Germany. 100 pp.
Clausen CA, Green F, Woodward BM, Evans JW, De Groot RC (2000) Correlation between oxalic acid production and copper tolerance in Wolfiporia cocos. Int. Biodeter. Biodegr. 46: 69-76.
Collet O (1992) Comparative tolerance of the brown-rot fungus Antrodia vaillantii (DC.:Fr.) Ryv. isolates to copper. Holzforschung 46: 293-298.
Conaf-Conama-Birf (1999a) Catastro y evaluacion de recursos vegetacionales nativos de Chile. Informe Regional Octava Region. Universidad Austral de Chile / Pontificia Universidad Catolica de Chile / Universidad Catolica de Temuco. Chile. 130 pp.
Conaf-Conama-Birf (1999b) Catastro y evaluacion de recursos vegetacionales nativos de Chile. Informe Nacional con Variables Ambientales. Universidad Austral de Chile / Pontificia Universidad Catolica de Chile / Universidad Catolica de Temuco. Chile. 89 pp.
D'Annibale A, Rosetto F, Leonardi V, Federici F, Petruccioli M (2006) Role of autochthonous filamentous fungi in bioremediation of a soil historically contaminated with aromatic hydrocarbons. Appl. Env. Microbiol. 72: 28-36.
Dashtban M, Schraft H, Syed TA, Qin W (2010) Fungal biodegradation and enzymatic modification of lignin. Int. J. Biochem. Mol. Biol. 1: 36-50.
Donoso CA, Becerra J, Martinez M, Garrido N, Silva M (2008) Degradative ability of 2,4,6-tribromophenol by saprophytic fungi Trametes versicolor and Agaricus augustus isolated from Chilean forestry. World J. Microbiol. Biotechnol. 24: 961-968.
D'Souza TM, Boominathan K, Reddy CA (1996) Isolation of laccase gene-specific secuences from white rot and brown rot fungi by PCR. Appl. Env. Microbiol. 62: 3739-3744.
Ferraz A, Guerra A, Mendonca R, Masarin F, Vicentin MP, Aguiar A, Pavan PC (2008) Technological advances and mechanistic basis for fungal biopulping. Enz. Microb. Technol. 43: 178-185.
Freitag M, Morrell JJ (1992) Decolorization of the polymeric dye Poly R-478 by wood-inhabiting fungi. Can. J. Microbiol. 38: 811-822.
Gadd GM (1993) Interactions of fungi with toxic metals. New Phytol. 124: 25-60.
Gadd GM (2004) Microbial influence on metal mobility and application for bioremediation. Geoderma 122: 109-119.
Gao D, Du L, Yang J, Wu W-M, Liang H (2010) A critical review of the application of white-rot fungi to environmental pollution control. Crit. Rev. Biotechnol. 30: 70-77.
Gramms G, Gunther Th, Fritsche W (1998) Spot tests for oxidative enzymes in ectomycorrhizal, wood-, and litter decaying fungi. Mycol. Res. 102: 67-72.
Green F, Clausen CA (2003) Copper tolerance of brown-rot fungi: time course of oxalic acid production, Int. Biodeter. Biodegr. 51: 145-149.
Guillen Y, Machuca A (2008) The effect of copper on the growth of wood-rotting and a bluestain fungus. World J. Microbiol. Biotechnol. 24: 31-37.
Hakala T, Maijala P, Konn J, Hatakka A (2004) Evaluation of novel wood-rotting polypores and corticoid fungi for the decay and biopulping of Norway spruce (Picea abies) wood. Enz. Microb. Technol. 34: 255-263.
Hernandez-Luna C, Gutierrez-Soto G, Salcedo-Martinez S (2008) Screening for decolorizing basidiomycetes in Mexico. World J. Microbiol. Biotechnol. 24: 465-473.
Hoffmann A (1998) Flora Silvestre de Chile Zona Central. 4th ed. Fundacion Claudio Gay. Chile. 254 pp.
Horak E (1979) Fungi, Basidiomycetes Agaricales y Gasteromycetes secotioides. In Guarrera SA, Gamundi IJ, Rabinovich D (Eds.) Flora Criptogamica de Tierra del Fuego. Vol. XI. FE- CYC, Buenos Aires, Argentina. 524 pp.
Illman B, Yang V (2004) Bioremediation and degradation of CCA-C-treated wood waste. In Proceedings of Environmental Impacts of Preservative-Treated Wood. Center of Environmental Solutions. Gainesville, FL, USA. 10 pp.
Kaarik A (1965) The identification of the myeelia of wood-decay fungi by their oxidation reactions with phenolic compounds. St. Forest. Suec. 31: 1-81.
Kirk K, Cullen D (1998) Enzymology and molecular genetics of wood degradation by white-rot fungi. In Young R, Akhtar M (Eds.) Environmentally Friendly Technologies for the Pulp and Paper Industry. Wiley. New York, USA. pp. 273-307.
Kjoller A, Miller M, Struvwe S, Wolters V, Pflug A (2000) Diversity and role of microorganisms. In Schulze ED (Ed.) Carbon and Nitrogen Cycling in European Forest Ecosystems. Springer. Berlin, Germany. pp. 382-402.
Krieglsteiner GJ (2000) Standerpilze: Gallert-, Rinden-, Stachelund Porenpilze. In Krieglsteiner GJ (Ed.) Die Grosspilze Baden-Wurttembergs. Vol. 1. Ulmer. Stuttgart, Germany. 629 pp.
Kuhad R, Singh A, Eriksson K (1997) Microorganisms and enzyme involved in the degradation of plant fiber cell walls. In Eriksson KEL (Ed.) Advances in Biochemical Engineering Biotechnology, Vol. 57. Springer. Berlin, Germany. pp. 45-125.
Lazo W (2001) Hongos de Chile. Atlas Micologico. Universidad de Chile. Santiago, Chile. 231 pp.
Lee KH, Wi SG, Singh AP, Kim YS (2004) Micromorphological characteristics of decayed wood and laccase produced by the brown rot fungus Coniophora puteana. J. Wood Se. 50: 281-284.
Magan N, Fragoeiro S, Bastos C (2010) Environmental factors and bioremediation of xenobiotics using white rot fungi. Mycobiology 38: 238-248.
Matheus DR, Bononi VLR, Machado KMG (2000) Biodegradation of hexachloro-benzene by basidiomycetes in soil contaminated with industrial residues. World J. Microbiol. Biotechnol. 16: 415-421.
Mendonca R, Jara JF, Gonzalez V, Elissetche JP, Freer J (2008) Evaluation of the white-rot fungi Ganoderma australe and Ceriporiopsis subvermispora in biotechnological applications. J. Ind. Microbiol. Biotechnol. 35: 1323-1330.
Minussi RC, de Moraes SG, Pastore GM, Duran N (2001) Biodecolorization screening of synthetic dyes by four white-rot fungi in a solid medium : possible role of siderophores. Lett. Appl. Microbiol. 33: 21-25.
Mujica F, Vergara C, Oehrens E (1980) Flora Fungosa Chilena. 2nd ed. Universidad de Chile. Ciencias Agricolas No 5, Santiago-Chile. 308 pp.
Murphy RJ, Levy JF (1983) Production of copper oxalate by some copper tolerant fungi. Trans. J. Brit. Mycol. Soc. 81: 165-168.
Okino LK, Machado KMG, Fabric C, Bonomi VLR (2000) Ligninolytic activity of tropical rainforest basidiomycetes. World J. Microbiol. Biotechnol. 16: 889-893.
Pelaez F, Martinez M, Martinez A (1995) Screening of 68 species of basidiomycetes for enzymes envolved in lignin degradation. Mycol. Res. 1: 37-42.
Pointing SB (1999) Qualitative methods for the determination of lignocellulolytic enzyme production by tropical fungi. Fungal Div. 2: 17-33.
Pointing SB (2001) Feasibility of bioremediation by white-rot fungi. Appl. Microbiol. Biotechnol. 57: 20-33.
Pointing SB, Bucher V, Vrijmoed L (2000) Dye decolorization by sub-tropical basidiomyceous fungi and the effect of metals on decolorizing ability. World J. Microbiol. Biotechnol. 16: 199-205.
Rajchenberg M (2006) Los Poliporos (Basidiomycetes) de los Bosques Andino Patagonicos de Argentina. Biblioteca Mycoligica. Cramer. Berlin, Germany. 300 pp.
Riis V, Babel W, Pucci OH (2002) Influence of heavy metals on the microbial degradation of diesel fuel. Chemosphere 49: 559-568.
Rodriguez-Couto S, Toco-Herrera JL (2006) Industrial applications of laccases: a review. Biotechnol. Adv. 24: 500-513.
Rubilar O, Tortella G, Cea M, Acevedo F, Bustamante M, Gianfreda L, Diez MC (2011) Bioremediation of a Chilean Andisol contaminated with pentachlorophenol (PCP) by solid substrate cultures of white-rot fungi. Biodegradation 22: 31-41.
Ryvarden L (1987) New and noteworthy Polypores from Tropical America. Mycotaxon 28: 525-541.
Ryvarden L (1991) Genera of Polypores. Nomenclature and Taxonomv. Synopsis Fungorum 5, Fungiflora, Norway. 363 pp.
Stalpers J (1978) Identification of wood-inhabiting Aphyllophorales in pure culture. St. Mycol. 16: 1-248.
Tham L, Matsuhashi S, Kume T (1999) Responses of Ganoderma lucidum to heavy metals. Mycoscience 40: 209-213.
Tortella GR, Rubilar O, Gianfreda L, Valenzuela E, Diez MC (2008) Enzymatic characterization of Chilean native woodrotting fungi for potential use in the bioremediation of polluted environments with chlorophenols. World J. Microbiol. Biotechnol. 24: 2805-2818.
Woodward B, De Groot R (1999) Tolerance of Wolfiporia cocos isolates to copper in agar media. Forest Prod. J. 49: 87-94.
Wright J, Deschamps J (1972) Basidiomycetes xilofagos de los bosques Andinopatagonicos. Rev. Inv. Agropec. INTA 9: 1-195.
Wright J, Alberto E (2006) Guia de los Hongos de la Region Pampeana II. Hongos sin Laminillas. Literature of Latin America. Buenos Aires, Argentina. 195 pp.
Yudith Guillen. Doctor in Forest Sciences, Universidad de Concepcion (UDEC), Chile. Professor-Researcher, Universidad Nacional Experimental de Guayana, Venezuela.
Gotz Palfner. Doctor in Natural Sciences, Ludwing Maximilians Universitat, Germany. Professor-Researcher, UDEC, Chile.
Angela Machuca. Doctor in Sciences, Universidade Estadual de Campinas, Brasil. Professor-Researcher, UDEC, Chile. Address: Department of Plant Science and Technology, Universidad de Concepcion, J. A. Coloma 0201, Los Angeles, Chile. e-mail: firstname.lastname@example.org
TABLE I COLLECTIONS OF BASIDIOMES OF WOOD-ROTTING FUNGI FROM DIFFERENT SUBSTRATES AND SITES IN SOUTHERN-CENTRAL CHILE Species Strain Herbary Code Site N [degrees] Agrocybe sp. 97 CONC-F0489 7 Anthracophyllum discolor (Mont.) 18 CONC-FO455 2 Singer Antrodia xantha (Fr.) Ryvarden * 139 CONC-FO509 11 Bjerkandera adusta (Willd. ex 30 CONC-F0403 2 Fr.) Karst B. adusta 62 CONC-FO470 2 B. adusta 119 CONC-FO499 1 B. adusta 122 CONC-FO500 1 Bondarzewia guaitecasensis 140 CONC-FO510 11 (Henn.) J.E. Wright apud Singer Clitocybula dusenii (Bresadola) 144 CONC-FO514 10 Singer Flammulina velutipes (Curtis) 95 CONC-F0488 7 Singer F. velutipes 98 CONC-FO490 7 F. velutipes 117 CONC-F0498 7 Ganoderma australe (Fr.) Pat. 28 CONC-F0462 2 G. australe 64 CONC-FO471 2 G. australe 86 CONC-FO480 4 G. australe 87 CONC-F0481 2 G. australe 100 CONC-FO492 5 G. australe 105 CONC-FO493 5 G. australe 142 CONC-FO512 8 Gloeophyllum abietinum (Fr.) P. 132 CONC-FO504 8 Karst * G. protractum (Fr.) Imaz. * 31 CONC-F0464 6 Gloeophyllum sp. 8 CONC-FO447 1 Gymnopilus spectabilis (Fr.) 137 CONC-FO507 6 Smith G. spectabilis 138 CONC-F0508 6 Lenzites betulina (Fr.) Fr. 20 CONC-FO457 2 L. betulina 22 CONC-F0458 2 L. betulina 27 CONC-F0461 2 L. betulina 34 CONC-F0465 2 L. betulina 67 CONC-FO472 2 L. betulina 111 CONC-F0496 5 L. betulina 131 CONC-FO503 8 Paxillus panuoides (Fr.) Fr. 83 CONC-FO479 3 Phellinus sp. 17 CONC-FO454 1 Phellinus sp. 42 CONC-F0468 4 Pleurotus ostreatus (Jacq. ex 91 CONC-F0485 7 Fr.) Kumm P. ostreatus 92 CONC-F0485 7 Schizophyllum commune Fr. 25 CONC-F0460 2 Serpula lacrymans (Wulfen) Karst. 141 CONC-FO511 4 Stereum hirsutum (Willd.) Gray 9 CONC-F0448 2 S. hirsutum 10 CONC-FO449 2 S. hirsutum 15 CONC-FO452 2 S. hirsutum 19 CONC-F0456 2 S. hirsutum 69 CONC-FO473 2 S. hirsutum 72 CONC-FO475 2 S. hirsutum 89 CONC-F0483 2 S. hirsutum 99 CONC-FO491 7 S. hirsutum 108 CONC-FO494 5 S. hirsutum 113 CONC-FO497 5 S. hirsutum 125 CONC-FO501 7 Stereum rameale (Pers.: Fr.) 71 CONC-FO474 2 Burt * Trametes versicolor (L.) Pilit 1 CONC-FO445 1 T. versicolor 7 CONC-F0446 1 T. versicolor 12 CONC-FO451 2 T. versicolor 24 CONC-FO459 2 T. versicolor 38 CONC-F0466 2 T. versicolor 73 CONC-F0476 4 T. versicolor 81 CONC-FO477 2 T. versicolor 88 CONC-F0482 2 T. versicolor 110 CONC-FO495 5 Species Substrate Agrocybe sp. Stem of Acer sp. Anthracophyllum discolor (Mont.) Unidentified log Singer Antrodia xantha (Fr.) Ryvarden * Stump of Pinus radiata Bjerkandera adusta (Willd. ex Unidentified log Fr.) Karst B. adusta Log of Eucalyptus nitens B. adusta Stem of Liquidambar styraciflua B. adusta Stem of Fraxinus excelsior Bondarzewia guaitecasensis Stem of Nothofagus obliqua (Henn.) J.E. Wright apud Singer Clitocybula dusenii (Bresadola) Log of Nothofagus obliqua Singer Flammulina velutipes (Curtis) Stem of Acer sp. Singer F. velutipes Unidentified log F. velutipes Stem of Acer sp. Ganoderma australe (Fr.) Pat. Unidentified wood G. australe Log of Eucalyptus nitens G. australe Stem of Prunus armeniaca G. australe Stem of Robinia pseudoacacia G. australe Unidentified stump G. australe Unidentified stump G. australe Log of Nothofagus obliqua Gloeophyllum abietinum (Fr.) P. Post of Cupressus sp. Karst * G. protractum (Fr.) Imaz. * Log of Pinus radiata Gloeophyllum sp. Stem of Prunus cerasifera Gymnopilus spectabilis (Fr.) Stump of Pinus radiata Smith G. spectabilis Stump of Pinus radiata Lenzites betulina (Fr.) Fr. Branch of Acacia melanoxylon L. betulina Unidentified log L. betulina Unidentified log L. betulina Unidentified log L. betulina log of Acacia melanoxylon L. betulina Unidentified log L. betulina Branch of Persea lingue Paxillus panuoides (Fr.) Fr. Burnt log of Pinus radiate Phellinus sp. Unidentified stump Phellinus sp. Stem of Peumus boldus Pleurotus ostreatus (Jacq. ex Stem of Prunus cerasifera Fr.) Kumm P. ostreatus Stem of Prunus cerasifera Schizophyllum commune Fr. Wood of Acacia melanoxylon Serpula lacrymans (Wulfen) Karst. Wood of Pinus radiata Stereum hirsutum (Willd.) Gray Unidentified branch S. hirsutum Stem of Eucalyptus nitens S. hirsutum Log of Acacia melanoxylon S. hirsutum Unidentified log S. hirsutum Bark of Eucalyptus nitens S. hirsutum Branch of Eucalyptus nitens S. hirsutum Unidentified log S. hirsutum Log of Acacia melanoxylon S. hirsutum Unidentified branch S. hirsutum Unidentified branch S. hirsutum Branch of Quercus palustris Stereum rameale (Pers.: Fr.) Branch of Aextoxicon punctatum Burt * Trametes versicolor (L.) Pilit Unidentified stump T. versicolor Unidentified stump T. versicolor Unidentified log T. versicolor Log of Eucalyptus nitens T. versicolor Unidentified log T. versicolor Stem of Aristotelia chilensis T. versicolor Unidentified log T. versicolor Unidentified log T. versicolor Unidentified log 1: Concepcion, UDEC university campus; 2: La Cantera and el Guindo Forest (native forest and plantation); 3: San Ignacio Estate, Quilacoya, Hualqui (plantation); 4: Concepci6n center; :5 Tumbes Park, Talcahuano (native forest and plantation); 6: Jorge Alessandri Park, route to Coronel (native forest and plantation); 7: Los Angeles, UDEC university campus; 8: San Lorenzo Estate, Santa Barbara (native forest); 9: Saltos del Laja Park, Laja (native forest); 10: Los Patos Sector, Nacimiento (native forest); 11: Collanmahuida Park, Florida (native forest). TABLE II DETECTION OF LIGNOCELLULOLYTIC ENZYME ACTIVITY AND TOLERANCE TO METAL IONS IN MEA MEDIUM OF SPECIES OF WOOD-ROT FUNGI COLLECTED IN SOUTHERN-CENTRAL CHILE Species Strain (a) CMC X G TA RBB Agrocybe sp. 97 + + - - - Anthracophyllum discolor 18 + + + + + Antrodia xantha 139 +/- + - - - Bjerkandera adusta 30 + + - - + B. adusta 62 + + - - + B. adusta 119 + + - - + B. adusta 122 +/- + - + + Bondarzewia guaitecasensis 140 +/- + + + + Cluoeybula dusenii 144 + + + + + Flammulina velutipes 95 + + - - + F. velutipes 98 + + - - - F. velutipes 117 + + - + - Ganoderma australe 28 + + - - + G. australe 64 +/- + + + + G. australe 86 + + - - + G. australe 87 + + + + + G. australe 100 + + - + + G. australe 105 + + - + + G. australe 142 + + + + + Gloeophyllum abietinum 132 + + - - - G. protractum 31 + + - - - Gloeophyllum sp. 8 +/- + - - - Gymnopilus spectabilis 137 + + - + + G. spectabilis 138 +/- + - + + Lenzites betulina 20 +/- + - + + L. betulina 22 + + - + + L. betulina 27 + + + + + L. betulina 34 + + + + + L. betulina 67 +/- + - + + L. betulina 111 + + + + + L. betulina 131 + + + + + Paxillus panuoides 83 +/- + - - + Phellinus sp. 17 +/- + + + + Phellinus sp. 42 + + + + + Pleurotus ostreatus 91 + + + + + P. ostreatus 92 + + + + + Schyzophyllum commune 25 + + - + + Serpula lacrymans 141 +/- + - - - Stereum hirsutum 9 +/- + - + + S. hirsutum 10 + + - + - S. hirsutum 15 + + + + + S. hirsutum 19 + + + + + S. hirsutum 69 + + - + - S. hirsutum 72 +/- + - + + S. hirsutum 89 + + - + - S. hirsutum 99 + + + + - S. hirsutum 108 +/- + - + + S. hirsutum 113 +/- + - + + S. hirsutum 125 + + + + + Siereum rameale 71 +/- + - - - Trametes versicolor 1 +/- + + + + T versicolor 7 + + - - + T versicolor 12 + + + + + T versicolor 24 + + + + + T versicolor 38 + + + + + T versicolor 73 + + + + + T. versicolor 81 + + + + + T versicolor 88 + + + + + T versicolor 110 + + + + + Wolfiporia cocos ATCC62778 +/- + - - - G. trabeum ATCC11539 + + - - - T versicolor DEBIO + + + + + G. australe A464 + + + + + GR (mm/day) Species PolyR WR Control (b) Agrocybe sp. - B 3.24 [+ or -] 0.03 Anthracophyllum discolor + W 1.45 [+ or -] 0.00 Antrodia xantha - B 5.82 [+ or -] 0.32 Bjerkandera adusta + W 4.90 [+ or -] 0.46 B. adusta + W 8.10 [+ or -] 0.10 B. adusta + W 3.94 [+ or -] 0.53 B. adusta + W 8.90 [+ or -] 0.03 Bondarzewia guaitecasensis + W 1.08 [+ or -] 0.16 Cluoeybula dusenii + W 1.32 [+ or -] 0.02 Flammulina velutipes + W 5.43 [+ or -] 0.08 F. velutipes - W 3.48 [+ or -] 0.39 F. velutipes - W 3.94 [+ or -] 0.05 Ganoderma australe + W 8.80 [+ or -] 0.04 G. australe + W 5.28 [+ or -] 0.04 G. australe + W 3.40 [+ or -] 0.03 G. australe - W 3.90 [+ or -] 0.14 G. australe - W 2.74 [+ or -] 0.36 G. australe - W 2.63 [+ or -] 0.04 G. australe + W 5.14 [+ or -] 0.20 Gloeophyllum abietinum - B 9.08 [+ or -] 0.35 G. protractum - B 2.52 [+ or -] 0.27 Gloeophyllum sp. - B 3.90 [+ or -] 0.16 Gymnopilus spectabilis + W 3.13 [+ or -] 0.00 G. spectabilis + W 3.38 [+ or -] 0.10 Lenzites betulina + W 5.67 [+ or -] 0.07 L. betulina + W 7.42 [+ or -] 0.26 L. betulina + W 6.37 [+ or -] 0.17 L. betulina + W 4.29 [+ or -] 0.12 L. betulina + W 6.02 [+ or -] 0.10 L. betulina + W 5.19 [+ or -] 0.06 L. betulina + W 5.62 [+ or -] 0.62 Paxillus panuoides - B 4.35 [+ or -] 0.25 Phellinus sp. + W 1.61 [+ or -] 0.31 Phellinus sp. + W 1.60 [+ or -] 0.05 Pleurotus ostreatus - W 5.52 [+ or -] 0.02 P. ostreatus - W 4.58 [+ or -] 0.35 Schyzophyllum commune - W 4.08 [+ or -] 0.l0 Serpula lacrymans - B 0.39 [+ or -] 0.05 Stereum hirsutum - W 6.98 [+ or -] 0.18 S. hirsutum - W 5.45 [+ or -] 0.22 S. hirsutum - W 6.15 [+ or -] 0.52 S. hirsutum + W 1.58 [+ or -] 10.02 S. hirsutum - W 5.50 [+ or -] 0.01 S. hirsutum + W 6.20 [+ or -] 0.30 S. hirsutum + W 2.99 [+ or -] 0.12 S. hirsutum - W 6.88 [+ or -] 0.05 S. hirsutum - W 6.90 [+ or -] 0.13 S. hirsutum - W 6.40 [+ or -] 0.70 S. hirsutum - W 6.75 [+ or -] 0.32 Siereum rameale - W 5.56 [+ or -] 0.08 Trametes versicolor + W 8.15 [+ or -] 0.75 T versicolor + W 10.6 [+ or -] 0.40 T versicolor + W 7.42 [+ or -] 0.12 T versicolor + W 11.10 [+ or -] 0.57 T versicolor + W 7.34 [+ or -] 0.01 T versicolor + W 5.41 [+ or -] 0.09 T. versicolor + W 7.02 [+ or -] 0.02 T versicolor + W 3.70 [+ or -] 0.31 T versicolor + W 8.60 [+ or -] 0.50 Wolfiporia cocos - B 11.36 [+ or -] 1.21 G. trabeum - B 4.16 [+ or -] 0.07 T versicolor + W 7.15 [+ or -] 0.20 G. australe + W 4.04 [+ or -] 0.72 GR (mm/day) Species 3mM Cu (c) Agrocybe sp. NG Anthracophyllum discolor 0.34 [+ or -] 0.0 (76) Antrodia xantha 3.23 [+ or -] 0.06 (44) Bjerkandera adusta 1.27 [+ or -] 0.07 (74) B. adusta 1.50 [+ or -] 0.36 (81) B. adusta 1.65 [+ or -] 0.02 (58) B. adusta 1.18 [+ or -] 0.12 (87) Bondarzewia guaitecasensis NG Cluoeybula dusenii 0.40 [+ or -] 0.03 (70) Flammulina velutipes NG F. velutipes NG F. velutipes NG Ganoderma australe 3.36 [+ or -] 0.30 (62) G. australe NG G. australe 1.35 [+ or -] 0.01 (60) G. australe 1.62 [+ or -] 0.17 (58) G. australe 2.10 [+ or -] 0.29 (23) G. australe 1.49 [+ or -] 0.07 (43) G. australe 1.87 [+ or -] 0.17 (64) Gloeophyllum abietinum 2.30 [+ or -] 0.27 (75) G. protractum 0.07 [+ or -] 0.00 (97) Gloeophyllum sp. 2.80 [+ or -] 0.40 (28) Gymnopilus spectabilis 0.93 [+ or -] 0.09 (70) G. spectabilis 1.44 [+ or -] 0.03 (57) Lenzites betulina NG L. betulina NG L. betulina NG L. betulina NG L. betulina NG L. betulina NG L. betulina NG Paxillus panuoides 1.63 [+ or -] 0.08 (62) Phellinus sp. 0.47 [+ or -] 0.05 (71) Phellinus sp. 0.56 [+ or -] 0.01 (65) Pleurotus ostreatus 0.38 [+ or -] 0.01 (93) P. ostreatus 1.03 [+ or -] 0.02 (77) Schyzophyllum commune NG Serpula lacrymans 0.60 [+ or -] 0.01 (0) Stereum hirsutum 0.50 [+ or -] 0.04 (93) S. hirsutum 0.59 [+ or -] 0.07 (89) S. hirsutum 0.71 [+ or -] 0.19 (88) S. hirsutum 0.58 [+ or -] 0.01 (63) S. hirsutum NG S. hirsutum 1.16 [+ or -] 0.14 (81) S. hirsutum 1.04 [+ or -] 0.09 (65) S. hirsutum 0.42 [+ or -] 0.06 (94) S. hirsutum 0.36 [+ or -] 0.01 (95) S. hirsutum 0.40 [+ or -] 0.14 (94) S. hirsutum 0.40 [+ or -] 0.10 (78) Siereum rameale 1.21 [+ or -] 0.03 (97) Trametes versicolor 0.28 [+ or -] 0.01 (80) T versicolor 2.15 [+ or -] 0.40 (80) T versicolor NG T versicolor 2.34 [+ or -] 0.04 (79) T versicolor 2.57 [+ or -] 0.84 (65) T versicolor 2.09 [+ or -] 0.12 (71) T. versicolor 2.34 [+ or -] 0.04 (67) T versicolor 1.60 [+ or -] 0.04 (57) T versicolor 1.42 [+ or -] 0.03 (83) Wolfiporia cocos 5.29 [+ or -] 0.63 (53) G. trabeum 0.68 [+ or -] 0.30 (84) T versicolor 1.15 [+ or -] 0.30 (84) G. australe 0.71 [+ or -] 0.11 (82) GR (mm/dy) Species 1mM C (d) Agrocybe sp. NG Anthracophyllum discolor NG Antrodia xantha 0.75 [+ or -] 0.04 (87) Bjerkandera adusta NG B. adusta 0.26 [+ or -] 0.04 (97) B. adusta NG B. adusta 1.22 [+ or -] 0.09 (86) Bondarzewia guaitecasensis NG Cluoeybula dusenii 0.32 [+ or -] 0.02 (76) Flammulina velutipes NG F. velutipes NG F. velutipes NG Ganoderma australe NG G. australe 0.67 [+ or -] 0.03 (87) G. australe NG G. australe 0.29 [+ or -] 0.03 (82) G. australe NG G. australe 0.26 [+ or -] 0.04 (90) G. australe 0.39 [+ or -] 0.06 (92) Gloeophyllum abietinum NG G. protractum NG Gloeophyllum sp. NG Gymnopilus spectabilis NG G. spectabilis NG Lenzites betulina NG L. betulina NG L. betulina NG L. betulina NG L. betulina NG L. betulina NG L. betulina NG Paxillus panuoides 1.27 [+ or -] 0.31 (71) Phellinus sp. NG Phellinus sp. NG Pleurotus ostreatus NG P. ostreatus NG Schyzophyllum commune NG Serpula lacrymans 0.66 [+ or -] 0.01 (0) Stereum hirsutum 0.15 [+ or -] 0.02 (98) S. hirsutum NG S. hirsutum 0.33 [+ or -] 0.06 (95) S. hirsutum NG S. hirsutum NG S. hirsutum NG S. hirsutum 0.40 [+ or -] 0.02 (87) S. hirsutum NG S. hirsutum NG S. hirsutum 0.11 [+ or -] 0.01 (98) S. hirsutum NG Siereum rameale NG Trametes versicolor NG T versicolor 0.88 [+ or -] 0.11 (92) T versicolor 1.03 [+ or -] 0.06 (86) T versicolor NG T versicolor NG T versicolor NG T. versicolor NG T versicolor NG T versicolor 1.11 [+ or -] 0.05 (87) Wolfiporia cocos 0.81 [+ or -] 0.39 (93) G. trabeum 0.44 [+ or -] 0.04 (89) T versicolor 0.82 [+ or -] 0.31 (89) G. australe NG (a) Number of collection of the strain, (b) growth rate (mm-day) in MEA medium without metals, and (c) with 3mM copper and (d) with 1mM cadmium. CMC: carboxymethylcellulose, X: xylan, G: guaiacol, TA: tannic acid, RBB: Remazol Brilliant Blue R, Poly-R 478, WR: wood-rot type, W: white, B: Brown, +: positive reaction with formation of coloration or discoloration halo of the substrate indicator, +/-: weak reaction, -: negative reaction, NG: no growth observed. Values of GR [+ or -] SD. Values in parenthesis indicate the percentage of growth inhibition with respect to the control (without metal).