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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.

SUMMARY

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.

RESUMEN

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.

RESUMO

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.

Introduction

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

Study area

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.

Conclusions

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.

ACKNOWLEDGMENTS

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.

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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: angmachu@udec.cl
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).
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Author:Guillen, Yudith; Palfner, Gotz; Machuca, Angela
Publication:Interciencia
Date:Nov 1, 2011
Words:8331
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