Cultural and ligninolytic activity studies of some polypores ( Basidiomycota ) from brazilian Atlantic Forest , São Paulo State , Brazil

(Cultural and ligninolytic activity studies of some polypores (Basidiomycota) from brazilian Atlantic Forest, São Paulo State, Brazil). During a fungi survey in the brazilian Atlantic Forest in São Paulo, São Paulo State, Brazil, polypores growing on decaying wood were collected and fragments of the basidiomata were used to obtain isolates in pure culture. A total of 37 isolates were obtained and 23 species were identified. Cultural features of Bjerkandera atroalba, Henningsia brasiliensis, Rigidoporus undatus, and Steccherinum reniforme were described for the first time. A total of 31 isolates were evaluated in terms of their ability to oxidize the Remazol brilliant blue R dye (RBBR) and guaiacol. The results of these tests indicate that all isolated species were associated with white rot in the wood. Laccase and peroxidase activities were detected by drop tests using solutions of α-naphthol and pyrogallol. Production of cellulases and siderophores was detected with carboxymethylcellulose (CMC) and chrome azurol S (CAS) agar, respectively.


Introduction
Morphology until the latter part of the last century was the main tool used to characterize and to develop the taxonomy and putative phylogeny of different fungi groups (Rajchenberg 2011).Agaricomycetes have been studied in culture for about 100 years, and published works such as Nobles (1965) made the first attempt to understand polypores on the basis of their cultural features studied in vitro.Her work stressed the importance of (i) ability to degrade wood (brown vs. white rot types) as a key element when considering generic delimitation and taxonomy, (ii) detailed macro-and microscopic features of cultures in vitro and (iii) mating system as a character of great importance that underlines the biology of each species.After this work, an increasing number of studies were developed recording those features for each taxon in the literature.Stalpers (1978) published a major work of cultural characteristics of Polyporales and Corticiales and Nakasone (1990) presented a cultural description of 277 wood-inhabiting basidiomycetes, Rajchenberg (2011) present a review of the available knowledge on nuclear behavior of the mycelium, mating system and number of nuclei of the basidiospores within polypore genera, among others.
For decades, the division based on the presence or absence of extracellular oxidase in cultures proposed by Nobles (1965) to differentiate the fungi with different capacities to degrade wood, white-rot fungi, which breakdown lignin and cellulose, and brown-rot fungi, which breakdown cellulose and hemicellulose, was recognized like a character that have diagnostic significance to separate different species of woodinhabiting fungi (Lundell et al. 2010).A recent phylogenetic study by Riley et al. (2014) indicates that this simple dichotomy of white rot vs. brown rot does not properly reflect the diversity of mechanisms by which wood-rotting fungi obtain nutrition.This study suggest that a more nuanced categorization scheme is needed to describe wood decay by species that degrade all cell wall polymers.
Nevertheless, the study of features of fungal cultures isolated in axenic conditions and the qualitative detection of enzymes causing the decomposition of lignin is still valuable as an identification tool, and a first approach to describe the underlying biology and biochemistry of the fungi (Nakasone 1990).This approach is also very useful to select isolates with potential use in biotechnological, biotransformation and bioremediation processes (Machado et al. 2005).
In this way, this study aimed: (1) to expand the knowledge about the biology of polypores presents in the Parque Estadual da Cantareira and Parque Estadual da Serra do Mar, São Paulo State, Brazil, with culture studies, and (2) to increase the knowledge of the enzymatic activity of some brazilian fungi, which have been poorly studied to date.

Materials and methods
Study of pure cultures -Specimens were collected from August 2011 to February 2015 in the Parque Estadual da Cantareira (PEC), (23°32'36"S, 46º37'59"W) and Parque Estadual da Serra do Mar (PESM) -Núcleo Santa Virginia (23º24' to 23º17'S, 45º03' W), São Paulo, Brazil.The species identification was based on macroscopic and microscopic characters of basidiomata.The specimens were deposited in the herbarium SP.Cultures were obtained from fresh basidiomata and grown in Malt Extract Agar (MEA).The species codes describing the cultures follow the Species Code of Nobles (1965) with the modifications summarized by Nakasone (1990).The isolates were preserved in sterile distilled water (Castellani 1967, Burdsall & Dorworth 1994) and deposited in the Coleção de Culturas de Algas, Cianobactérias e Fungos (CCIBt) of the Instituto de Botânica, São Paulo, Brazil.
Enzymatic test -All the enzymatic tests were inoculated with a 5 mm diam.agar plug, always in triplicate.Plates were incubated at 25 °C in the dark for 14 days.
Phenol-oxidases production (Laccase s.s.) -was Detected by formation of a reddish zone in the culture medium containing 0.2% sugarcane bagasse powder, 0.01% guaiacol (Sigma) and 1.6% agar (Nishida et al. 1998).The drop test (Stalpers 1978) was carried out on the same plate using: (a) α -naphthol solution to detect Laccase (0.1 M in 96% ethanol) and (b) pyrogallol solution to detect Lignin Peroxidase (equal parts of 4% hydrogen peroxide and 1% pyrogallol, freshly prepared).The production of Laccase and Lignin Peroxidase was detected by formation of a purple and a yellowish-brown zone in the culture medium, respectively.
Comments: the velvety to woolly mat and unusually downy, white to yellowish-brown, the dimitic hyphal system with generative hyphae clamped and skeletal hyphae in the aerial mat are features of this species studied in culture, which is consistent with the observations made by Stalpers (1978).
Species code: 2. 3.4.34.36.40.41.53.54.60.Comments: Molecular analysis confirmed that Tyromyces atroalbus (Rick) Rajchenb.belongs to Bjerkandera P. Karst and is separated from Tyromyces P. Karst, which is supported by the morphological features, such as the darkening of the basidiomata when dried and distinctly monomitic hyphal system (Westphalen et al. 2015).This study presents the first description of the culture of Bjerkandera atroalba characterized by the formation of chlamydospores and a monomitic hyphal system with generative hyphae with clamps.This features are shared with the culture of B. adusta (Willd.)P. Karst.(as Polyporus adustus Willd.), another species of the genus, described by Nobles (1965).This results differ with descriptions of cultures of species of Tyromyces in which Stalpers (1978) described the presence of skeletal hyphae.This results presents additional data that supports the transfer of this species to Bjerkandera.
Description of culture in Neves (1998).
Comments: the fast growth, the downy to felty mat and the presence of exudates hyaline to yellowish in most colonies characterizes this species in culture.Although the hyphal system of the basidioma is trimitic, in culture it was only possible to find generative hyphae with clamps and brown skeletal hyphae.These results differ from those presented by Neves (1998) Description of culture in Rajchenberg (1983) as Megasporoporia setulosa (Henn.)Rajchenb.
Species code: 2. 3.4.8.32.36.38.40.43.44.50.54.60.Comments: this culture presents a dextrinoid reaction in the skeletal hyphae, which is a feature that generally have been used to identify species of this genus (Ryvarden 2004).The same reaction was observed by Neves (1998)  Comments: Fomitella supina is morphologically similar in culture with species of Fomitopsis P. Karst., except by the production of white rot in the former and the production of chlamydospores in the latter (Stalpers 1978, Neves 1998).
Description of culture in Nobles (1965).
Comments: the culture of this species is characterized in culture by white and then dark brown mat with small floccose areas.Microscopically the presence of hyaline to yellowish generative hyphae, simple septate, hyaline to brown skeletal hyphae, globose to ovoid chlamydospores and irregular cells forming a pseudoparenchyma are characteristic.Ganoderma australe (Fr.)Pat., Bull.Soc.Mycol.
Species code: 2. 3.8.10.32.37.39.(40).46.47.50.54.55.Comments: this species is very variable in culture, feature observed in the three different isolates obtained from different basidiomata.But it usually can be recognized by the slow growth, cracking the agar medium and wrinkling the colony surface.Besides of the mat white and the abundant sphaerical cuticular cells, features that were also observed by Bettucci & Guerrero (1971) and Yeh et al. (2000).
Micromorphological characters -Advancing zone: generative hyphae simple septate, hyaline, straight, branched, with smooth and thin-walled, 1.5-3.0µm diam.Aerial mycelium: as in the advancing zone.Pseudoparenchyma: hyphae differentiated, with thickened walls presents in the crusty areas.Submerged mycelium: as in the advancing zone.Species code: 2.6.10.32.36.(37).38.45.46.53.54.Comments: Henningsia brasiliensis has been closely associated with the genus Rigidoporus Murrill with regard to their morphological characteristics.In culture, it is possible to relate the two genera by the coloration of the mat and the formation of pseudoparenchyma.But in the culture, H. brasiliensis could be separated microscopically by the absence of cystidia, when compared with the two species of Rigidoporus studied here: R. lineatus (Pers.)Ryv. and R. microporus (Fr.)Overeem).
Comments: the cultural features of our specimen are quite similar to those found by Stalpers (1978).
The white mat with salmon pores after 3-5 weeks, encrusted skeletal hyphae and variably present cystidia sometimes present, apically somewhat swollen are characteristics of this species in culture.Description of culture in Lombard et al. (1960).
Comments: the rapid growth on MEA, absence of clamps, generative hyphae often encrusted and absence of cystidia are characteristics of this species in culture.The results presented here agree with those reported by Lombard et al. (1960).
Comments: the cottony and white mat, the dimitic hyphal system with clamped generative hyphae, branched skeletal hyphae and abundant arthroconidia characterizes the culture of this species.Neves (1998) observed the presence of crystals in the cultures studied, this results differ from our observations and largely with those made by Stalpers (1978).Description of culture in Nobles (1965).
Comments: the strong orange to pinkish-salmon color of the culture is similar to the color of the basidioma.This is a very important characteristic for easy recognition of the species in culture.Our results agree with those presented by Nobles (1965), Bettucci & Guerrero (1971) and Wright & Deschamps (1975).Neves (1998) observed the formation of chlamydospores and rhomboid crystals near the mat surface, characteristics absent in the cultures studied in this work.Rigidoporus lineatus (Pers.)Ryvarden, Norw.J. Bot. 19: 236 (1972) Description of culture in Wright & Deschamps (1975).
Comments: Tinctoporellus epimiltinus showed similar growth and microscopic characteristics of Grammothele subargentea (Speg.)Rajchenb.in culture medium (Rajchenberg 1983).Both change the color of the culture medium, making it reddish and form a plectenchyma.The basidioma of both species exhibit a similar hyphal system, with dextrinoid skeletal hyphae and the production of white rot in the substrate.In addition, different species of the genus Grammothele Berk.& M.A. Curtis have also been transferred or synonymized with Porogramme (Pat.)Pat.species (see Soares et al. 2015), which also indicates the narrow morphological and ecological relationship of these three genus and the need for taxonomic and phylogenetic revision with more specialized techniques like cytological and sexual studies as commented by Rajchenberg (1983).
Cultures examined: BRASIL.Enzymatic test -A total of 37 specimens were isolated from basidiomata growing in decaying wood and classified into 23 species.Of all the isolated obtained, 31 were qualitatively evaluated in terms of their ligninolytic activity (table 1).All species were associated with production of a white rot with regard to the presence of Phenol-oxidases that was confirmed for almost all specimens tested.In this test only had negative results: A. semisupina (RBBR), S. flavipora (all tests), I. tropicalis (Guaiacol), S. reniforme (Laccase and RBBR) T. sector (Guaiacol) and T. atroalbus (laccase, peroxidase and RBBR).These species were also associate with production of a white rot from data obtained in the literature (Stalpers 1978, Larsen & Lombard 1988, Alexander et al. 1989).Currently it is known that negative results in this tests may be related to the lack of specificity of the enzymes in the oxidation of substrates tested or absence of expression of the total ligninolytic system due to the culture conditions used (Platt et al. 1985).Production of cellulolytic enzymes was detected in 18 of the 31 isolates tested.In all the isolates the growth was very poor.These results agree with the literature, since the expression of the total cellulolytic activity could differ due to the culture conditions (Arantes & Milagres 2009).
, who described the presence of skeletal and connective hyphae in the culture.