Fomitopsis betulina (formerly Piptoporus betulinus): the Iceman’s polypore fungus with modern biotechnological potential

Higher Basidiomycota have been used in natural medicine throughout the world for centuries. One of such fungi is Fomitopsis betulina (formerly Piptoporus betulinus), which causes brown rot of birch wood. Annual white to brownish fruiting bodies of the species can be found on trees in the northern hemisphere but F. betulina can also be cultured as a mycelium and fruiting body. The fungus has a long tradition of being applied in folk medicine as an antimicrobial, anticancer, and anti-inflammatory agent. Probably due to the curative properties, pieces of its fruiting body were carried by Ötzi the Iceman. Modern research confirms the health-promoting benefits of F. betulina. Pharmacological studies have provided evidence supporting the antibacterial, anti-parasitic, antiviral, anti-inflammatory, anticancer, neuroprotective, and immunomodulating activities of F. betulina preparations. Biologically active compounds such as triterpenoids have been isolated. The mushroom is also a reservoir of valuable enzymes and other substances such as cell wall (1→3)-α-d-glucan which can be used for induction of microbial enzymes degrading cariogenic dental biofilm. In conclusion, F. betulina can be considered as a promising source for the development of new products for healthcare and other biotechnological uses.


Introduction
In 1991, a mummified body was discovered in the Val Senales glacier in Italy. The man (named Ӧtzi the Iceman), who lived 5300 years ago, carried two fragments of a fruiting body of Fomitopsis betulina (formerly Piptoporus betulinus). Some scientists believe that Ӧtzi might have used the fungus for medical purposes (Capasso 1998) and, although the idea arouses some controversy (Pöder 2005), the long tradition of the use of F. betulina in folk medicine is a fact (Reshetnikov et al. 2001;Wasser 2010). Infusion from F. betulina fruiting bodies was popular, especially in Russia, Baltic countries, Hungary, Romania for its nutritional and calming properties. Fungal tea was used against various cancer types, as an immunoenhancing, anti-parasitic agent, and a remedy for gastrointestinal disorders (Grienke et al. 2014;Lucas 1960;Peintner and Pöder 2000;Semerdžieva and Veselský 1986;Shamtsyan et al. 2004). Antiseptic and anti-bleeding dressings made from fresh F. betulina fruiting body were applied to wounds and the powder obtained from dried ones was used as a painkiller (Grienke et al. 2014;Papp et al. 2015;Rutalek 2002).
In the present paper, we have shown the current knowledge of the fungus F. betulina, including its lifestyle, chemical composition, and potential in biotechnology.
Abstract Higher Basidiomycota have been used in natural medicine throughout the world for centuries. One of such fungi is Fomitopsis betulina (formerly Piptoporus betulinus), which causes brown rot of birch wood. Annual white to brownish fruiting bodies of the species can be found on trees in the northern hemisphere but F. betulina can also be cultured as a mycelium and fruiting body. The fungus has a long tradition of being applied in folk medicine as an antimicrobial, anticancer, and anti-inflammatory agent. Probably due to the curative properties, pieces of its fruiting body were carried by Ötzi the Iceman. Modern research confirms the health-promoting benefits of F. betulina. Pharmacological studies have provided evidence supporting the antibacterial, anti-parasitic, antiviral, anti-inflammatory, anticancer, neuroprotective, and immunomodulating activities of F. betulina preparations. Biologically active compounds such as triterpenoids have been isolated. The mushroom is also a reservoir of valuable enzymes and other substances such as cell wall (1→3)-α-d-glucan which can be used for induction of microbial enzymes degrading cariogenic dental biofilm. In conclusion, F. betulina can be considered as thicker walls. No primordia or fruiting bodies of this species were found in vitro (Petre and Tanase 2013). Basidiospores are smooth, hyaline, thin-walled, and cylindrical (Han and Cui 2015;Han et al. 2016;Kim et al. 2005;Schwarze 1993).
The birch polypore grows mainly as a saprophyte on dead trees and occasionally as a parasite of living trees. It occurs in northern temperate forests and parks in Europe, North America, and Asia. The host range of the fungus is restricted exclusively to birch species, e.g. Betula pendula Roth., B. pubescens Ehrh., B. papyrifera Marsh., and B. obscura Kotula (Schwarze 1993;Žižka et al. 2010).

Wood decay
Wood rotting fungi are traditionally divided into white and brown rot species based on the structure and composition of residual wood. Brown rot fungi extensively degrade the carbohydrate fraction of lignocellulose but, in contrast to white rot fungi, leave lignin, although in a modified form. In these fungi, chemical depolymerization of cellulose, which precedes and supports its enzymatic degradation, is very important. They lack ligninolytic peroxidases and usually some other enzymes such as processive cellobiohydrolases used for degradation of crystalline cellulose, but contain H 2 O 2 -generating oxidases and Fe 3+ -and quinone-reducing enzymes used for non-enzymatic depolymerization of polysaccharides (Arantes and Goodell 2014; Baldrian and Valášková 2008;Hori et al. 2013). Modern phylogenetic evidence suggest, however, that there is no sharp distinction between the two groups of fungi (Hori et al. 2013;Riley et al. 2014).
Fomitopsis betulina is one of the most common brown rot species but its wood-decaying mechanism has been tested only fragmentarily (Meng et al. 2012) and is still poorly understood. As other fungi of this type, it degrades wood to yield brown, cubical cracks easily broken down. Many factors, including microflora or compounds present in wood, contribute to this complex process (Przybył and Żłobińska-Podejma 2000;Song et al. 2016;Zarzyński 2009). Shang et al. (2013) showed that wood samples decayed by F. betulina lost 57% of dry weight (dw) and 74% of holocellulose after 30 days, whereas the fungus growing on wheat straw causes 65% loss of dw within 98 days of culture (Valášková and Baldrian 2006a). A set of enzymes of F. betulina involved in the degradation of lignocellulose was characterized in detail by Valášková and Baldrian (2006a, b). The fungus growing on straw produced enzymes with wide substrate specificities: (1→4)-β-endoglucanase, β-glucosidase, (1→4)-β-endoxylanase, (1→4)-β-endomannanase, (1→4)-β-xylosidase, and (1→4)-β-mannosidase. The activities of ligninolytic enzymes and cellobiose dehydrogenase for oxidoreductive cleavage of cellulose were not detected. Similar results were obtained in liquid cultures by Vĕtrovský et al. (2013). When F. betulina grew in nature, β-glucosidase and β-mannosidase activity was associated with the fruiting bodies while endopolysaccharidases were detected in colonized wood (Valášková and Baldrian 2006a).

Cultivation
Carpophores of F. betulina from natural habitats or mycelium and culture liquid from submerged cultures were used as raw material to obtain extracts and bioactive substances with medicinal properties (Table 1) (Lomberh et al. 2002). Studies concerning the mycelium growth rate in the presence of various substances (metals, dyes) were conducted mainly on agar media or in liquid cultures (Baldrian and Gabriel 2002;Dresch et al. 2015;Hartikainen et al. 2016). The yield of F. betulina mycelium was established in liquid cultures with addition of some agricultural wastes in the studies of Krupodorova and Barshteyn (2015). The enzymatic activity of F. betulina was studied in laboratory conditions on agar media (Krupodorova et al. 2014), in liquid cultures (Vĕtrovský et al. 2013), on wheat straw (Valášková and Baldrian 2006a, b), and on Betula sp. wood samples (Reh et al. 1986;Shang et al. 2013).
There are limited data on small-or large-scale cultivation of this species in which carpophores could be obtained in controlled conditions. The first such report referring to outdoor log cultivation of F. betulina on Betula davurica Pallas originated from Korea (Ka et al. 2008). Logs with a diameter of 8-18 cm and length of 107-135 cm were inoculated and then cultured in natural conditions. The yield obtained was in the range from 212 to 1298 g fresh weight (1-2 mushrooms per log). Development of fruiting bodies took an average of 18 months. The ratio of log yield was estimated at 2.8-6.1%. The only report on indoor production of F. betulina fruiting bodies was given by Pleszczyńska et al. (2016). In the study, four strains of F. betulina isolated from natural habitats were applied. Their mycelia were inoculated into birch sawdust supplemented with organic additives. Mature fruiting bodies weighing from 50 to 120 g were obtained from only one strain, after 3-4 months of the cultivation in artificial conditions (Fig. 1c). The biological efficiency ranged from 12 to 16%. It was shown that extracts isolated from cultivated and naturally grown F. betulina fruiting bodies had comparable biological activity (Table 1).
Although some authors considered young specimens of F. betulina edible (Wasson 1969), the fungus value is not the result of nutritional but therapeutic properties. The overview of the available literature concerning medical potential of birch polypore was presented in Table 1. Referring to the folk uses of the birch polypore, most of the presented research was based on crude extracts, which often have greater bioactivity than isolated constituents at an equivalent dose. This phenomenon is explained by mostly synergistic interactions between compounds present in mixtures. Furthermore, extracts often contain substances that inhibit multi-drug 83 Page 4 of 12 Kandefer-Szerszeń and  Ether extracts polyporenic acid (suggested) Kandefer-Szerszeń and Kawecki (1974) nucleic acids (RNA and DNA)  Page 5 of 12 83    Zwolińska (2004) 83 Page 6 of 12  Blumenberg and Kessler (1963) Page 7 of 12 83 resistance and therefore further increase the effectiveness of the active substances. Particularly noteworthy among the wide variety of biological activities of F. betulina extract, are properties proved in in vivo studies, e.g. the efficacy of water and ethanol extracts in treatment of the genital tract in dogs (Utzig and Samborski 1957;Wandokanty et al. 1954Wandokanty et al. , 1955 or mice protection from lethal infection with the TBE virus by water, ethanol, and ether extracts (Kandefer-Szerszeń et al. 1981;Kawecki 1974, 1979). The broad spectrum of antiviral and antimicrobial activity of F. betulina extracts proved by a number of research teams in different models based on different techniques deserves special attention as well (see references cited in Table 1).
Recently, Stamets (2011Stamets ( , 2014 has invented formulations prepared from different medicinal mushrooms including F. betulina, which are useful in preventing and treating viral and bacterial diseases, i.e. herpes, influenza, SARS, hepatitis, tuberculosis, and infections with E. coli and S. aureus . Some pure compounds corresponding to the bioactivity of the birch polypore were also identified (Fig. 2). They belong to several chemical classes but the greatest attention was paid to small molecular weight secondary metabolites, especially triterpenoids. Kamo et al. (2003) isolated several triterpenoid carboxylic acids with a lanostane skeleton, e.g. polyporenic acids and their derivatives (Table 1). In in vivo tests, the substances suppressed TPA-induced mouse ear inflammation up to 49-86% at the dose of 0.4 µM/ear. Alresly et al.
In their search for fungal antimicrobial substances, Schlegel et al. (2000) isolated another valuable compound-piptamine, N-benzyl-N-methylpentadecan-1-amine from submerged culture of F. betulina Lu 9-1. It showed activity against gram-positive bacteria (MIC,  Wandokanty et al. (1954;1955) Tumor size reduction and inhibition of bleeding from the genital tract minimum inhibitory concentration, values in the range from 0.78 to 12.5 µg/ml) and yeasts including Candida albicans (MIC 6.25 µg/ml). Polysaccharides from higher basidiomycota mushrooms have been usually considered to be the major contributors of their bioactivity. However, birch polypore polysaccharides have not yet been sufficiently explored, in terms of either the structure or pharmacological activity. It is known that the Fomitopsis cell wall contains (1→3)-β-d-glucans in an amount of ca. 52% dw (Jelsma and Kreger 1978;Grün 2003). They are built from β-d-glucopyranose units connected with (1→3)-linkages in the main chain, with (1→3)-β-d linked side branches. However, there are no reports about their biological activities. Another polysaccharide isolated from the birch polypore was water-insoluble, alkali-soluble (1→3)-α-d-glucan. Although α-glucans are believed to be biologically inactive, its carboxymethylated derivative showed moderate cytotoxic effects in vitro (Wiater et al. 2011).

Miscellaneous applications
With the knowledge of the mechanisms of action of brown rot decay, there are possibilities of new applications of these fungi in biotechnology. The enzymatic and non-enzymatic apparatus for lignocellulose degradation can be used for bioprocessing of biomass towards fuels and chemicals (Arantes et al. 2012;Giles and Parrow 2011;Ray et al. 2010). Brown rot fungi, including F. betulina, were tested for bioleaching of heavy metals (Cu, Cr, and As) from wood preservatives due to accumulation of metal-complexing oxalic acid (Sierra Alvarez 2007). Production of biomass degrading enzymes, for instance cellulases, hemicellulases, amylases, etc., was also studied (Krupodorova et al. 2014;Valášková and Baldrian 2006a, b).

Conclusions and outlook
The F. betulina fungus has been widely used and appreciated in folk medicine, and modern pharmacological studies have confirmed its potential indicating significant antimicrobial, anticancer, anti-inflammatory, and neuroprotective activities. The possibility of successful cultivation thereof in artificial conditions additionally promotes the applicability of the fungus. However, compared with other polypore fungi, the research on F. betulina is less developed; for instance, little is known about its lifestyle, including the wood degradation strategy. Moreover, most of the bioactivity studies have been performed using crude extracts; hence, only a few of the effects have been associated with the active substances identified, e.g. antibacterial activities with piptamine or polyporenic acids. With a few exceptions, we still do not know the mechanisms underlying the biological activities. Verification of biological activities in in vivo and clinical studies is also required. The further research could contribute to better exploitation of the F. betulina application potential.