Colony Growth and Bioactivity of the European
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Dresch et al. AMB Express (2015) 5:4 DOI 10.1186/s13568-014-0093-0 ORIGINAL ARTICLE Open Access Fungal strain matters: colony growth and bioactivity of the European medicinal polypores Fomes fomentarius, Fomitopsis pinicola and Piptoporus betulinus Philipp Dresch1, Maria Nives D´Aguanno2, Katharina Rosam1, Ulrike Grienke3, Judith Maria Rollinger3 and Ursula Peintner1* Abstract Polypores have been applied in traditional Chinese medicine up to the present day, and are becoming more and more popular worldwide. They show a wide range of bioactivities including anti-cancer, anti-inflammatory, antiviral and immuno-enhancing effects. Their secondary metabolites have been the focus of many studies, but the importance of fungal strain for bioactivity and metabolite production has not been investigated so far for these Basidiomycetes. Therefore, we screened several strains from three medicinal polypore species from traditional European medicine: Fomes fomentarius, Fomitopsis pinicola and Piptoporus betulinus. A total of 22 strains were compared concerning their growth rates, optimum growth temperatures, as well as antimicrobial and antifungal properties of ethanolic fruit body extracts. The morphological identification of strains was confirmed based on rDNA ITS phylogenetic analyses. Our results showed that species delimitation is critical due to the presence of several distinct lineages, e.g. within the Fomes fomentarius species complex. Fungal strains within one lineage showed distinct differences in optimum growth temperatures, in secondary metabolite production, and accordingly, in their bioactivities. In general, F. pinicola and P. betulinus extracts exerted distinct antibiotic activities against Bacillus subtilis and Staphylococcus aureus at minimum inhibitory concentrations (MIC) ranging from 31-125 μgmL−1; The antifungal activities of all three polypores against Aspergillus flavus, A. fumigatus, Absidia orchidis and Candida krusei were often strain-specific, ranging from 125-1000 μgmL−1. Our results highlight that a reliable species identification, followed by an extensive screening for a ‘best strain’ is an essential prerequisite for the proper identification of bioactive material. Keywords: Fungal strain selection; Temperature optimum; Wood-rotting fungi; Antimicrobial activity of fungal extracts; Phylogeny Introduction health food supplements. They show a wide range of Polypores are a diverse group of Agaricomycetes (Basid- bioactivities including anti-cancer, anti-inflammatory, iomycota) with tough poroid hymenophores. These fungi antiviral and immuno-enhancing effects (Grienke et al. have been extensively applied in traditional Chinese 2014; Molitoris 2005; Paterson 2006). The large number medicine (TCM) up to the present day (Chang 2000; of scientific studies focusing on their bioactivity (Khatun Chang and Wasser 2012; Hobbs 1995; Ying et al. 1987) et al. 2011; Lo and Wasser 2011; Xu et al. 2010; Zhao and are becoming more and more popular also in other 2013; Zhong and Xiao 2009; Zhou et al. 2011), secondary parts of the world where they are used as a source for metabolite production (Hwang et al. 2013; You et al. medicinal compounds and therapeutic adjuvants, or as 2013) and genomics (Floudas et al. 2012; Liu et al. 2012) is therefore not surprising. * Correspondence: [email protected] Secondary metabolites of medicinal polypores have 1 Institute of Microbiology, University of Innsbruck, Technikerstraße 25, 6020 been the focus of many studies (Grienke et al. 2014; Innsbruck, Austria Full list of author information is available at the end of the article Zjawiony 2004), but the importance of fungal strain for © 2015 Dresch et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Dresch et al. AMB Express (2015) 5:4 Page 2 of 14 bioactivity and metabolite production has not been inves- properties or their metabolic fingerprint. We therefore tigated so far. Strain-specific differences in bioactivities investigated strain-specific differences of three medicinal (on the function of macrophages) were detected for differ- polypore species of the traditional European medicine. ent strains of the Ascomycete Ophiocordyceps sinensis, The applications of these polpores in traditional medicine, whose fruit bodies are highly praised as traditional remedy and knowledge on their metabolite profile have been ex- (Meng et al. 2013). Distinct strain differences are also tensively reviewed by Grienke et al. (2014). known for anamorphic ascomycetes, e.g. for the secondary When using defined commercially available culture metabolite production of Fusarium avenaceum,where strains, interpretation of the results is often difficult due substrate is playing an additional important role in the to lack of information, e.g. on the environment, substrate, regulation of secondary metabolite biosynthesis (Sørensen or on the fruit body morphology. We therefore used newly and Giese 2013). isolated strains, and extensively documented their habitat, Fungal medicinal polypore species belonging to the substrate, and the morphology of fruit bodies. This also same genus differ clearly in their metabolite profile (Hseu allowed for a comparison of strains based on both, bioac- et al. 1996; Lv et al. 2012). tivities of fruit body extracts and the physiological proper- Species delimitation of polypores was long based upon ties of cultures in vitro. macro-morphological and ecological characters. However, Physiological properties are often used for species de- these classical morphological species concepts have proven limitation because they reflect genetic distance: Fomes to be too wide for many important medicinal polypore strains belonging to different species have different species, e.g. for Ganoderma lucidum, G. applanatum,or physiological properties, like colony growth responses Laetiporus sulphureus (Banik et al. 2010; Banik et al. 2012; to temperature (McCormick et al. 2013). The question Hseu et al. 1996, Ota et al. 2009). Polypore species, de- whether also strains within one species, e.g. F. fomentar- scribed upon morphology as generalists on several hosts, ius, differ in growth responses to temperature was not have often turned out to consist of multiple phylogenetic addressed so far. (cryptic) lineages, and the description of new species if The main aim of this work was to study the influence often necessary within such species complexes (Skaven of fungal strain (isolate) of three selected, medicinal Seierstad et al. 2013). This makes comparison of studies polypores on their antimicrobial bioactivity and physio- working on metabolites from medicinal polypores forming logical properties. The bioactivity was assessed for ethano- a morphological species complex especially critical and lic extracts from the same fruit bodies, which had been difficult. It is therefore essential to document, from which used for isolation of mycelial cultures. Bioactivity was habitat and substrate the fruit body was obtained, to tested against potentially pathogenic fungi and bacteria. deposit a voucher in a public mycological collection and a Physiological properties of mycelial cultures (colony growth culture in a public culture collection, in order to check or rate and optimum temperature) were investigated under to re-identify the fungus. Moreover, it is also recom- standard in vitro conditions. All polypore strains used were mended to sequence barcoding regions of the investigated characterized by rDNA ITS sequences, and phylogenetic fungal material, in order to allow for a later verification of analyses were carried out to verify species identification. the identification of the fungus. The potential influence of geographic provenance and A recent study on the secondary metabolites of the substrate on strain evolution and properties are discussed. medicinal polypore Laricifomes officinalis was carried out in an exemplary way by also including rDNA ITS Material and methods sequences of the used strain (Hwang et al. 2013). But Organism collection, identification, culture screening for optimal strains is rarely carried out, and F. fomentarius, F. pinicola, and P. betulinus fruit bodies strain differences within polypore species are usually were mainly collected in Tyrol, Austria in 2013. Fruit not taken into account, although it is well known that a body vouchers were deposited in the Mycological large variety of existing alleles are promoting outcrossing Collection of the Herbarium Innsbruck (IB), cultures of polypores; genetic exchange in dikaryons by somatic were deposited in the Jena Microbial Resource Collection recombination creates additional new combinations of al- (JMRC), sequences were deposited in GenBank. Voucher leles (Peabody et al. 2000). This implies that ecologically numbers, GenBank accession numbers, JMRC collection relevant genetic variation permanently occurs within numbers, plant hosts, as well as habitat information are dikaryotic fungal individuals, allowing for evolutionary given in Table 1. adaptation and thus for the evolution of physiologically Sterile techniques were used to obtain cultures from the distinct strains. context tissue. Small pieces of context tissue (2.0 mm3) To study polypore species with known bioactivities were excised from each basidiome, plated on 2-3 % w/v allows for an