Molecular systematics of Keratinophyton: the inclusion of species formerly referred to Chrysosporium and description of four new species

Four new Keratinophyton species (Ascomycota, Pezizomycotina, Onygenales), K. gollerae, K. lemmensii, K. straussii, and K. wagneri, isolated from soil samples originating from Europe (Austria, Italy, and Slovakia) are described and illustrated. The new taxa are well supported by phylogenetic analysis of the internal transcribed spacer region (ITS) region, the combined data analysis of ITS and the nuclear large subunit (LSU) rDNA, and their phenotype. Based on ITS phylogeny, within the Keratinophyton clade, K. lemmensii is clustered with K. durum, K. hubeiense, K. submersum, and K. siglerae, while K. gollerae, K. straussii and K. wagneri are resolved in a separate terminal cluster. All four new species can be well distinguished from other species in the genus based on phenotype characteristics alone. Ten new combinations are proposed for Chrysosporium species which are resolved in the monophyletic Keratinophyton clade. A new key to the recognized species is provided herein. Supplementary Information The online version contains supplementary material available at 10.1186/s43008-021-00070-2.


INTRODUCTION
Keratinophyton is a genus of microscopic fungi (Ascomycota, Onygenales, Onygenaceae) comprising species that live mostly on the remains of hair and feather in soil as saprotrophs (Cano and Guarro 1990;Hubka et al. 2016;Sutton et al. 2013;Vidal et al. 2000). Formerly, they were classified in Aphanoascus mainly based on the presence of ascomata (cleistothecia) composed of a membranous peridium (Cano and Guarro 1990;Cano et al. 2002). In a review employing a phenotypic and phylogenetic approach, Cano et al. (2002) accepted 18 Aphanoascus species which all have sexual morphs. Only recently, the polyphyletic status of Aphanoascus s. lat. has been resolved by Sutton et al. (2013) who established the genus Keratinophyton encompassing and redisposing six species, namely K. durum, K. hispanicum, K. multisporum, K. punsolae, K. saturnoideum and the type species K. terreum. Ascospores of Keratinophyton species are characterized by a conspicuous equatorial rim and pitted wall, while Aphanoascus species have reticulate ascospores without a rim (Sutton et al. 2013). Within Keratinophyton, only K. multiporum is related to a Malbranchea asexual morph (Cano and Guarro 1990), while the remaining known species have a Chrysosporium asexual morph. In addition to the above mentioned species, the monophyletic Keratinophyton clade currently encompasses at least 11 species known only as asexual morphs (Cano and Guarro 1990;Hubka et al. 2016;Sharma and Shouche 2017;Liang et al. 2009;van Oorschot 1980;Vidal et al. 2000;Vidal et al. 2002;Zhang et al. 2016;Zhang et al. 2017). Sharma and Shouche (2017) introduced a new species, Keratinophyton turgidum, based on the morphology of its chrysosporium-like aleurioconidia and ITS locus phylogeny. The same authors stated that all species in this monophyletic clade which have a Chrysosporium asexual morph require redisposing in the genus Keratinophyton.
The presence of this large group of ubiquitous and keratinolytic species is rather common especially in areas with high animal activity that results in transfer of the keratinous material (fur, hairs, etc.) to the soil (Papini et al. 1998;Vidal et al. 2000). The following reports confirm their world-wide distribution and occurrence in different habitats usually associated with soil environments, e.g. soil in city parks (Papini et al. 1998;Vidyasagar et al. 2005), flower pots (Singh et al. 2009), sand in children's sandpits (Labuda et al. 2008), mud (Zaki et al. 2005), poultry farms (Anbu et al. 2004;Cano and Guarro 1990), marshy meadows, salt pans, desert, cultivated or uncultivated soils (Cano and Guarro 1990;Chmel and Vláčilíková 1977;Deshmukh 2004;Deshmukh et al. 2008;Han et al. 2013;Javorekova et al. 2012;Zhang et al. 2016;Zhang et al. 2017) and river sediments (Ulfig et al. 1997;Vidal et al. 2000;Vidal et al. 2002). In general, these fungi are rarely reported as animal pathogens, and in fact, only two species C. echinulatum and C. pannicola (formerly known as C. evolceanui) have been involved in mycoses (Hajsig et al. 1974;Cabanes et al. 2014;Hubka et al. 2016).
During a microbiological survey of environmental samples (soil and compost) in July 2019, several interesting Chrysosporium asexual morphs were isolated. These isolates were phenotypically similar to those previously isolated from the same samples in August 2015 by one us (R.L.). These isolates were designated BiMM-F76, BiMM-F77 (also strain RL-07, isolated in July 2019), BiMM-F78 (also strains RL-05 and RL-06, isolated in July 2019), and BiMM-F250. All strains were further characterized in terms of morphology, physiology, and molecular phylogeny. Phylogenetically informative sequences were obtained from the internal transcribed spacer (ITS) region and the nuclear large subunit (LSU) rDNA. Overall, the resulting data revealed that these isolates represent novel species of the genus Keratinophyton, and they were illustrated for the first time in this paper.

Sample collection and isolation of the fungi
A sample of a garden soil in Vieste (Italy) was collected in July 2004, one of a forest soil in Tatranská Lomnica (The Slovak Republic) in August 2011, and one of compost from an agricultural base at the Institute of Agrobiotechnology (IFA Tulln, Austria) in August 2015. All three samples were taken from the surface layer (3-5 cm deep), dried, and stored in plastic bags in a fridge (5-8°C) until the time of analysis (August 2015 and July 2019). Isolation of the keratinophilic fungi was performed as described previously (Javoreková et al. 2012). Each sample was divided into 10 subsamples. The subsamples (20 g each) were poured into Petri dishes and soaked with antibiotic solution containing 0.5 g cycloheximide and 0.1 g chloramphenicol. Sterile defatted horse hair fragments (10 pieces of ca 2.0 cm per plate) were used as baits. The Petri dishes were then incubated at laboratory temperature (23-25 ± 1°C), under ambient daylight, for a period of 2-3 months and remoistened with sterile deionized water when necessary. The Petri dishes were checked weekly for the presence of fungi, and isolates were cultured on Sabouraud 4% dextrose agar (SDA; Merck, Darmstadt, Germany) supplemented with 0.5 g cycloheximide and 0.05 g chloramphenicol. Pure cultures were then transferred onto potato dextrose agar [PDA; Van Waters and Rogers (VWR) International, Leuven, Belgium]. The preliminary identification of the resulting keratinophilic fungi was carried out based on their phenotypic characteristics according to van Oorschot (1980) and Vidal et al. (2000Vidal et al. ( , 2002.

Morphological analysis
For phenotypic determination, the strains were transferred (three-point inoculation with a needle) to PDA, Malt Extract Agar (MEA; Merck, Darmstadt, Germany), and SDA, and incubated for 14 d in the dark at 25°C. Christensen's urea agar (Sigma-Aldrich, St Louis, MO, USA) was used for additional physiological and biochemical characteristics (25°C, 14 d, in the dark). Corn Meal Agar (CMA; Oxoid, Basingstoke, UK), Potato Carrot Agar (PCA) (Samson et al. 2010) and Emerson YpSs agar (Atlas 1946) were used for stimulation of sexual reproduction (at 20°, 25°, and 28°C, for up to 3 months in the dark).
Colony size (mm), colony structure and characteristics were noted after 14 d (on PDA, MEA, SDA, PYE, YpSs, CMA, and PCA). However, the cultivation was extended up to 3 months to observe and record changes in pigmentation of the colonies as well as to determine the onset of sexual reproduction. In order to determine the optimal and minimum/maximum temperatures for growth, PDA, MEA and SDA plates were incubated at 5°, 8°, 10°, 12°, 15°, 18°, 20°, 25°, 28-32°, 35°, and 37°C, and the growth rate was measured on the 14th day of cultivation. For comparative descriptions of the macroscopic and microscopic characteristics, PDA was used according to Vidal et al. (2002), Hubka et al. (2016) and Sharma and Shouche (2017).
For determination of microscopic traits, PDA was used after 14-18 d. Conidiophore and conidia formation were observed in situ under low magnification (50-100x). Details of conidiophores, conidia (aleurioconidia) and other microscopic structures, such as width of hyphae, were observed in Melzer's reagent and lactic acid with cotton blue. Photomicrographs were taken in Melzer's reagent and lactic acid with cotton blue using phase and Nomarski contrast optics on an Olympus BX51 microscope with Olympus DP72 camera and QuickPHOTO Micro 3.0 software. Photographs of the colonies were taken with a Sony DSC-RX100.
Scanning electron microscopy (SEM) was performed on a JEOL JSM-6380 LV microscope (JEOL, Tokyo, Japan). Fungal samples were prepared according to a simplified method (Samson et al. 1979). Pieces of colonies (ca. 3 × 5 mm) growing on PDA were fixed in 6% glutaraldehyde overnight in the refrigerator (ca. 20 h), then dehydrated in 2-methoxyethanol for 10 min. This was followed by critical point drying and gold coating in a BAL-TEC SCD 050 Sputter Coater. The samples were observed with spot size 35-39 and accelerating voltage 20-23 kV.
Dried fungarium specimens deposited as holotypes in the collections of the Mycological Department, National Museum in Prague, Czech Republic (PRM); ex-type cultures were deposited in the Bioactive Microbial Metabolites (BiMM) Fungal Collection, UFT-Tulln in Austria and in the Culture Collection of Fungi in Prague (CCF).

Keratinolytic activity
Keratinolytic activity was tested by placing a few sterilized blond hairs of a 5 y old child on a PDA plate 1 cm away from the point of inoculation (van Oorschot 1980). Ability to digest keratin was observed after 21 d of incubation at 25°C in the dark. In addition, a hair perforation test was also performed following de Hoog et al.
(2020) using 25 mL water containing 2-3 drops 10% yeast extract (YEW). The hairs were examined microscopically after 14 and 21 d of the inoculation at 25°C in the dark. At the end of the incubation period, a few pieces of hair were taken out from the testing media (PDA and YEW). The overgrowing fungus was deactivated with 70% ethanol and then removed from the hair surface mechanically in a stream of a tap water. The degree of hair digestion-degradation (keratinolytic activity) was assessed in the light microscope under 100x and 400x magnification. For the observation and microphotography of the hairs, water was used as mounting fluid. Intensity of degradation of the hair was estimated on a scale of 0 to 4 (Marchisio et al. 1994): 0 = no degradation; 0-1 = light degradation on the cuticle; 1 = moderate degradation on the cuticle and/or rare formation of boring hyphae; 2 = degradation of cuticle and cortex, with about 20% degradation of the hair; 3 = degradation of cuticle and cortex, with about 50% degradation of the hair; 4 = degradation of cuticle and cortex, with about 80% degradation of the hair. The photomicrographs of the hairs were taken using a Motic BA 310 microscope with Motic Image Plus 3.0 software. The final microscopic pictures were black-and-white inverted.
DNA extraction, PCR amplification and sequencing DNA was extracted using a standard cetyltrimethyl ammonium bromide (CTAB) procedure, as described previously (Doyle and Doyle 1987). The internal transcribed spacer (ITS) region was amplified with primers ITS1-F (Gardes and Bruns 1993) and ITS4 (White et al. 1990) using Taq-polymerase. The D1/D2 domains of the largesubunit (28S) rRNA gene (LSU) were amplified and sequenced using the primer pair ITS1/TW14 (White et al. 1990;Mori et al. 2000). All reactions were performed in an Eppendorf Gradient MasterCycler (Eppendorf, Hamburg, Germany). Conditions for amplification of ITS and LSU domains: 95°C for 5 min; 35 cycles of 95°C for 30 s, 54°C for 30 s, 72°C for 90 s, and finally 5 min at 72°C. The PCR products were sequenced with the same primers used for the PCR amplifications (Microsynth, Balgach, Switzerland). All sequences obtained in this study were deposited in GenBank nucleotide database (Table 1).

Morphological analyses and keratin degradation
The results of the morphological analyses are given for each novel species under the Taxonomy section below. Temperature dependent growth of the new Keratinophyton species on PDA, MEA and SDA after 14 d are provided in Table S1a-c. Briefly, K. lemmensii grew better than the other three new species on the same type of media and at the same incubation temperatures. All species showed good growth at 20-25°C on all three media.
Ability to digest keratin after 21 d was observed in all four new species on both testing media (PDA and YEW). However, a value of attack intensity on the hair according to the scale of Marchisio et al. (1994) differed substantially amongst the species. It was very strong in K. gollerae and K. straussii (=4), moderate in K. wagneri (=2), and weak in K. lemmensii (= 0--1) (Fig. 10).

Phylogenetical analysis
The phylogenetic tree of ITS dataset (n = 32) was 551 bp in length which had 286 variable and 200 parsimony-informative sites. ITS phylogeny indicated the presence of six terminal clusters in the monophyletic Keratinophyton clade with high bootstrap support and low interspecific sequence divergence (Fig. 1a). Keratinophyton saturnoideum and K. minutisporosum formed a basal branch to the clade. Isolate BiMM-F76 (K. lemmensii sp. nov.) was close to K. durum (with 99% ITS and 95% LSU similarity) and clustered also with K. hubeiense and K. submersum. In addition, K. straussii sp. nov., K. saturnoideum as a basal branch (Fig. 1b). Differently from the ITS phylogeny, K. durum was placed in a different cluster from K. submersum and K. lemmensii in the concatenated loci phylogeny (Fig. 1b).

TAXONOMY
The phylogenetic analyses strongly supported the recent distinct classification of the species previously classified as Chrysosporium and only known from asexual morphs into two phylogenetically different genera, Aphanoascus and Keratinophyton (Sharma and Shouche 2017; Sutton et al. 2013). Species described in Chrysosporium which were resolved in a monophyletic clade with Keratinophyton are therefore combined into Keratinophyton in the present paper and provided together with four new Keratinophyton species. The main distinguishing phenotypic characteristics of the four new species were compared with those in the other members of the genus that are also unable to produce ascomata ( Description: Sexual morph not observed on any of the media used. Asexual morph on PDA. Vegetative mycelium of hyaline, septate, smooth-walled, sparsely to pronouncedly branched hyphae, often at right angles, 1.0-5.0 μm diam. Racquet hyphae present. Conidia (aleurioconidia), hyaline, white in mass, thinwalled, mostly smooth to finely roughened, some also verrucose (light microscope) and irregularly ornamented with minute warts (SEM). Terminal and lateral conidia born on main fertile hyphae or from side branches of variable length, sessile or on short protrusions, occasionally only very slightly swollen and of variable length, solitary, 1-3 (− 5) per conidiogenous cell, obovate to clavate, mostly 1-celled, (3.5-)5.0-7.0(− 10.0) x (1.5-)2.0-2.5(− 3.0) μm (mean = 5.2 ± 0.9 × 2.2 ± 0.2 μm, n = 120). Intercalary conidia not observed. Chlamydospores not observed.
Culture characteristics: Colonies on PDA 20-22 mm diam at 25°C, after 14 d, powdery to downy (mealy), with abundant sporulation, white to creamy, flat, umbonate at the centre, with regular colony margin submersed into agar, reverse white to slightly yellowish, no pigment or exudate produced. At 30°C, no growth (germination only). Colonies on SDA 23-25 mm diam at 25°C, after 14 d, morphology similar to when on PDA with more floccose colony margin and more yellowish colonies, with dark yellow reverse. At 30°C, no growth (no germination). Colonies on MEA 14-16 mm diam at 25°C, after 14 d, morphology similar to PDA with more floccose colonies and with yellow reverse. At 30°C, no growth (no germination). Colonies on CMA and PCA attaining 15-20 mm diam at 25°C, after 21 d, white, granular, with good sporulation, reverse yellowish. No ascomata observed after prolonged incubation (3 months). The optimum temperature for growth on PDA, SDA and MEA 15-25°C (Table S1a-c). Minimum growth (microcolonies to 1 mm in diam) at 10°C. Germination of the conidia observed at 8°C. The maximum temperature for growth on PDA 29°C, while 27°C and 28°C on MEA and SDA, respectively (microcolonies to 1 mm diam). Keratinolytic activity very strong (Fig. 10b), with hair attack intensity = 4. Urease activity negative (after 14 d of incubation).
Diagnosis: Keratinophyton gollerae molecularly can be distinguished from other Keratinophyton species by ITS locus analysis. Combination of the following phenotypic features can be used to differentiate this fungus from other species in the genus: (1) obovoid-clavate and smooth to finely roughened conidia, (2) No growth at Notes: Based on a search of NCBI GenBank nucleotide database, the closest hit for K. gollerae using the ITS sequence is K. minutisporosum (as Chrysosporium minutisporosum CBS 101577; GenBank acc. KT155616), with identity = 487/543 (90%) and gaps 11/543 (2%). Phenotypically, K. gollerae can be readily distinguished from the K. minutisporum by its smooth to finely roughened larger conida (5-7 × 2-2.5 μm vs. 3-4 × 1.5-3.5 μm), dark yellow colony reverse at 25°C on PDA. Based on ITS phylogeny (Fig. 1a), K. gollerae formed a cluster together with K. straussii and K. wagneri, and it can be differentiated by its inability to grow at 30°C, narrower and mostly smooth to finely roughened conidia, and its slower growth at 25°C on PDA. Moreover, in comparison with K. straussii, K. gollerae grows substantially faster at 15°C (on PDA and SDA) and its conidia germinate at 8°C (see Table S1a-c).
Culture characteristics: Colonies on PDA 24-28 mm diam at 25°C, after 14 d, powdery to downy (mealy), with abundant sporulation, white to very slightly creamy yellowish, flat, slightly elevated (umbonate) remaining powdery at the centre, with irregular margin, reverse white with slightly yellowish centre, no pigment or exudate produced. At 30°C, 15-20 mm diam after 14 d, white to creamy yellowish, flat, powdery to downy (mealy) with very good sporulation, and with white to yellowish reverse. Colonies on SDA 16-20 mm diam at 25°C, after 14 d, morphology as on PDA with dark yellow reverse. In age (after 5 wk) yellow pigment produced and colony reverse becoming bright reddish yellow to orange. At 30°C, 15-20 mm diam after 14 d, white to creamy yellowish, umbonate, with strong sporulation, and with yellowish reverse. Colonies on MEA 18-20 mm diam at 25°C, after 14 d, morphology as on PDA with more floccose and yellowish. At 30°C, 5-10 mm diam Labuda et al. IMA Fungus (2021) 12:17 after 14 d, slightly umbonate, floccose to granular, with very good sporulation white to yellowish, and with yellow reverse. Colonies on CMA and PCA 18-20 mm diam at 25°C, after 21 d, white, granular, good sporulation, reverse yellowish. No ascomata observed after prolonged incubation (3 months). The optimum temperature for growth on PDA, SDA and MEA 20-25°C (Table S1a-c). Minimum growth (microcolonies to 1-2 mm diam) at 10°C. No germination of the spores at 8°C. The maximum temperature for growth 32°C (microcolonies to 1-2 mm diam). Keratinolytic activity very strong (Fig. 10c), with hair degradation intensity = 4. Urease activity negative (after 14 d of incubation).
Diagnosis: Keratinophyton straussii molecularly can be distinguished from other Keratinophyton species by ITS locus analysis. Phenotypically, it can be differentiated by combination of the ability to grow at 30°C, white to creamy colonies with white to yellowish revers at 25°C on PDA and conidia morphology (obovoid to clavate and verrucose) ( Table 2).
Additional material examined: Italy: Vieste, from garden soil, isolated from different sub-samples, July 2019, Notes: Based on a search of the NCBI GenBank nucleotide database, the closest hit for K. straussii using the ITS sequence was K. minutisporosum (as Chrysosporium minutisporosum CBS 101577; GenBank acc. KT155616), with identity = 489/543 (90%) and gaps 10/ 543 (1%). Two species can be differentiated from each other based on growth rate and colony reverse at 25°C on PDA (Table 2). Additionally, K. straussii differs from K. wagnerii by its ability to grow at 30°C and strong keratinolytic activity. For the morphological differences between K. gollerae and K. straussii, see under K. gollerae. Additional strains RL-05 and RL-06 grew relatively better (to 5 mm larger diam) than the ex-type culture at 30°C.
Culture characteristics: Colonies on PDA 25-30 mm diam at 25°C, after 14 d, powdery to downy (mealy), with abundant sporulation, white to slightly yellowish, flat, slightly elevated (umbonate) and more floccose at the centre, margin irregular, reverse white with slightly yellowish centre, no pigment or exudate produced. At 30°C, 4-8 mm diam after 14 d, white, floccose with poor sporulation, and with yellowish reverse. Colonies on   (Fig. 10d), with hair attack intensity = 2. Urease activity negative (after 14 d of incubation). Diagnosis: K. wagneri molecularly can be distinguished from other Keratinophyton species by ITS locus analysis. Phenotypically, it can be differentiated by combination of the growth rate at 30°C and conidia size (4.0-8.0 × 2.5-4.0 μm) and morphology (obovoid to clavate, verrucose) ( Table 2).
All four new species are readily distinguished from the other taxa in the genus Keratinophyton, based on phenotypical characteristics such as growth at high temperature and/or conidial morphology (Cano and Guarro 1990;Cano and Guarro 1994;Currah 1985). The most important species-specific phenotypic distinguishing characteristics are found as morphology of conidia (shape, surface and dimensions) and growth rate at 30°C after 14 d on PDA.

KEY TO SPECIES OF KERATINOPHYTON
This key is modified from that of Cano et al. (2002). The given data for source and origin represent the type strains of the related species. In our study we confirmed ten species required transfer. The monophyletic genus Keratinophyton is now extended and includes 25 species including ten species known from sexual morphs (Sutton et al. 2013; and this paper) and 15 species which are currently known only from asexual morphs (including the recently described K. turgidum (Sharma and Shouche 2017). The species known only from the asexual morphs can be distinguished by particular combinations of their morphological traits (colony colour and growth rate, growth response at higher/lower temperatures, as well as morphology of conidia) and differences in the ITS regions (Fig. 1a, Table 2).

Ecology and distribution
Almost all known Keratinophyton species have been isolated from soil or soil-like substrates, such as river sediments, compost and sand (Table 1; Cano and Guarro 1990;Sharma and Shouche 2017;Labuda et al. 2008;Liang et al. 2009;van Oorschot 1980;Vidal et al. 2000;Vidal et al. 2002). Hubalek (2000) provided a list of keratinolytic fungi associated with free-living mammals and birds of which Keratinophyton pannicola (as Chrysosporium evolceanui) has been isolated from a variety of animals, different species of rodents in Australia, Czechia, Germany, the UK, and the former Yugoslavia; a rabbit in Canada; and from birds in Australia (Queensland), Czechia, India, and the former Yugoslavia. Keratinophyton durum (as Aphanoascus durus) has been isolated from a hedgehog in Ivory Coast, and K. terreum (as Aphanoascus terreus) has been found associated with a variety of rodents in Czechia, Germany, India, Nigeria, Romania, and the former Yugoslavia and, and further birds in Australia (Queensland) Czechia, India, the USA, and the former Yugoslavia (Hubalek 2000). To the best of our knowledge, there is only a single report of a human clinical isolate belongs to K. echinulatum (CCF 4652 = CBS 141178) from the sole of the foot of a 35year-old woman in Czechia (Hubka et al. 2016). However, Hubka et al. (2016) indicated that the etiological significance of this fungus was unclear, and they concluded that the infection was actually caused by another dermatophyte, which was not isolated or was overgrown by K. echinulatum. A few other cases have been published in a small range of animals including Keratinophyton pannicola (as Chrysosporium pannicola) from skin of a dog in former Yugoslavia (Hajsig et al. 1974;van Oorschot 1980) and from a case of keratomycosis in a horse (Grahn et al. 1993).
In her review on Chrysosporium and related genera in Onygenaceae, Sigler (2003) stated that some reports concerning Chrysosporium species as etiological agents must be viewed with caution, in case the isolated fungus has neither been identified to species level nor documented well enough to confirm the aetiology. In the follow-up list of medically relevant species provided by Sigler (2003), no species is mentioned as being currently affiliated within the genus Keratinophyton, while K. pannicola (as C. pannicola) is included in the Atlas of Clinical Fungi (de Hoog et al. 2020) as a concern in skin infections. Even though the keratinophilic fungi were considered as potential pathogens by several researchers (Rippon 1982;Papini et al. 1998); they rarely cause infections. Therefore, soil is proposed as an epidemiological and probably also an evolutionary link, that relates geophilic, zoophilic, and anthropophilic keratinophilic fungi (Papini et al. 1998). Interestingly, during a mycological investigation of the soil samples in the present study, a high prevalence of geophilic dermatophytes such as Nannizzia gypsea from Italy (collected in 2004), a cooccurrence of Arthroderma uncinatum with Aphanoascus keratinophilus (as Chrysosporium keratinophilum) from the Slovak Republic (collected in 2011), and Arthroderma terrestre along with abundant A. uncinatum from Austria (collected in 2015) were noted (data not shown).
As the members Keratinophyton are considered as typical soil-borne fungi (Cano and Guarro 1990;Cano et al. 2002;Sutton et al. 2013) and there is no solid evidence of pathogenicity, it is likely that previously reported animal-associated cases reflect environmental transmissions from soil to the animals during activities in contact with soil. The ability of these fungi to persist and survive in the soil was observed also during the present study, as in case of K. straussii, the type strain was isolated 11 years after sampling in 2004, and two more strains (RL-05 and RL-06) representing the same taxon were isolated in a repeated study even 15 years after the sampling. Likewise, a second strain (RL-07) used for the description of K. wagneri and the type of K. gollerae (BiMM-F250) were both isolated 8 years after the samples were collected.
The degree of keratin degradation by the novel strains described here varied. It was very strong in both K. gollerae and K. straussii compared to other tested strains, attacking the cuticle and cortex of hairs with about 50-80% degradation. In addition to keratin degradation, keratinolytic fungi share common properties with dermatophytes (Marchisio et al. 1994;Mitola et al. 2002). Even though some of these fungi can grow at 37°C (Fig. 1a), potential pathogenicity to homeothermic vertebrates (mammals and birds) by these fungi seems highly unlikely because of some presumably missing pathways in their metabolism. Instead, their strong keratinolytic ability might be providing a competitive advantage in the nature to acquire nutrients from hair and may have potential in industry for the production of proteolytic enzymes to degrade keratinous materials (hairs, fur, feathers, etc.). Furthermore, these fungi represent a yet unexplored possible source of new bioactive compounds as there is not much known of these properties in the genus (Kushwaha and Guarro 2000).