Morphological analyses and keratin degradation
The results of the morphological analyses were given for each novel species under the taxonomy section. Temperature dependent growth of the new Keratinophyton species on PDA, MEA and SDA after 14 days were provided in Table S1a‒c. Briefly, K. lemmensii growth better than the other three new Keratinophyton species with inoculations on the same media and at the same incubation temperatures. All species showed good growth at 20 °C‒25 °C on all three types of media.
Ability to digest keratin after 21 days was observed in all four novel species on both testing media (PDA and YEW). However, a value of attack intensity on the hair according to scale of Marchisio et al. (1994) substantially differed among the species; being very strong in K. gollerae and K. straussii (=4), moderate in K. wagneri (=2) and weak in K. lemmensii with a value of 0-1 (Fig. 10).
Phylogenetical analysis
The phylogenetic tree of ITS dataset (n=32) was 551 bp 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 basal branched to the clade. The isolate BiMM-F76 (named as K. lemmensii sp. nov.) was found close to K. durum (with 99% ITS and 95% LSU similarity) clustered also with K. hubeiense and K. submersum. In addition, K. straussii sp. nov., K. gollerae sp. nov., and K. wagneri sp. nov., represented by the ex-type cultures BiMM-F78, BiMM-F250 and BiMM-F77, respectively, were resolved in a separate terminal cluster-lineage. A concatenated phylogeny of ITS and LSU sequences (n=22) was 1094 bp length and included 354 variable and 224 parsimony-informative sites. According to combined data analysis, four clusters were found in the Keratinophyton clade and K. saturnoideum formed a basal branch (Fig. 1b). Differently from the ITS phylogeny, K. durum was found in a different cluster than K. submersum and K. lemmensii in the concatenated loci phylogeny (Fig. 1b).
Taxonomy
Keratinophyton lemmensii Labuda, Bernreiter, Kubátová & Schüller sp. nov.
MycoBank: MB833632. (Figs 2 and 3).
Etymology: Named in honour of Professor Marc Lemmens, Laboratory of Plant Protection (IFA, BOKU, AT), an expert in the fungal plant pathology.
Diagnosis: Presence of filiform often sinusoidal conidia and abundant arthroconidia, production of bright yellow pigment on PDA and good growth at 30 °C. Sequence data from ITS separate this taxon from all other Keratinophyton species.
Type: Austria: Tulln and der Donau, from compost soil at IFA Tulln, Aug. 2015, R. Labuda (PRM 952498-holotype; BiMM-F76 = CCF 6359 - ex-type cultures). ITS sequence, GeneBank MN633082; LSU sequence, GeneBank MT874998.
Description: Sexual morph not observed on any of the media used in the present study. Asexual morph on PDA. Vegetative mycelium consisting of hyaline, smooth-walled, septate, sparsely branched hyphae, 1.5‒5.0 µm diam. Racquet hyphae observed. Conidia (aleurioconidia), hyaline, white in mass, thin-walled, smooth to sparsely irregularly ornamented with minute warts. Terminal and lateral conidia born on main fertile hyphae as sessile or on short protrusions, solitary, 1‒3 (5) per conidiogenous cell, obovate to clavate, 1-celled, (3.0‒)4.5‒6.5(‒7.5) x (1.5‒)2.0‒2.5(‒4.0) µm (mean = 4.9±0.8 x 2.4±0.4 µm, n= 120), and filiform, often sinusoidal, 1- to 2-celled, 25‒35(‒40) µm long conidia also present. Intercalary conidia (arthroconidia) present, 10‒15 µm long. Chlamydospores not observed.
Culture characteristics: Colonies on PDA 28‒35 mm diam at 25 °C, after 14 days, floccose, with good sporulation, white, flat, slightly elevated (umbonate) at the center, with irregular margin, reverse lemon yellow, soluble pigment bright yellow, a few small clear to yellow-orange exudate droplets produced. At 30 °C, 38‒45 mm diam after 14 days, white, flat, floccose and radially sulcate with good sporulation only at the center, and with lemon yellow reverse. Colonies on SDA 28‒35 mm diam at 25 °C, after 14 days, morphology similar to PDA, without exudate and with pale yellow reverse. At 30 °C, 30‒32 mm diam after 14 days, white, flat, floccose with good sporulation, with pale yellow reverse. Colonies on MEA 20‒25 mm diam at 25 °C after 14 days, morphology similar to PDA, exudate absent, and pale-yellow reverse. At 30 °C, 18‒20 mm diam after 14 days, white, floccose and radially sulcate, with good sporulation, and with pale yellow reverse. Colonies on CMA and PCA, 45‒50 mm diam at 25 °C, after 21 days, white, flat and spread with poor sporulation, reverse white. No ascomata observed after prolonged incubation (3 months). The optimum temperature for growth on PDA, SDA and MEA was between 25‒30 °C (Table S2a‒c). Minimum growth (1-2 mm diam) was observed at 8 °C. The maximum temperature for growth was 32 °C (microcolonies to 1 mm diam). Keratinolytic activity very weak (Fig. 10a), with hair attack intensity = 0-1. Urease activity positive (after 3 days of incubation).
Notes: Based on a search of NCBI GenBank nucleotide database, the closest hit for K. lemmensii using the ITS sequence is K. durum (FMR5651; Genbank acc. AJ439434; identities = 568/577 (98%), gaps 0/577 (0%). Phenotypically, K. lemmensii is unique and differs from the relatives in the same clade (K. durum, K. hubeiense, K. submersum, and K. siglerae) by the combination of the following features: (1) presence of long filiform often sinusoidal uni- to bicellular conidia (up to 40 µm), (2) white, moderately fast growing colonies (28‒35 mm diam, on PDA at 25 °C), (3) production of lemon yellow pigment on PDA at 25 °C, (4) minimum (8 °C) and maximum (32 °C) growth temperature, (5) very weak keratin digestion after 21 d. Along with foregoing features, K. lemmensii can be directly distinguished from the phylogenetically closest K. durum asexual morph also by the presence of numerous arthroconidia which are completely missing in the latter species (Cano and Guarro 1990; Currah 1985).
Keratinophyton gollerae Labuda, Bernreiter, Kubátová, Schüller & Strauss sp. nov.
MycoBank: MB 833633. (Figs. 4 and 5).
Etymology: Named in honour of Dr. Sabine Strauss-Goller, the Department of Applied Genetics and Cell Biology, Fungal Genetics and Genomics Laboratory (BOKU, AT), an expert in the fungal genetics and indoor mold analytics.
Diagnosis: Production of slender and mostly smooth-walled conidia, no growth at 30 °C, and strong keratinolytic ability. Sequence data from ITS separate this taxon from all others Keratinophyton species.
Type: The Slovak Republic: Tatranská Lomnica, from forest soil, Jul. 2019, R. Labuda (PRM 952499 - holotype; BiMM-F250 = CCF 6360 – ex-type cultures). ITS sequence, GeneBank MN633084; LSU sequence, GeneBank MT874997.
Description: Sexual morph not observed on any of the media used. Asexual morph on PDA. Vegetative mycelium consisting of hyaline, septate, smooth-walled, sparsely to pronouncedly branched hyphae, often in right angles, 1.0‒5.0 µm diam. Racquet hyphae observed. Conidia (aleurioconidia), hyaline, white in mass, thin-walled, mostly smooth to finely roughened, some also verrucose (under light microscope) and irregularly ornamented with minute warts (visible under 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 x 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 days, powdery to downy (mealy), with abundant sporulation, white to creamy, flat, umbonate at the center, 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 days, morphology similar to 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 days, 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 days, 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 was between 15 and 25 °C (Table S2a‒c). Minimum growth (microcolonies to 1 mm in diam.) was observed at 10 °C. Germination of the spores was observed at 8 °C. The maximum temperature for growth on PDA was 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 days of incubation).
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; GeneBank acc. KT155616), with identity = 487/543 (90%) and gaps 11/543 (2%). Phenotypically, K. gollerae can be readily distinguished from the two other species K. straussii and K. wagneri, in the same clade 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 spores germinate at 8 °C (see Table S1a‒c).
Keratinophyton straussii Labuda, Bernreiter, Kubátová & Schüller sp. nov.
MycoBank: MB 833634. (Figs. 6 and 7).
Etymology: Named in honour of Professor Joseph Strauss, a head of the Department of Applied Genetics and Cell Biology, founder of the Fungal Genetics and Genomics Laboratory (BOKU, AT), an expert in the fungal genetics, epigenetics and functional genomics.
Diagnosis: Coarsely roughened conidia produced from swollen conidiogenous cells, optimal growth at 30 °C, with strong keratinolytic ability. Sequence data from ITS separate this taxon from other Keratinophyton species.
Type: Italy: Vieste, from garden soil, Aug. 2015, R. Labuda (PRM 952500 - holotype; BiMM-F78 = CCF 6361 – ex-type cultures). ITS sequence, GeneBank MN633081; LSU sequences, GeneBank MT874996.
Description: Sexual morph not observed on any of the media used. Asexual morph on PDA. Vegetative mycelium consisting of hyaline, septate, smooth-walled, sparsely to pronouncedly branched hyphae, usually in right angles, 1.5‒4.0 µm diam. Racquet hyphae observed. Conidia (aleurioconidia), hyaline, white to yellowish in mass, thin-walled and regularly ornamented with minute warts (visible under SEM) and coarsely roughened (under light microscope). Terminal and lateral conidia born on main fertile hyphae or from side branches of variable length, sessile or on short protrusions, commonly slightly swollen and of variable length, solitary, 1‒3 (5) per conidiogenous cell, obovate to clavate, 1-celled, (3.5‒)4.5‒5.0(‒6.5) x (2.0‒)2.5-3.0(‒3.5) µm (mean = 4.9±0.4 x 2.6±0.2 µm, n = 120), very rarely 2- to 3-celled, up to 12 µm large aleurioconida also present. Intercalary conidia not observed. Chlamydospores not observed.
Culture characteristics: Colonies on PDA 24‒28 mm diam at 25 °C, after 14 days, powdery to downy (mealy), with abundant sporulation, white to very slightly creamy yellowish, flat, slightly elevated (umbonate) remaining powdery at the center, with irregular margin, reverse white with slightly yellowish center, no pigment or exudate produced. At 30 °C, 15‒20 mm diam after 14 days, white to creamy yellowish, flat, powdery to downy (mealy) with very good sporulation, and with white to yellowish reverse. Colonies on SDA attaining 16‒20 mm diam at 25 °C, after 14 days, morphology similar to PDA with dark yellow reverse. In age (after 5 weeks) yellow pigment is produced and colony reverse becomes bright reddish-yellow to orange. At 30 °C, 15‒20 mm diam after 14 days, white to creamy yellowish, umbonate, with strong sporulation, and with yellowish reverse. Colonies on MEA 18‒20 mm diam at 25 °C, after 14 days, morphology similar to PDA with more floccose and yellowish. At 30°C, 5‒10 mm diam after 14 days, 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 days, white, granular, good sporulation, reverse yellowish. No ascomata observed after prolonged incubation (3 months). The optimum temperature for growth on PDA, SDA and MEA was between 20 and 25 °C (Table S1a‒c). Minimum growth (microcolonies to 1-2 mm diam) was observed at 10 °C. No germination of the spores was observed at 8 °C. The maximum temperature for growth was 32 °C (microcolonies to 1-2 mm diam). Keratinolytic activity very strong (Fig 10c), with hair degradation intensity = 4. Urease activity negative (after 14 days of incubation).
Notes: Based on a search of NCBI GenBank nucleotide database, the closest hit for K. straussii using the ITS sequence is K. minutisporosum (as Chrysosporium minutisporosum CBS 101577; GeneBank acc. KT155616), with identity = 489/543 (90%) and gaps 10/543 (1%). Phenotypic similarities see under K. wagneri.
Additional material examined: Italy: Vieste, from garden soil, isolated from different sub-samples, July 2019, R. Labuda, strain RL-05 (ITS sequence, MT898644; LSU sequence, MT898648) and strain RL-06 (ITS sequence, MT898645; LSU sequence, MT898649) referring to personal collection of RL. At 30 °C they grew relatively better (up to 5 mm larger in diam) than the ex-type culture.
Keratinophyton wagneri Labuda, Bernreiter, Kubátová & Schüller sp. nov.
MycoBank: MB 833635. (Figs. 8 and 9).
Etymology: Named in honour of Professor Martin Wagner, Head of Unit for Food Microbiology and Head of Institute for Food Safety, Food Technology and Veterinary Public Health, University of Veterinary Medicine, Vienna (Austria), an expert in the veterinary microbiology.
Diagnosis: Robust and coarsely roughed conidia produced from non-swollen conidiogenous cells, none to very limited growth at 30 °C, and production of pink pigment on PCA after prolonged incubation (3 weeks). Sequence data from ITS separate this taxon from all others Keratinophyton species.
Type: The Slovak Republic: Tatranská Lomnica, from forest soil, Aug. 2015, R. Labuda (PRM 952501 - holotype; BiMM-F77 = CCF 6362 – ex-type cultures). ITS sequence, GeneBank MN633083; LSU sequence, GeneBank MT874999.
Description: Sexual morph not observed on any of the media used. Asexual morph on PDA. Vegetative mycelium consisting of hyaline, septate, smooth-walled, sparsely to pronouncedly branched hyphae, 2.0‒6.0 µm diam. Racquet hyphae present. Conidia (aleurioconidia), hyaline, white to yellowish in mass, thin-walled and regularly ornamented with minute warts with minute warts (visible under SEM) and coarsely roughened (under light microscope). Terminal and lateral conidia born on main fertile hyphae or from side branches of variable length, sessile or on short protrusions, occasionally swollen and of variable length, solitary, 1‒4 (10) per conidiogenous cell, obovate to clavate, single celled, (4.0‒) 5.5‒6.5 (‒8.0) x (2.5‒) 3.0‒3.5(‒4.0) µm (mean = 5.7±0.4 x 3.2±0.2 µm, n= 120), rarely 2-celled, up to 12 µm large ones also present. Intercalary conidia not observed. Chlamydospores not observed.
Culture characteristics: Colonies on PDA 25‒30 mm diam at 25 °C, after 14 days, powdery to downy (mealy), with abundant sporulation, white to slightly yellowish, flat, slightly elevated (umbonate) and more floccose at the center, with irregular margin, reverse white with slightly yellowish center, no pigment or exudate produced. At 30 °C, 4‒8 mm diam after 14 days, white, floccose with poor sporulation, and with yellowish reverse. Colonies on SDA 14‒18 mm diam at 25 °C, after 14 days, morphology similar to PDA. In age, yellowish-brown (amber) pigment is produced and colony reverse becomes dark reddish-brown (after 4 weeks). At 30 °C, no growth or only microcolonies were observed. Colonies on MEA 18‒22 mm diam at 25 °C, after 14 days, morphology similar to PDA with more yellowish colonies. At 30 °C, no growth or only micro-colonies were observed. Colonies on CMA and PCA 20‒25 mm diam at 25 °C, after 21 days, white to yellowish, granular, good sporulation, reverse yellowish. Pinkish pigment production observed after 3-4 weeks on PCA (in both tested strains). No ascomata observed after prolonged incubation (3 months). The optimum temperature for growth on PDA, SDA and MEA was between 20 and 25 °C (Table S2a‒c). Minimum growth (1-2 mm diam) was observed at 10 °C, and germination of majority of the spores was observed at 8 °C. The maximum temperature for growth was 31 °C (1‒3 mm diam). Keratinolytic activity weak to moderate (Fig 10d), with hair attack intensity = 2. Urease activity negative (after 14 days of incubation).
Notes: Based on a search of NCBI GenBank nucleotide database, the closest hit for K. wagneri using the ITS sequence is K. minutisporosum (as Chrysosporium minutisporosum CBS 101577; Genebank: KT155616); with identity = 486/541 (90%) and gaps 11/541 (2%). Morphologically, K. straussii and K. wagneri seem to be very similar, however, they can be distinguished based on (1) size of conidia (av. = 4.9 x 2.5 µm vs 5.7 x 3.2 µm), (2) growth at 30 °C on PDA (15‒20 mm vs 3‒4 mm diam), (3) morphology of conidiogenous cells (commonly vs non– to occasionally swollen), (4) colony pigmentation on SDA after prolonged incubation (bright orange vs dark brown), and (5) keratinolytic ability after 3 weeks (very strong vs moderate). In addition, the production of a pinkish pigment on PCA after 3-4 weeks (at 20 °C and 25 °C) has been observed only in K. wagneri. Moreover, conidia of this species are more coarsely roughed (warty) than those in K. straussii (Fig. 8 c‒e).
Additional material examined: The Slovak Republic: Tatranská Lomnica, from forest soil, isolated from a different sub-sample, July 2019, R. Labuda, RL-07 (ITS sequence, MT903275; LSU sequence, MT903309) referring to personal collection of RL, phenotypically identical with the ex-type culture.
The main distinguishing phenotypic characteristics of the four new species compared with the other asexual members of the genus Keratinophyton are outlined in the Table 2.
Table 2 Comparison of the key phenotypic characteristics of Keratinophyton spp.
Species
|
Growth at 30 °C on PDA*
|
Colony Color Growth/ Reverse
on PDA at 25 °C, after 14d**
|
Conidial
Shape
|
Conidial dimensions
(µm)
|
Conidial
Surface
|
Intercalary conidia
|
References
|
K. gollerae sp. nov.
|
None
|
White to creamy, 20‒22 mm/white to yellowish
|
Obovoid to clavate
|
5.0‒7.0 x 2.0‒2.5
|
Smooth to finely roughened
|
Absent
|
This study
|
K. lemmensii sp. nov.
|
Present (good)
|
White, 28‒35 mm/lemon yellow
|
Clavate to filiform
|
3.0‒40 µm (1- to 2-celled)
|
Smooth
|
Present
|
This study
|
K. straussii sp. nov.
|
Present (good)
|
White to creamy,
24‒28 mm/white to yellowish
|
Obovoid to clavate
|
4.5‒5.0 x 2.5‒3.0
|
Verrucose
|
Absent
|
This study
|
K. wagneri sp. nov.
|
Present (inhibited)
|
White to yellowish,
25‒30 mm/white to yellowish
|
Obovoid to clavate
|
4.0‒8.0 x 2.5‒4.0
|
Verrucose
|
Absent
|
This study
|
K. clavisporum
|
Present
(inhibited)ᵜ
|
White, 53 mm (26 °C)/red-brown
|
Clavate to long -ellipsoidal
|
5.0‒10 x 2.5‒5.0
|
Smooth
|
Absent
|
Zhang et al. 2017
|
K. echinulatum
|
Present (good)
|
Yellow to pale orange yellow, 28‒45 mm/orange yellow
|
Obovoid to clavate
|
4.5‒7.0 x 2.5‒4.0
|
Echinulate
|
Present
|
Crous et al. 2016
|
K. fluviale
|
Present (good)
|
White to yellowish white, 60‒70mm (30 °C)/brownish orange
|
Obovate, clavate, nearly ellipsoidal or pyriform
|
3.5‒15 x 2.0‒3.0 (1-and 2-celled)
|
Verrucose
|
Present
(very rare)
|
Vidal et al. 2000
|
K. qinghaiense
|
Present (good) ᵜ
|
White to yellowish, 30 mm (7days)/yellowish
|
Clavate to cylindrical
|
3.6‒13 x1.8‒3.6
|
Smooth
|
Present
|
Han et. al. 2013
|
K. hubeiense
|
Present
(inhibited)ᵜ
|
Grey white to white, 65‒67 mm/reverse yellowish
|
Obovoid to ellipsoidal
|
2.2‒4.3 x 1.6‒3.2
|
Smooth
|
Absent
|
Zhang et al. 2016
|
K. linfenense
|
Present (good)
|
White to cream, 72 mm (30 °C)/white to light yellow
|
Ellipsoidal to fusiform, also clavate
|
3.2‒5.4 x-1.4‒2.2
|
Smooth
|
Absent
|
Liang et al. 2009
|
K. minutisporosum
|
Present (inhibited)
|
White to yellowish white,
55‒70 mm/white
|
Pyriform or subglobose, also clavate
|
3.0‒4.0 (‒11) x 1.5‒3.5
|
Verrucose
|
Present
(very rare)
|
Vidal et al. 2002
|
K. pannicola
|
Present (good)
|
White to pale yellow,
20‒38 mm (PYE***)/pale brown
|
Obovoid to clavate
|
6.0‒11 x 3.5‒4.5
|
Verrucose
|
Present
(less abundant)
|
Oorschot 1980
|
K. siglerae
|
Present (good)
|
Griseous orange, 15‒20 mm (21 days)/ pale brown
|
Cylindrical to clavate
|
5.0‒30 x 2.0‒3.5
1-and 2-celled
|
Smooth to slightly verrucose
|
Present
|
Cano and Guarro 1994
|
K. submersum
|
Present (inhibited)
|
Yellowish white, 50‒60mm/yellowish white
|
Clavate, also pyriform, obovoid and subglobose
|
4.0‒35 x 2.5‒5.0
(1- to 4-celled)
|
Smooth to verrucose-thick-walled
|
Present (in old cultures)
|
Vidal et al. 2002
|
K. turgidum
|
Present (good)
|
White, 50‒55 mm (SGA at 28 °C)/pale brown
|
Pyriform to oval
|
5.0‒7.0 x 3.5‒5.0
|
Smooth
|
Present
|
Crous et al. 2017
|
*if not stated other medium; ** if not stated otherwise; *** PYE, phytone yeast extract agar; **** CZA, Czapek agar; ᵜ Yanfeng Han personal communication
Table S1 a Temperature dependent growth of the new Keratinophyton species (in mm) on PDA after 14 days
Species
|
Temperature (°C)
|
8*
|
10
|
12
|
15*
|
18
|
20
|
25*
|
28
|
29
|
30*
|
31*
|
32
|
K. lemmensii
|
1‒2
|
4‒5
|
7‒9
|
10‒14
|
-
|
25‒27
|
28‒35
|
-
|
-
|
38‒45
|
32‒38
|
M‒1
|
K. gollerae
|
SG‒M
|
M‒1
|
2‒6
|
15‒18
|
-
|
18‒20
|
20‒22
|
11‒12
|
M‒1
|
SG
|
-
|
-
|
K. straussii
|
nSG
|
1‒2
|
4‒5
|
7‒9
|
18-20
|
18‒22
|
24‒28
|
-
|
-
|
15‒20
|
6‒8
|
1‒2
|
K. wagneri
|
SG
|
1‒2
|
8‒10
|
6‒8
|
18-20
|
20‒25
|
25‒30
|
-
|
-
|
3‒4
|
2‒3
|
M‒1
|
*crucial distinctive growth temperatures; SG, spore germination; nSG, no spore germination; M, microcolonies
Table S1 b Temperature dependent growth of the new Keratinophyton species (in mm) on MEA after 14 days
Species
|
Temperature (°C)
|
8*
|
10
|
12
|
15
|
18
|
20
|
25
|
28
|
29
|
30*
|
31*
|
32
|
K. lemmensii
|
1‒2
|
2‒4
|
5‒7
|
7‒10
|
-
|
15‒17
|
20‒25
|
-
|
-
|
18‒20
|
12‒15
|
SG
|
K. gollerae
|
SG-M
|
M
|
1‒2
|
5‒8
|
-
|
10‒12
|
14‒16
|
M
|
nSG
|
-
|
-
|
-
|
K. straussii
|
nSG
|
M
|
M
|
4‒7
|
10‒12
|
10‒12
|
18‒20
|
-
|
-
|
5‒10
|
3‒4
|
M‒1
|
K. wagneri
|
SG
|
M
|
M‒2
|
3‒5
|
10‒13
|
12‒15
|
18‒20
|
-
|
-
|
M
|
M
|
SG
|
*crucial distinctive growth temperatures; SG, spore germination; nSG, no spore germination; M, microcolonies
Table S1 c Temperature dependent growth of the new Keratinophyton species (in mm) on SDA after 14 days
Species
|
Temperature (°C)
|
8*
|
10
|
12
|
15*
|
18
|
20
|
25
|
28
|
29
|
30*
|
31*
|
32
|
K. lemmensii
|
1‒2
|
6‒7
|
8‒10
|
7‒10
|
-
|
24‒26
|
28‒35
|
-
|
-
|
30‒32
|
25‒30
|
M‒1
|
K. gollerae
|
SG‒M
|
M‒1
|
M-1
|
20‒22
|
-
|
12‒17
|
23‒25
|
M‒2
|
SG
|
SG
|
nSG
|
-
|
K. straussii
|
nSG
|
M‒1
|
2‒3
|
3‒4
|
-
|
14‒16
|
16‒20
|
-
|
-
|
15‒20
|
1‒2
|
nSG
|
K. wagneri
|
SG
|
M‒1
|
1‒3
|
3‒4
|
-
|
12‒14
|
14‒18
|
-
|
-
|
M‒1
|
SG‒M
|
nSG
|
*crucial distinctive growth temperatures; SG, spore germination; nSG, no spore germination; M, microcolonies
All four new species are readily distinguished from the other taxa in the genus Keratinophyton, known only as Chrysosporium asexual morphs, based on phenotypical characteristic alone. From the sexual Keratinophyton species, they principally differ by their inability to form ascomata in culture (Cano et al. 2002), but also by overall phenotypic characters, 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 morphology of conidia (shape, surface and dimensions) and growth response to 30 °C exposure after 14 days on PDA. An identification key for Keratinophyton species modified from Cano et al. (2002) with addition of the new species is provided.
Dichotomous key to the species of Keratinophyton (asexual morphs) modified from Cano et al. (2002).
1
2 (1)
3 (2)
4 (3)
5 (2)
6 (5)
7 (5)
8 (7)
9 (1)
10 (9)
11 (9)
12 (11)
13 (12)
|
Conidial surface smooth to finally roughed............................................... 2
Conidial surface verrucose to echinulate.................................................... 9
Growth at 30 °C absent............................................................... K. gollerae
Growth at 30 °C inhibited............................................................................ 3
Growth at 30 °C present (good).................................................................. 5
Colony reverse red-brown..................................................... K. clavisporum
Colony reverse in other colors.................................................................... 4
Intercalary conidia absent, 2.2‒4.3 x 1.6‒3.2 µm conidia........ K. hubeiense
Intercalary conidia present, 4.0‒35 x 2.5‒5.0 µm conidia..... K. submersum
Cylindrical to filiform 1- to 2-celled conidia present................................... 6
Cylindrical to filiform 1- to 2-celled conidia absent.................................... 7
Colony white with yellow reverse............................................ K. lemmensii
Colony griseous orange with brownish reverse........................... K. siglerae
Intercalary conidia present......................................................................... 8
Intercalary conidia absent........................................................ K. linfenense
Clavate to cylindrical conidia, 3.6‒13 x 1.8‒3.6 µm.............. K. qinghaiense
Pyriform to oval conidia, 5.0‒7.0 x 3.5‒5.0 µm......................... K. turgidum
Growth at 30 °C inhibited.......................................................................... 10
Growth at 30 °C present (good)................................................................ 11
Obovoid to clavate conidia, 4.0‒8.0 x 2.5‒4.0 µm..................... K. wagneri
Pyriform to subglobose conidia, 3.0‒4.0 x 1.5‒3.5 µm.. K. minutisporosum
Colonies yellow to orange, conidia echinulate..................... K. echinulatum
Colonies white to creamy (yellowish), conidia verrucose........................ 12
Intercalary conidia absent, conidia 4.5‒5.0 x 2.5‒3.0 µm.......... K. straussii
Intercalary conidia present, conidia more than 5.0 µm in length............ 13
Conidia usually up to 3.0 µm wide................................................ K. fluviale
Conidia usually more than 3.0 µm wide.................................... K. pannicola
|
New combinations
The phylogenetic analyses strongly supported the recent distinct classification of the species previously classified as Chrysosporium asexual morphs into two phylogenetically different sexual genera, namely Aphanoascus and Keratinophyton (Crous et al. 2017; Sutton et al. 2013). In this study, ten new combinations are proposed for Chrysosporium asexual morphs resolved in a monophyletic clade affiliated to the latter genus. Hence, the following Chrysosporium species C. clavisporum, C. echinulatum, C. evolceanui, C. fluviale, C. hubeiense, C. linfenense, C. minutisporosum, C. siglerae, C. submersum and C. qinghaiense are revised in the genus Keratinophyton H.S. Randhawa & R.S. Sandhu.
Keratinophyton clavisporum (Zhang, Han & Liang) Labuda & Bernreiter, comb. nov.
Basionym: Chrysosporium clavisporum Zhang, Han & Liang, Phytotaxa 303: 177 (2017).
Type: G80.1 from plant root soil, China.
MycoBank: MB833653
Keratinophyton echinulatum (Hubka, Mallátová, Cmoková & Kolařík) Labuda & Bernreiter, comb. nov.
Basionym: Chrysosporium echinulatum Hubka, Mallátová, Cmoková & Kolařík, Persoonia 36: 410 (2016).
Type: CCF 4652 = CBS 141178 = UAMH 11824, from sole of the foot, Czechia.
MycoBank: MB833636
Keratinophyton pannicola (Corda) Labuda & Bernreiter, comb. nov.
Basionym: Capillaria pannicola Corda, Icon. Fung. (Prague) 1: 10 (1837).
≡ Sporotrichum pannicola (Corda) Rabenh., Deutschl. Krypt.-Fl. (Leipzig) 1: 78 (1844)
≡ Chrysosporium pannicola (Corda) Oorschot & Stalpers, Stud. Mycol. 20: 43 (1980)
= Chrysosporium evolceanui (Randhawa & Sandhu) Garg, Sabouraudia 4: 262 (1966)
= Trichophyton evolceanui Randhawa & Sandhu, Mycopath. Mycol. Appl. 20: 232 (1963)
Type: CBS 116.63 = ATCC 22400 = IHEM 4436 = IMI 147545 = NCPF 489 = RV 26475 = UAMH 1275, from soil, India.
MycoBank: MB8333643
Keratinophyton fluviale (Vidal & Guarro) Labuda & Bernreiter, comb. nov.
Basionym: Chrysosporium fluviale Vidal & Guarro, Mycol. Res. 104: 245 (2000).
Type: CBS 100809 = FMR 6005 =IMI 378764, from river sediments, Spain.
MycoBank: MB8333637
Keratinophyton hubeiense (Zhang, Han & Liang) Labuda & Bernreiter, comb. nov.
Basionym: Chrysosporium hubeiense Zhang, Han & Liang, Phytotaxa 270: 213 (2016).
Type: EM66601, from soil under the chicken feather, China.
MycoBank: MB833638
Keratinophyton linfenense (Liang, Liang & Han) Labuda & Bernreiter, comb. nov.
Basionym: Chrysosporium linfenense Liang, Liang & Han, Mycotaxon 110: 67 (2009).
Type: GZAC H31, from rhizosphere soil, China.
MycoBank: MB833639
Keratinophyton minutisporosum (Vidal & Guarro) Labuda & Bernreiter, comb. nov.
Basionym: Chrysosporium minutisporosum Vidal & Guarro, Stud. Mycol. 47: 205 (2002).
Type: CBS 101577 = FMR 6096 from river mouth sediment, Spain.
MycoBank: MB833640
Keratinophyton siglerae (Cano & Guarro) Labuda & Bernreiter, comb. nov.
Basionym: Chrysosporium siglerae Cano & Guarro, Mycotaxon 51: 75 (1994)
Type: UAMH 6541 = FMR 3066 = IMI 336467, from garden soil, Spain.
MycoBank: MB833641
Keratinophyton submersum (Vidal & Guarro) Labuda & Bernreiter, comb. nov.
Basionym: Chrysosporium submersum Vidal & Guarro, Stud. Mycol. 47: 200 (2002)
Type: CBS 101575 = FMR 6088, from river mouth sediment, Spain
MycoBank: MB833642
Keratinophyton qinghaiense (Han, Wang, Liang & Liang) Labuda & Bernreiter, comb. nov.
Basionym: Chrysosporium qinghaiense Han, Wang, Liang & Liang, Mycosystema 32: 607 (2013).
Type: GZUIFR 11, from farmland soil, China.
MycoBank: MB833655