New Records of Four Species Belonging to Eurotiales from Soil and Freshwater in Korea.

Four strains of Penicillium and Talaromyces species are described and illustrated in an inventory of fungal species belonging to Eurotiales. The strains, CNUFC-DDS17-1, CNUFC-DDS27-1, CNUFC-PTM72-1, and CNUFC-YJW3-31, were isolated from soil and freshwater samples from South Korea. Based on their morphological characteristics and sequence analyses by the combined β-tubulin and calmodulin gene, the CNUFC-DDS17-1, CNUFC-DDS27-1, CNUFC-PTM72-1, and CNUFC-YJW3-31 isolates were identified as Penicillium pasqualense, Penicillium sanguifluum, Talaromyces apiculatus, and Talaromyces liani, respectively. The designated strains were found to represent a previously undescribed species of Korean fungal biota. In this study, detailed morphological descriptions and phylogenetic relationships of these species are provided.

Introduction

Soil and freshwater provide unique environments for the growth of fungi. Fungal communities perform essential functions in the biogeochemical cycles of these ecosystems. In soils, they act as agents governing soil carbon cycling, plant nutrition, and pathology [1]. Freshwater nourishes diverse habitats such as fallen leaves, plant litter, decaying wood, aquatic plants and insects, and soils for fungi. Fungi act in the energy flow, food-web, nutrient transformations, and self-purification in the freshwater ecosystem [2].

The genera Penicillium and Talaromyces (Trichocomaceae, Eurotiales), belonging to the Eurotiomycetes, are of high environmental and biotechnological relevance [3]. These genera are abundant in contaminated foods, fruits, indoor environments, air, soil, and water [4-6]. They also produce a variety of useful secondary metabolites including anticancer and antifungal compounds, extracellular enzymes, and harmful mycotoxins [7-9]. Previously, Talaromyces was thought to be related to teleomorphic Penicillium subgenus Biverticillium and other genera, but recently both have been classed as separate genera containing both sexual and asexual species with the introduction of single name nomenclature [10,11]. The species belonging to both these genera were identified on the recommendations of the polyphasic species concept, using morphological and molecular phylogenetic analyses [11,12].

The genus Penicillium was first described by Link and included asexual fungi bearing Penicillium-like fruiting bodies [13]. New species have since been added to the already 354 accepted Penicillium species [8,14,15]. Members of this genus were divided into 26 sections using the polyphasic approach [16]. To the best of our knowledge, over 100 Penicillium species have been reported from Korea [17,18]. Some of the new species discovered in Korea were isolated from various environmental samples such as P. daejeonium from grape and Schisandra fruit, P. koreense from soil, P. samsonianum from stems and leaves of Viscum album var. coloratum, P. jejuense from marine environments of Jeju Island, P. punicae from pomegranate (Punica granatum) fruit, P. aquaticum and P. acidum from freshwater [16,19-22]. Section Citrina represents one of these sections clade and are abundant worldwide [23]. Among the specified 39 species and 17 new species accepted in the section Citrina, only 11 species have been reported from Korea [24,25].

The genus Talaromyces was introduced by Benjamin for teleomorphic Penicillium species with T. vermiculatus as the type species [26]. These species were characterized taxonomically by their sexual morphology, having cleistothecial or gymnothecial ascomata, unitunicate 8-spored asci, and unicellular ascospores with or without equatorial crests. Currently, the genus accepts 110 species which are divided into seven sections, i.e., Bacillispori, Heici, Islandici, Purpurei, Subinflati, Talaromyces, and Trachyspermi [6]. Recently, several new Talaromyces species were discovered from various environmental habitats [6,15,27,28]. To date, only 21 Talaromyces species have been reported from Korea [17,29-31]. Among the reported species, only six have been well described in Korea, these species are not reported before [30]. Talaromyces apiculatus and Talaromyces liani are classified in section Talaromyces. The diversity of the genus Penicillium and Talaromyces in Korea remains unknown using both molecular and morphological analyses.

During investigation of the fungal species inhabiting soil and freshwater, two Penicillium and two Talaromyces species were identified. The objective of this study was to perform morphological and molecular analyses to characterize the undescribed Penicillium and Talaromyces species in Korea: P. pasqualense, P. sanguifluum, T. apiculatus and T. liani belonging to the Eurotiales.

Materials and methods

Isolation of fungal strains from water and soil samples

Soil samples were collected from Dongdo (eastern islet) of Dokdo Island (37°14′21.3″ N, 131°52′04.4″ E) and under the rhizosphere of a pine tree located at Geumgol Mountain, Jin Island (Jindo). Freshwater samples were collected from Yeosu stream in a small falcon tube. Serial-dilution plating methods were employed using potato dextrose agar (PDA; Becton, Dickinson and Co., Sparks, MD) and malt extract agar (MEA; Becton). Pure isolates were obtained by picking individual colonies of varied morphologies and transferring them to PDA plates and subculturing until pure mycelia were obtained. All pure isolates were maintained in PDA slant tubes and in 20% glycerol at −80 °C at the Environmental Microbiology Laboratory Fungarium, Chonnam National University, Gwangju, Korea.

P. pasqualense, P. sanguifluum, T. apiculatus, and T. liani strains isolated in our study were designated CNUFC-DDS17-1 and CNUFC-DDS17-2, CNUFC-DDS27-1, and CNUFC-DDS27-2, CNUFC-PTM72-1 and CNUFC-PTM72-2, CNUFC-YJW3-31 and CNUFC-YJW3-32, respectively. The isolates, CNUFC-DDS17-1, -DDS17-2, -DDS27-1, and -DDS27-2 were isolated from soil of Dokdo Island; CNUFC-PTM72-1 and -PTM72-1 from soil of Geumgol Mountain; CNUFC-YJW3-31 and -YJW3-32 from freshwater.

DNA extraction, PCR, and purification

Genomic DNA was extracted using the Solg TM Genomic DNA Prep Kit (Solgent Co. Ltd., Daejeon, Korea). The internal transcribed spacer (ITS)-rDNA region, β-tubulin gene (BenA), and calmodulin (CaM) gene were amplified with the primer pairs ITS1/ITS4 [32], Bt2a/Bt2b [33], and Cmd5/Cmd6 [5], respectively. The PCR amplification reaction mixture was set up according to Nguyen et al. [30]. An Accuprep PCR Purification Kit (Bioneer Corp., Daejeon, Korea) was employed for PCR purification. DNA sequencing was performed using an ABI 3700 Automated DNA sequencer (Applied Biosystems Inc., Foster City, CA).

Molecular analysis

Fungal sequences were aligned using Clustal_X version 2.1 [34] and edited with Bioedit version 7.2.6.0 [35]. Maximum likelihood (ML) was constructed using MEGA 6 software [36]. Sequences of CNUFC-DDS17-1, CNUFC-DDS17-2, CNUFC-DDS27-1, CNUFC-DDS27-2, CNUFC-PTM72-1, CNUFC-PTM72-2, CNUFC-YJW3-31, and CNUFC-YJW3-32 were deposited in the NCBI database under the accession numbers shown in Table 1.

Table 1.

Taxa, collection numbers, sequences, and GenBank accession numbers used in this study.

		GenBank accession no.
Taxon name	Collection no. (isolate no.)	BenA	CaM
Penicllium aurantiacobrunneum	CBS 126228 (T)	JN606702	JN606522
P. cairnsense	CBS 124325 (T)	JN606693	JN606512
P. chrzaszii	CBS 217.28 (T)	JN606758	JN606423
P. citrinum	CBS 139.45 (T)	GU944545	GU944638
P. cosmopolitanum	CBS 126995 (T)	JN606733	JN606472
P. decaturense	CBS 117509 (T)	JN606685	JN606413
P. godlewskii	CBS 215.28 (T)	JN606768	JN606443
P. manginii	CBS 253.31 (T)	JN606651	JN606381
P. miczynskii	CBS 220.28 (T)	JN606706	JN606526
P. neomiczynskii	CBS 126231 (T)	JN606705	JN606523
P. nothofagi	CBS 130383 (T)	JN606732	JN606507
P. pancosmium	CBS 276.75 (T)	JN606790	JN606446
P. pasqualense	CBS 126330 (T)	JN606673	JN606394
P. pasqualense	CBS 124327	JN606672	JN606392
P. pasqualense	CBS 122402	JN606674	JN606393
P. pasqualense	CNUFC-DDS17-1	MK204815	MK204817
P. pasqualense	CNUFC-DDS17-2	MK204816	MK204818
P. quebecense	CBS 101623 (T)	JN606700	JN606509
P. roseopurpureum	CBS 266.29 (T)	JN606838	JN606556
P. sanguifluum	CBS 127032 (T)	JN606819	JN606555
P. sanguifluum	CBS 118024	JN606833	JN606537
P. sanguifluum	CBS 643.73	JN606853	JN606576
P. sanguifluum	CNUFC-DDS27-1	MK204819	MK204821
P. sanguifluum	CNUFC-DDS27-2	MK204820	MK204822
P. sizovae	CBS 413.69 (T)	GU944535	GU944618
P. sucrivorum	CBS 135116 (T)	JX141015	JX141506
P. sumatrense	CBS 281.36 (T)	JN606639	JN606368
P. ubiquetum	CBS 126437 (T)	JN606800	JN606460
P. waksmanii	CBS 230.28 (T)	JN606779	JN606431
P. westlingii	CBS 231.28 (T)	JN606718	JN606500
Talaromyces aculeatus	CBS 289.48 (T)	KF741929	KF741975
T. apiculatus	CBS 101366	KF741910	KF741932
T. apiculatus	CBS 312.59 (T)	KF741916	KF741950
T. apiculatus	CNUFC-PTM72-1	MK204823	MK204825
T. apiculatus	CNUFC-PTM72-2	MK204824	MK204826
T. aurantiacus	CBS 314.59 (T)	KF741917	KF741951
T. australis	CBS 137102 (T)	KF741922	KF741971
T. bacillisporus	CBS 296.48 (T)	AY753368	KJ885262
T. derxii	CBS 412.89 (T)	JX494305	KF741959
T. duclauxii	CBS 322.48 (T)	JX091384	KF741955
T. flavovirens	CBS 102801 (T)	JX091376	KF741933
T. francoae	CBS 113134 (T)	KX011489	KX011501
T. funiculosus	CBS 272.86 (T)	JX091383	KF741945
T. liani	CBS 118434	KM066139	KP453744
T. liani	CBS 225.66 (T)	JX091380	KJ885257
T. liani	CNUFC-YJW3-31	MK204811	MK204813
T. liani	CNUFC-YJW3-32	MK204812	MK204814
T. kendrickii	CBS 136666 (T)	KF741921	KF741967
T. marneffei	CBS 388.87 (T)	JX091389	KF741958
T. purpurogenus	CBS 286.36 (T)	JX315639	KF741947
T. stellenboschiensis	CBS 135665 (T)	JX091605	JX140683
T. stollii	CBS 408.93 (T)	JX315633	JX315646
T. verruculosus	CBS 388.48 (T)	KF741928	KF741974
T. viridis	CBS 114.72 (T)	JX494310	KF741935
T. viridulus	CBS 252.87 (T)	JX091385	KF741943

Bold letters indicate isolates and accession numbers described in our study.

CBS: Centraalbureau voor Schimmelcultures (Utrecht, The Netherlands); CNUFC: Chonnam National University Fungal Collection (Gwangju, South Korea); T: ex-type strain.

Morphological studies

The isolated strains CNUFC-DDS17-1, CNUFC-DDS27-1, CNUFC-PTM72-1, and CNUFC-YJW3-31 were cultured on Czapek yeast autolysate agar (CYA), Blakeslee’s MEA, and yeast extract sucrose agar (YES) [5]. The plates were incubated at 25 °C in the dark for one week. To observe fine fungal structures, the isolates were fixed in 2.5% paraformaldehyde-glutaraldehyde in 0.05 M phosphate buffer (pH 7.2) for 2 h, and then washed with cacodylate buffer (Junsei Chemical Co. Ltd., Kyoto, Japan). Cellular membranes were preserved by fixing the samples in 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA) diluted in cacodylate buffer for 1 h. Samples were then washed again in cacodylate buffer, dehydrated in graded ethanol (Emsure, Darmstadt, Germany) and isoamyl acetate (Junsei Chemical Co. Ltd.), and dried in a fume hood. Finally, samples were sputter-coated with gold and observed under a Hitachi S4700 field emission scanning electron microscope at the Korea Basic Science Institute, Gwangju, Korea. Samples were also observed under an Olympus BX51 microscope with DIC optics (Olympus, Tokyo, Japan) by mounting in a lactophenol solution (Junsei Chemical Co. Ltd.).

Results

Molecular phylogenetic analysis

The results constructed by ML analyses based on combined BenA and CaM sequences analysis of the isolates respectively belonging to Penicillium and Talaromyces are shown in Figures 1 and 2. A BLASTn search was conducted using the ITS, BenA, and CaM sequences of CNUFC-DDS17-1, CNUFC-DDS27-1, CNUFC-PTM72-1, and CNUFC-YJW3-31 isolates. The ITS region of isolates CNUFC-DDS17-1, CNUFC-DDS27-1, CNUFC-PTM72-1, and CNUFC-YJW3-31 show 99.3% (543/547 bp), 98.9% (520/526 bp), 98.6% (348/353 bp), and 100% (537/537 bp) sequence similarities with P. pasqualense CBS 126330 (JN617676), P. sanguifluum CV1856 (JX140865), T. apiculatus CBS 101366 (KF741977), and T. liani CBS 225.66 (MH858781). BLASTn analysis of BenA of CNUFC-DDS17-1, CNUFC-DDS27-1, CNUFC-PTM72-1, and CNUFC-YJW3-31 showed similarities of 97.8% (402/411 bp), 100% (413/413 bp), 98.9% (373/377 bp), and 98.4% (358/364 bp), respectively, with P. pasqualense CBS122402 (JN606674), P. sanguifluum CBS 643.73 (JN606853), T. apiculatus CBS 312.59 (JX091378), and T. liani CBS 225.66 (JX091380). Similarly, BLASTn analysis of CaM of CNUFC-DDS17-1, CNUFC-DDS27-1, CNUFC-PTM72-1, and CNUFC-YJW3-31 showed similarities of 98.1% (418/426 bp), 99.8% (504/505 bp), 98.7% (441/447 bp), and 99.3% (446/449 bp) with P. pasqualense CBS 122402 (JN606393), P. sanguifluum CBS 118020 (JN606536), T. apiculatus CBS 101366 (KF741932), and T. liani CBS 225.56 (KJ885257), respectively.

Figure 1.

Phylogenetic tree of Penicillium pasqualense CNUFC-DDS17-1 and CNUFC-DDS17-2, Penicillium sanguifluum CNUFC-DDS27-1 and CNUFC-DDS27-2, and related species based on maximum likelihood analysis of the combined datasets for BenA and CaM. The sequence of Penicillium corylophilum was used as an out group. Numbers at the nodes indicate the bootstrap values (>50%) from 1000 replicates. The bar indicates the number of substitutions per nucleotide. The study isolates are shown in bold red and blue.

Figure 2.

Phylogenetic tree of Talaromyces apiculatus CNUFC-PTM72-1 and CNUFC-PTM72-2, Talaromyces liani CNUFC-YJW3-31 and CNUFC-YJW3-32, and related species based on maximum likelihood analysis of the combined datasets for BenA and CaM. Sequence of Talaromyces bacillisporus was used as an out group. Numbers at the nodes indicate the bootstrap values (>50%) from 1000 replicates. The bar indicates the number of substitutions per nucleotide. The study isolates are shown in bold red and blue.

In the combined BenA and CaM sequence tree, the isolates CNUFC-DDS17-1, CNUFC-DDS27-1, CNUFC-PTM72-1, and CNUFC-YJW3-31 were identical to P. pasqualense, P. sanguifluum, T. apiculatus, and T. liani, respectively (Figures 1 and 2).

Taxonomy

Taxonomy of CNUFC-DDS17-1

Houbraken, Frisvad & Samson, Studies in Mycology 70: 108 (2011) (Table 2, Figure 3).

Table 2.

Morphological characteristics of CNUFC-DDS17-1 compared with the reference strain of Penicillium pasqualense.

Character	CNUFC-DDS17-1	P. pasqualensea
Conidiophores	Biverticillate, triverticillate conidiophores, measured 100–300 μm long, 4.5–5 μm wide	Predominantly symmetrically biverticillate, 200–400 μm, smooth, 2.5–3.0 μm wide
Metuale	Few metuale, 10–18.9 × 2.5–3.2 μm	Metulae in a divergent vertical terminal, 11–17 × 2.5–3.5 μm, branches longer up to 25 μm
Phialides	Ampulliform, 5.9−10.2 × 2.5−3.5 μm	Ampulliform, 7.5–10 × 2.5–3.5 μm
Conidia	Globose to subglobose, 2.3–3.1 µm in diameter	Globose to subglobose, spinose, 2.5–3.5 µm in diameter
Sclerotia	Absent	Orange or brown

From the description by Houbraken et al. [24].

Figure 3.

Morphology of Penicillium pasqualense. (A,D) Colonies on yeast extract sucrose agar (YES); (B,E) Colonies on Blakeslee’s malt extract agar (MEA); (C,F) Colonies on Czapek yeast autolysate agar (CYA); (A–C: obverse view, D–F: reverse view); (G–I) Conidiophores; (J) Conidia (Scale bars: G = 15 μm, H–I = 10 μm, J = 5 μm).

Description: Colonies on YES were dark beige with white mycelium, no soluble pigment, weak to moderate sporulation, and reached 20–26 mm in diameter after 7 d at 25 °C. Colonies on MEA were pale green, with good sporulation, and velvety to floccose texture. Colonies grew slow, reaching 25–35 mm in diameter after 7 d at 25 °C. Colonies were dark brown, with no soluble pigment, entire margins, and floccose colony texture at the center on CYA, reaching 25–30 mm in diameter. There were no formations of asci or ascospores. The conidiophores were predominantly symmetrically biverticillate, triverticillate (few) with additional branches and divergent terminal verticals of metulae, measured 10–18.9 × 2.5–3.2 μm. Phialides were ampulliform and measured 5.9–10.2 × 2.5–3.5 μm in diameter. Conidia were dark green or dark-blue green in color, globose to subglobose, and spinose and measured 2.3–3.1 μm in diameter.

Taxonomy of CNUFC-DDS27-1

(Sopp) Biourge, La Cellule 33: 105 (1923) (Table 3, Figure 4).

Table 3.

Morphological characteristics of CNUFC-DDS27-1 compared with the reference strain of Penicillium sanguifluum.

Character	CNUFC-DDS27-1	P. sanguifluuma
Conidiophores	Monoverticillate, 17–50 μm	Monoverticillate, 15–50 µm
Metuale	Not observed	Branched-like metulae scarcely formed, strongly vesiculate (10–) 13–18 × 2.0–3.0 μm at base
Phialides	Ampulliform, 5.5–8.3 × 2.0–3.0 μm	Ampulliform, terminally, and subterminally formed, 6.5–8 × 2–3 μm
Conidia	Globose to subglobose, smooth to finely roughened, 2.0–2.5 µm in diameter	Globose to subglobose, smooth to finely roughened, 2.0–2.5 μm in diameter
Sclerotia	Absent	Absent

From the description by Houbraken et al. [24].

Figure 4.

Morphology of Penicillium sanguifluum. (A,D) Colonies on yeast extract sucrose agar (YES); (B,E) Colonies on Blakeslee’s malt extract agar (MEA); (C,F) Colonies on Czapek yeast autolysate agar (CYA); (A–C: obverse view, D–F: reverse view); (G–J) Conidiophores; (K) Conidia (Scale bars: G = 20 μm, H–I = 10 μm, J = 5 μm, K = 2 μm).

≡Citromyces sanguifluus Sopp, Skrifter udgivne af Videnskabs-Selskabet i Christiania. Mathematisk-Naturvidenskabelig Klasse 11: 115 (1912).

Description: Colonies on YES were pale yellow with white mycelium, sparse sporulation, reaching 25–35 mm in diameter after 7 d at 25 °C. On MEA, colonies were muted greenish-blue at the center to white mycelium, colony texture floccose, sporulation sparse, and reaching 25–30 mm in diameter after 7 d at 25 °C. Colonies on CYA grew slow, reaching 25–30 mm in diameter at 25 °C, were pale beige to grey green at the center, had absent to moderate sporulation, and an entire or irregular margin. Sclerotia absent. Conidiophores were monoverticillate with short stipes and measured to be 17–50 μm. Phialides were ampulliform and measured 5.5–8.3 × 2.0–3.0 μm. Conidia were grey-green in color, globose to subglobose and measured 2.0–2.5 μm in diameter.

Taxonomy of CNUFC-PTM72-1

R.A. Samson, N. Yilmaz & J.C. Frisvad, Studies in Mycology 70: 174 (2011) (Table 4, Figure 5).

Table 4.

Morphological characteristics of CNUFC-PTM72-1 compared with reference strain of Talaromyces apiculatus.

Character	CNUFC-PTM72-1	Talaromyces apiculatusa
Conidiophores	Biverticillate, 61–141 × 2.3–3.5 μm	Biverticillate (100–)150–350 × 2.5–3 μm
Phialides	Flask-shaped phialides, three to five per metulae, 7.5–10 × 2.3–3.6 μm	Flask-shaped phialides, three to five per metulae, 9–11 × 2.5–3.5 μm
Conidia	Rough-walled to echinulate, globose, 3.3–4.6 × 3.3–4.3 μm.	Rough-walled to echinulate, globose, 3–4(–5.5)×3–4(–5) μm
Ascomata	Not observed	Not observed

From the description by Yilmaz et al. [11].

Figure 5.

Morphology of Talaromyces apiculatus. (A,D) Colonies on yeast extract sucrose agar (YES); (B,E) Colonies on Blakeslee’s malt extract agar (MEA); (C,F) Colonies on Czapek yeast autolysate agar (CYA); (A–C: obverse view, D–F: reverse view); (G–J) Conidiophores; (K) Conidia (Scale bars: G–J = 20 μm, K = 5 μm).

≡Penicillium aculeatum var. apiculatum Abe, S., J. Gen. Appl. Microbiol., Tokyo 2: 124 (1956).

Description: Colonies on CYA were moderately deep, had radial, low margins, white mycelia, floccose texture, absent to sparse sporulation, no soluble pigment, and reached 22–23 mm in diameter after 7 d at 25 °C. On MEA, colonies were moderately deep, slightly raised at the center, had white mycelia, floccose texture, no soluble pigment, their reverse was brownish orange, and they reached 24–25 mm in diameter. On YES, colonies were moderately deep, low margins, white mycelia, no sporulation, no soluble pigment, and reaching 30–32 mm in diameter. Conidiophores were monoverticillate and measured to be 61–141 × 2.3–3.5 μm. Phialides were flask-shaped and measured 7.5–10 × 2.3–3.6 μm. Conidia were globose with rough echinulate walls and measured 3.3–4.6 × 3.3–4.3 μm in diameter.

Taxonomy of CNUFC-YJW3-31

(Kamyschko) N. Yilmaz, J.C. Frisvad & R.A. Samson, Studies in Mycology 78: 266 (2014) (Table 5, Figure 6).

Table 5.

Morphological characteristics of CNUFC-YJW3-31 compared with reference strain of Talaromyces liani.

Character	CNUFC-YJW3-31	Talaromyces liania
Conidiophores	Monoverticillate and biverticillate	Monoverticillate and biverticillate
Phialides	Acerose, three to six per metula, 10–17.5 × 2–3.5 μm	Acerose, three to six per metulae, 9–20 × 2–3.5 μm
Conidia	Globose to ellipsoidal, 2.5–4.0×2–3.5 μm.	Ellipsoidal, 2.5–4(–4.5)×2–3.5 μm.
Ascomata	Yellow, globose to subglobose, 110–429.5 × 109–428.5 μm	Yellow to orange red, globose to subglobose, 150–550 × 150–545 μm.
Asci	Globose to subglobose or slightly ellipsoidal, 9.5–11.5 × 8.0–10.0 μm	9–13 × 7.5–11 μm
Ascospores	Ellipsoidal, spiny, 3.5 –5.5 × 3.0–4.5 μm	Ellipsoidal, spiny, 4–6 × 2.5–4 μm

From the description by Yilmaz et al. [11].

Figure 6.

Morphology of Talaromyces liani. (A,D) Colonies on yeast extract sucrose agar (YES); (B,E) Colonies on Blakeslee’s malt extract agar (MEA); (C,F) Colonies on Czapek yeast autolysate agar (CYA); (A–C: obverse view, D–F: reverse view); (G) Ascomata on MEA; (H) Asci and ascopsores; (I) Ascopsores; (J) Conidiophore; (K) Conidia (Scale bars: G = 100 μm, H–K = 20 μm).

≡Penicillium liani Kamyschko, Not. Syst. Crypt. Inst. Bot. Acad. Sci. USSR 86: 115 (1962).

Description: Colonies were light yellow, with sparse sporulation, no soluble pigment, and their reverse was pastel yellow on CYA after 7 d at 25 °C. On MEA, colonies formed abundant yellow ascomata, had sparse sporulation, no soluble pigments, and their reverse was greyish yellow. Colonies were deep yellow on their reverse, had poor sporulation, white mycelium, and no soluble pigments on YES after 7 d at 25 °C. Conidiophores were monoverticillate and biverticillate, 2.0–4.5 μm wide. Phialides were acerose, three to six per metula, and measured 10–17.5 × 2–3.5 μm. Conidia were globose to ellipsoidal and measured 2.5–4.0 × 2–3.5 μm. Ascomata occurred within 7 d on MEA at 25 °C, were yellow in color and from globose to subglobose shape, and measured 110–429.5 × 109–428.5 μm. Asci were globose to subglobose or slightly ellipsoidal and measured 9.5–11.5 × 8.0–10.0 μm. Ascospores were ellipsoidal, spiny, and measured 3.5 –5.5 × 3.0–4.5 μm.

Discussion

The use of molecular study based on DNA sequences has dramatically increased the identification of fungal species [37]. DNA sequences, especially those of ITS-5.8S rDNA, have become important features for the rapid identification of fungi [38]. Although ITS sequences are well-accepted barcodes and place Penicillium species into their respective sections, more than 50% of the species could be erroneously linked as many species share the same ITS sequences. This led to the use of secondary markers, such as BenA and CaM, for the correct identification of Penicillium species in section Citrina. These markers act as a hub covering larger databases for the diverse genus Penicillium. Similarly, phylogenetic analyses of BenA genes are imperative for identification of Talaromyces species [11]. Here, we discuss the phylogeny and morphological characteristics of Penicillium and Talaromyces species and compare them to the most closely related species.

There were differences observed in the colony diameter for P. pasqualense and P. sanguifluum in comparison to previous descriptions. Colony diameter on MEA and YES media was shown to be different from the previously described P. pasqualense species (YES: 25–35 mm; MEA: 25–30) and (YES: 40–45 mm; MEA: 30–40) [8,24]. However, cultures on the other media were similar to the described species. In comparison to P. sanguifluum described by Visagie et al. [8], colony growth was shown to be restricted on CYA to 15–26 (vs. 7–16) mm at 25 °C. Also, no sclerotia production was observed for P. pasqualense [8,24]. These differences could be attributed to different soil conditions, seasons of sample collection, or different climate conditions. Another reason could be that the present isolated strain is a sub-cultured strain, because most species produce sclerotia in their first isolated strains [24].

Species of section Citrina have a cosmopolitan distribution and are commonly isolated from soil, but are also isolated from foods, leaf litter, and indoor air. Previously P. pasqualense and P. sanguifluum were reported to be isolated from various habitats; soils, indoor air environments, ants, and chestnuts [8,24]. Members belonging to Penicillium section Citrina are valuable owing to their ability to produce a variety of metabolites including citrinins and citreoviridins [23]. Many specific extrolites were extracted and identified from P. pasqualense and P. sanguifluum such as pyrenocines, indol alkaloids, PAS, bisanthrons, roseopurpurin, β -hydroxycurvularin, dehydrocurvularin, curvularin, FOSI, FYKS, SNIT, TIDL, and VERN [24]. The members of section Citrina can grow at temperatures ranging from 23 to 26 °C [24]. In this study, the two isolates had slow growth at lower temperatures (5–10 °C) and optimum growth at 25–30 °C, whereas growth was restricted at higher temperatures 35–37 °C for P. pasqualense and P. sanguifluum.

In comparison to T. apiculatus [11], the present isolate CNUFC-PTM72-1 shows similar morphological characteristics with small length conidiophores. The morphological characteristics of the T. liani isolate studied were generally similar to those previously described by Yilmaz et al. [11]. However, our observations showed that the size of asci and ascomata of isolate CNUFC-YJW3-31 were smaller than previously reported. Sexual reproduction structures were only observed on MEA. There were previously only reports of T. apiculatus and T. liani from soil samples [11,39]. This is the first isolation of T. liani from freshwater. T. apiculatus produces various bioactive extrolites such as macrocyclic polylactones (apiculides) NG-011, NG-012 (potentiators of nerve growth factors); BK223-A (=NG-012), BK-223B, BK-223C, 15G256B, and 15G256a-2 (act as antifungal) and bioxanthracene B, whereas no available information on the metabolites produced by T. liani yet [11].

Four species were identified, namely P. pasqualense, P. sanguifluum, T. apiculatus, and T. liani, successfully combining molecular and morphological analyses in this study. The main focus was to describe the diversity of Penicillium and Talaromyces species from soil and freshwater in Korea. The soil ecosystem has a tremendous impact on environmental sustainability in forestry, agriculture, and horticulture. Therefore, it is necessary to identify fungal species from soil and to characterize their role in the environment. Thus, our future studies would include studying the species ecological roles, such as the production of extracellular enzyme activity, antibacterial and antifungal activities, as well as the production of secondary metabolites.