Isolation and Characterization of Four Unreported Penicillium Species Isolated from the Freshwater Environments in Korea

Abstract

The fungal species of the genus Penicillium can be found across a diverse array of environments. The infrageneric classification of the genus Penicillium has been studied with comparison of morphological and phylogenetical features, derived into two subgenus, 32 sections, and 89 series. In this study, 11 fungal strains were isolated from freshwater environments, plant litter, and nearby substrates in Korea and were identified as previously unreported species. The internal transcribed spacer (ITS) regions, β-tubulin (BenA), calmodulin (CaM), and RNA polymerase II subunit (RPB2) genes were analyzed for phylogenetic analyses. A neighbor-joining tree was then constructed using the concatenated DNA sequences, and the strains were compared with closely related species of the genus Penicillium. The strain clustered into distinct phylogenetic lineages, confirming their classification as P. contaminatum, P. jinfoshanicum, P. xuanhanense, and P. soppii. NNIBRFG40229 exhibits monoverticillate conidiophores with flask-shaped phialides, characteristic of P. contaminatum; NNIBRFG1595 presents divaricate conidiophores, consistent with P. jinfoshanicum; NNIBRFG5602 shows a velutinous texture with orange pigmentation, resembling P. xuanhanense; and NNIBRFG4602 shows biverticillate conidiophores with cylindrical metulae, corresponding to P. soppii. This study provides the first report of these species in Korea, enhancing taxonomic understanding.

1. Introduction

The genus Penicillium is a widely recognized and prevalent fungal genus found in a vast array of environments, including soil, plants, air, indoor spaces, and various food items [1–3]. Due to its widespread presence across the globe, this species has been isolated from various environments [4]. In 1809, the name of Penicillium is derived from “penicillus”, which means “little brush” [5]. The classification of Penicillium was started in 1901 introducing a three subgeneric classification including Aspergilloides, Biverticillium, and Eupenicillium [6]. Since its first introduction, the genus Penicillium has undergone multiple changes in classification systems from a classification system based on cultural characteristics to a classification system based on phylogenetic analysis [1,7,8]. Following the recent classification system, the teleomorphs or anamorphs of Penicillium had been changed into a system based on DNA sequence data deriving the genus Penicillium into two subgenera, 32 sections, and 89 series [9]. In the genus Penicillium, infrageneric ranks such as subgenera and sections are crucial for organizing its numerous species, which aids in understanding their evolutionary relationships and practical applications. This classification stabilizes the structure of the genus, enabling more straightforward identification and study of its many species [9]. At the time of this writing, approximately 535 species were discovered in the genus Penicillium from all over the world [10]. In Korea, native species belonging to the genus Penicillium are being studied and reclassified according to the principle of “one fungus-one name” [11]. Korean Penicillium species records in GenBank were inaccurate due to the reliance of ITS sequence data, thus reevaluation was conducted by using the concatenated sequences of not only ITS regions, but also BenA, CaM, and RPB2 sequences to analysis for a more precise identification [12]. The aim of this study was to investigate the diversity of Penicillium strains from diverse environments in Korea, and the isolated strains were identified for the potential research. The morphological and molecular characteristics of isolated strains were recorded.

2. Materials and methods

2.1. Sample collection and fungal isolation

Samples from soil, water, and plants were collected from various locations in Korea (Table 1). Fungi were then isolated from the collected samples using the serial dilution method as described in a previous study [13]. Various fungal strains were isolated from different provinces. From the isolates, 11 fungal strains were chosen and then used for morphological, cultural, and phylogenetical analyses. Each strain was deposited at the Nakdonggang National Institute of Biological Resources under the accession numbers NNIBRFG25747, NNIBRFG40229, NNIBRFG1595, NNIBRFG1963, NNIBRFG5170, NNIBRFG44548, NNIBRFG48023, NNIBRFG5602, NNIBRFG5768, NNIBRFG25960, and NNIBRFG4602, respectively (Table 1).

Table 1.

Information of Penicillium isolates used in this study.

Species	Strain	Source	Year of isolation	Location
Penicillium contaminatum	NNIBRFG25747	Water	2019	Sincheon-ri, Hanbando-myeon, Yeongwol-gun, Gangwon, South Korea (37° 13′ 35″ N 128° 20′ 18″ E)
	NNIBRFG40229	Plant litter	2022	Sinjeom-ri, Yongmun-myeon, Yangpyeong-gun, Gyeonggi, South Korea (37° 32′ 42″ N 127° 35′ 2.45″ E)
Penicillium jinfoshanicum	NNIBRFG1595	Sediment	2016	Deoksan-ri, Daedeok-myeon, Gimcheon-si, Gyeongbuk, South Korea (35° 55′ 52.1″ N 127° 54′ 23″ E)
	NNIBRFG1963	Sediment	2016	Bugok-ri, Cheongsong-eup, Cheongsong-gun, Gyeongbuk, South Korea (36° 26′ 22.8″ N 129° 5′ 25.7″ E)
	NNIBRFG5170	Sediment	2019	Singung-ri, Naebuk-myeon, Boeun-gun, Chungbuk, South Korea (36° 30′ 35″ N 127° 38′ 5″ E)
	NNIBRFG44548	Sediment	2022	Mangmi-ri, Jipyeong-myeon, Yangpyeong-gun, Gyeonggi, South Korea (37° 25′ 54″ N 127° 39′ 50″ E)
	NNIBRFG48023	Water	2022	Dae-ri, Yeonghae-myeon, Yeongdeog-gun, Gyeongbuk, South Korea (36° 32′ 6″ N 129° 15′ 39″ E)
Penicillium xuanhanense	NNIBRFG5602	Water	2018	Singung-ri, Naebuk-myeon, Boeun-gun, Chungbuk, South Korea (36° 30′ 35″ N 127° 38′ 5″ E)
	NNIBRFG5768	Sediment	2019	Cheongpyeong-ri, Buksan-myeon, Chuncheon-si, Gangwon, South Korea (37° 59′ 1″ N 127° 49′ 5″ E)
	NNIBRFG25960	Water	2020	Jikdong-ri, Sohol-eup, Pocheon-si, Gyeonggi, South Korea (37° 44′ 56.3″ N 127° 9′ 57.8″ E)
Penicillium soppii	NNIBRFG4602	Cypripedium macranthum	2018	Gohan-ri, Gohan-eup, Jeongseon-gun, Gangwon, South Korea (37° 8′ 57.4″ N 128° 54′ 10.8″ E)

2.2. Cultural and morphological characterization

To observe cultural characteristics, the isolates were cultured on four different media. The strains were cultured at three points on potato dextrose agar (PDA; Difco, Detroit, MI), malt extract agar (MEA; Difco, Detroit, MI), Czapek yeast extract agar (CYA; MB Cell, Seoul, South Korea), and yeast extract sucrose agar (YES; yeast extract, 4 g; sucrose, 20 g; KH₂PO₄, 1 g; MgSO₄, 0.5 g; agar, 15 g; distilled H₂O, 1000 mL), then incubated at 25 °C for seven days [14]. The cultures were maintained in darkness, and various characteristics were observed, including the size, color, and shape of the mycelium, as well as morphological features such as conidiophore, stipe, metula, phialide, conidia, and the arrangement of conidia. A light microscope (BX-50; Olympus, Tokyo, Japan) was used to study the morphological properties.

2.3. Genomic DNA extraction, PCR amplification, and sequencing

Total genomic DNA from each strain was collected from growing colony on PDA and extracted using the HiGene™ Genomic DNA Prep Kit (Biofact, Daejeon, South Korea) according to the manufacturer’s instructions. The internal transcribed spacer (ITS) regions, β-tubulin (BenA), calmodulin (CaM), and RNA polymerase II subunit (RPB2) genes were amplified using the primer pairs ITS1F/ITS4, Bt2a/Bt2b, CMD5/CMD6, and RPB2-5f/RPB2-7cR, respectively [15–18]. Amplification was confirmed by electrophoresis using HP Agarose (BIOPURE, Cambridge, MA) 1.0% gels. Amplified products were purified using ExoSAP-IT (Thermo Fisher Scientific, Waltham, MA) and sequencing services were provided by Macrogen (Seoul, South Korea).

2.4. Phylogenetic analyses

Taxa for phylogenetic analysis and outgroups were chosen according to previous study [10], and additional related species were added according to the Basic Local Alignment Search Tool (BLAST) results. Sequences were downloaded from the National Center for Biotechnology Information (NCBI) database (Table 2). Phylogenetic trees were constructed from the concatenated sequences of the ITS regions, BenA, CaM, and RPB2 using the neighbor-joining (NJ) method in MEGA version 11.0 [19,20]. The evolutionary distance matrices for the NJ analysis were generated according to Kimura’s two-parameter model with bootstrap values based on 1000 replications [21].

Table 2.

GenBank accession numbers used for phylogenetic analyses in this study.

Species	Strain number	GenBank accession number
		ITS	BenA	RPB2	CaM
Penicillium aurantioviolaceum	CBS 137777T	KM189756	KM089005	KM089779	KM089392
Penicillium austroafricanum	CBS 137773T	KM189610	KM088854	KM089628	KM089241
Penicillium austrosinicum	CGMCC 3.18410T	KX885061	KX885041	KX885032	KX885051
Penicillium cainii	DAOM 239914T	JN686435	JN686366	MT156346	JN686389
Penicillium cartierense	CBS 137956T	KM189564	KM088804	KM089576	KM089189
Penicillium chroogomphum	CBS 136204T	KC594043	KP684056	MN969167	KP684057
Penicillium contaminatum	CBS 346.59T	KM189782	KM089032	KM089806	KM089419
Penicillium contaminatum	NNIBRFG25747	PQ771855	PQ772837	PQ772848	PQ772859
Penicillium contaminatum	NNIBRFG40229	PQ771856	PQ772838	PQ772849	PQ772860
Penicillium crocicola	CBS 745.70T	KM189581	KJ834445	JN406535	KM089210
Penicillium exsudans	HMAS 248735T	KX885062	KX885042	KX885033	KX885052
Penicillium fusisporum	CBS 137463T	KF769424	KF769400	MN969117	KF769413
Penicillium grevilleicola	CBS 137775T	KM189630	KM088874	KM089648	KM089261
Penicillium guanacastense	DAOM 239912T	JN626098	JN625967	KX961295	JN626010
Penicillium jejuense	CBS 138646T	KF818464	KF818461	KF818467	KF818470
Penicillium jinfoshanicum	CS12-10T	OQ870813	OR051074	OR051425	OR051253
Penicillium jinfoshanicum	NNIBRFG1595	PQ771857	PQ772839	PQ772850	PQ772861
Penicillium jinfoshanicum	NNIBRFG1963	PQ771858	PQ772840	PQ772851	PQ772862
Penicillium jinfoshanicum	NNIBRFG5170	PQ771859	PQ772841	PQ772852	PQ772863
Penicillium jinfoshanicum	NNIBRFG44548	PQ771861	PQ772843	PQ772854	PQ772865
Penicillium jinfoshanicum	NNIBRFG48023	PQ771860	PQ772842	PQ772853	PQ772864
Penicillium lenticrescens	CBS 138215T	KJ775675	KJ775168	MN969123	KJ775404
Penicillium mallochii	DAOM 239917T	JN626104	JN625973	KX961296	JN626016
Penicillium maximae	CBS 134565T	EU427298	KC773795	MN969126	KC773821
Penicillium meliponae	CBS 142495T	MF278315	MN969418	LT854653	LT854648
Penicillium roseoviride	CBS 267.35T	KM189549	KM088787	KM089559	KM089172
Penicillium sclerotiorum	IMI 40569T	JN626132	JN626001	JN406585	JN626044
Penicillium soppii	CBS 226.28T	AF033488	MN969399	JN406606	KJ867002
Penicillium soppii	NNIBRFG4602	PQ771865	PQ772847	PQ772858	PQ772869
Penicillium thomii	CBS 225.81T	KM189560	KM088799	KM089571	KM089184
Penicillium valentinum	CBS 172.81T	KM189550	KM088788	KM089560	KM089173
Penicillium xuanhanense	CS31-04T	OQ870873	OR051222	OR062086	OR051396
Penicillium xuanhanense	NNIBRFG5602	PQ771863	PQ772845	PQ772856	PQ772867
Penicillium xuanhanense	NNIBRFG5768	PQ771862	PQ772844	PQ772855	PQ772866
Penicillium xuanhanense	NNIBRFG25960	PQ771864	PQ772846	PQ772857	PQ772868
Penicillium yezoense	CBS H-21863T	KM189553	KM088792	KM089564	KM089177
Hamigera avellanea	CBS 295.48T	AF454075	EU021664	EU021627	EU021682

ITS: internal transcribed spacer regions; BenA: β-tubulin gene; RPB2: RNA polymerase II subunit gene; CaM: calmodulin gene.

^TType strain. The strains isolated in this study are indicated in bold.

3. Results

3.1. Phylogenetic analysis

The phylogenetic relationships of the isolated Penicillium species were assessed through BLASTn sequence similarity searches and phylogenetic tree reconstruction based on concatenated sequence datasets of four loci (ITS, BenA, CaM, and RPB2). BLASTn analysis revealed that strains NNIBRFG25747 and NNIBRFG40229 shared high sequence similarity with P. contaminatum CBS 345.52^T exhibiting high sequence similarity across multiple loci (ITS: 99.7–99.8%, BenA: 100%, CaM: 99.8%, and RPB2: 100%; bootstrap support = 100%), while NNIBRFG1595, NNIBRFG1963, NNIBRFG5170, NNIBRFG44548, and NNIBRFG48023 were closely related to P. jinfoshanicum CS12-10^T, showing high sequence similarity (ITS: 100%, BenA: 99.7–99.8%, CaM: 99.4–99.8%, and RPB2: 99.9–100%; bootstrap support = 100%). Similarly, NNIBRFG5602, NNIBRFG5768, and NNIBRFG25960 exhibited strong sequence similarity to P. xuanhanense CS31-04^T (sequence similarity for ITS = 100%, BenA = 100%, CaM = 100%, and RPB2 = 100%; bootstrap support = 100%), and NNIBRFG4602 showed a close phylogenetic relationship with P. soppii CBS 226.28^T, sharing high sequence similarity (ITS: 100%, BenA: 100%, CaM: 100%, and RPB2: 99.7%; bootstrap support = 100%). The phylogenetic tree, reconstructed using the NJ method, demonstrated that these isolates formed well-supported monophyletic groups with their respective reference strains. The concatenated alignment consisted of 1883 nucleotides (ITS: 352 bp, BenA: 366 bp, CaM: 404 bp, and RPB2: 761 bp), with consistently high bootstrap support values, confirming the taxonomic placement of the isolates (Figure 1).

Figure 1.

Neighbor-joining phylogenetic tree based on a combined dataset of partial sequences of ITS regions, BenA, RPB2, and CaM sequences, the different series of Penicillium, namely, Thomiorum, Sclerotiorum, and Soppiorum. Hamigera avellanea CBS 295.48^T was used as an outgroup. The numbers above/below the branches indicate bootstrap values (>70%) obtained from 1000 replicates. The unrecorded Penicillium species in Korea are highlighted in bold red. Bar = 0.02 substitutions per nucleotide position.

3.2. Taxonomy

Penicillium contaminatum Houbraken, Studies in Mycology 78: 419 (2014) [MB#809962]

Strains NNIBRFG25747 and NNIBRFG40229 were found to be morphologically identical, and they clustered together with P. contaminatum CBS 345.52^T in respect to the molecular phylogeny. Thus, in this study, only the cultural and morphological characteristics of strain NNIBRFG40229 were described in this study since they were identical.

Cultural characteristics: On PDA at 25 °C for 7 d: moderately deep and radially sulcate with circular colonies having white mycelium; dull green conidia; margins entire; texture is velvety; no exudate present; no soluble pigments detected; coloration of reverse is white and pale (Figure 2(A)). On MEA at 25 °C for seven days: moderately deep and radially sulcate with circular colonies having white mycelium; dull green conidia; margins entire; texture is velvety to floccose; no exudate present; no soluble pigments detected; coloration of reverse is beige (Figure 2(A)). On CYA at 25 °C for seven days: moderately deep and radially sulcate with circular colonies having white mycelium; grey green conidia; margins entire to slightly irregular; texture is velvety; no exudate present; no soluble pigments detected; coloration of reverse is beige to yellow (Figure 2(A)). On YES at 25 °C for seven days: moderately deep and radially sulcate with circular colonies having white mycelium; dull grey conidia; margins entire; texture is velvety; no exudate present; no soluble pigments detected; coloration of reverse is beige (Figure 2(A)). Colony diameters after seven days at 25 °C are as follows: PDA 46–48; MEA 43–45; YES 44–48; CYA 45–46 (Figure 2).

Figure 2.

Morphological characteristics of Penicillium contaminatum NNIBRFG40229. (A) Colonies after seven days at 25 °C, from left to right (top row) PDA obverse, MEA obverse, CYA obverse, YES obverse; (bottom row) PDA reverse, MEA reverse, CYA reverse, YES reverse; (B, C) Conidiophore consists of the stipe (black arrows) and phialide (red arrows); (D) Conidia; scale bars = 10 µm.

Morphological characteristics: Conidiophores monoverticillate (Figure 2(B,C)); stipes rough walled, 155–230 × 2.6–3.6 µm (Figure 2(B,C)); phialides ampulliform with short narrow neck, 9.1–11.2 × 2.5–3.5 µm (Figure 2(B,C)); conidia long irregular columns, ellipsoidal, 3.3–4.2 × 2.8–3.3 µm (Figure 2(D)).

Habitat: Plant litter in freshwater.

Specimen examined: Sinjeom-ri, Yongmun-myeon, Yangpyeong-gun, Gyeonggi, South Korea; April 14 2022; NNIBRFG40229 (ITS = PQ771856; BenA = PQ772838; CaM = PQ772860; RPB2 = PQ772849).

Note: Penicillium contaminatum was initially reported in 2014, isolated from the contaminant in the UK [22]. Comparing the Korean P. contaminatum NNIBRFG40229 and CBS 345.52^T, the cultural characteristics from MEA, CYA, and YES media are similar, but P. contaminatum isolated in Korea tends to grow slower than CBS 345.52^T grown on CYA (45–46 vs. 42–55 mm), MEA (43–45 vs. 46–50 mm), and YES (44–48 vs. 51–57 mm) (Figure 2(A)) [22]. For the morphological characteristics, both strains exhibit short, narrow, flask-shaped phialides at the ends of monoverticillate conidiophores (Figure 2(B,C)), producing long irregular columns or ellipsoidal conidia (Figure 2(D)) [22].

Penicillium jinfoshanicum X.C. Wang & W.Y. Zhuang, J. Fungi 9 (12, no. 1150): 79 (2023) [MB#571549]

Strains NNIBRFG1595, NNIBRFG1963, NNIBRFG5170, NNIBRFG44548, and NNIBRFG48023 were found to be morphologically identical, and they clustered together with P. jinfoshanicum CS12-10^T in respect to the molecular phylogeny. Thus, in this study, only the cultural and morphological characteristics of strain NNIBRFG1595 were described in this study since they were identical.

Cultural characteristics: On PDA at 25 °C for seven days: plain and protuberant at centers with circular colonies having white mycelium; dull green conidia; margins entire; texture is velutinous; no exudate present; no soluble pigments detected; coloration of reverse is cream to yellow (Figure 3(A)). On MEA at 25 °C for seven days: plain with nearly circular colonies having white mycelium; dull green conidia; margins slightly irregular; texture is velutinous to floccose; no exudate present; no soluble pigments detected; coloration of reverse is cream (Figure 3(A)). On CYA at 25 °C for seven days: protuberant at centers and radially sulcate with circular colonies having white mycelium; dull green conidia; margins entire; texture is velutinous; no exudate present; no soluble pigments detected; coloration of reverse is cream to yellow (Figure 3(A)). On YES at 25 °C for seven days: radially sulcate with nearly circular colonies having white mycelium; margins fimbriate; texture is velutinous to floccose; no exudate present; no soluble pigments detected; coloration of reverse is yellow (Figure 3(A)). Colony diameters after seven days at 25 °C are as follows: PDA 45–46; MEA 40–42; CYA 46–48; YES 56–57 (Figure 3).

Figure 3.

Morphological characteristics of Penicillium jinfoshanicum NNIBRFG1595. (A) Colonies after seven days at 25 °C, from left to right (top row) PDA obverse, MEA obverse, CYA obverse, YES obverse; (bottom row) PDA reverse, MEA reverse, CYA reverse, YES reverse; (B, C) Conidiophore consists of the stipe (black arrows) and phialide (red arrows); (D) Conidia; scale bars = 10 µm.

Morphological characteristics: Conidiophores monoverticillate, occasionally divaricate (Figure 3(B,C)); stipes rough walled, 60–170 × 3.2–3.7 µm (Figure 3(B,C)); phialides acerose to ampulliform, tapering into very thin neck, 9.0–13.2 × 2.5–3.3 µm (Figure 3(B,C)); conidia narrow ellipsoidal, smooth walled, 3.4–3.8 × 2.5–3.3 µm (Figure 3(D)).

Habitat: Sediment in freshwater.

Specimen examined: Gam-cheon, Deoksan-ri, Daedeok-myeon, Gimcheon-si, Gyeongbuk, South Korea; March 23 2016; NNIBRFG1595 (ITS = PQ771857; BenA = PQ772839; CaM = PQ772861; RPB2 = PQ772850).

Note: Penicillium jinfoshanicum was first reported in 2023, isolated from the soil in China [23]. Comparing the Korean P. jinfoshanicum NNIBRFG1595 and CS12-10^T, the cultural characteristics from PDA, MEA, CYA, and YES media are similar; however, the strain NNIBRFG1595 exhibits slower growth on PDA (45–46 vs. 49–51 mm), but faster growth on YES (56–57 vs. 52–53 mm) (Figure 3(A)) [23]. For the morphological characteristics, both strains exhibit short, flask-shaped phialides at the ends of monoverticillate conidiophores (Figure 3(B,C)), producing narrow ellipsoidal of conidia (Figure 3(D)) [23].

Penicillium xuanhanense X.C. Wang & W.Y. Zhuang, J. Fungi 9 (12, no. 1150): 127 (2023) [MB#571574]

Strains NNIBRFG5602, NNIBRFG5768, and NNIBRFG25960 were found to be morphologically identical, and they clustered together with P. xuanhanense CS31-04^T in respect to the molecular ­phylogeny. Thus, in this study, only the cultural and morphological characteristics of strain NNIBRFG40229 were described in this study since they were identical.

Cultural characteristics: On PDA at 25 °C for seven days: plain with circular colonies having white mycelium; grayish green conidia; margins entire; texture is velutinous; no exudate present; no soluble pigments detected; coloration of reverse is yellow to orange (Figure 4(A)). On MEA at 25 °C for seven days: protuberant at centers with circular colonies having white mycelium; dull green conidia; margins entire; texture is velutinous; no exudate present; no soluble pigments detected; coloration of reverse is yellow to red brown (Figure 4(A)). On CYA at 25 °C for seven days: plain and radially sulcate with circular colonies having white mycelium; dull green conidia; margins entire; texture is velutinous; no exudate present; no soluble pigments detected; coloration of reverse is orange to yellow (Figure 4(A)). On YES at 25 °C for seven days: radially sulcate with circular colonies having white mycelium; dull green conidia; margins entire; texture is velutinous; no exudate present; no soluble pigments detected; coloration of reverse is white to red (Figure 4(A)). Colony diameters after seven days at 25 °C are as follows: PDA 22–25; MEA 29–33; CYA 19–21; YES 29–33 (Figure 4).

Figure 4.

Morphological characteristics of Penicillium xuanhanense NNIBRFG5602. (A) Colonies after seven days at 25 °C, from left to right (top row) PDA obverse, MEA obverse, CYA obverse, YES obverse; (bottom row) PDA reverse, MEA reverse, CYA reverse, YES reverse; (B, C) Conidiophore consists of the stipe (black arrows) and phialide (red arrows); (D) Conidia; scale bars = 10 µm.

Morphological characteristics: Conidiophores monoverticillate or divaricate (Figure 4(B,C)); stipes smooth to rough walled, 44.0–145.2 × 2.6–3.3 µm (Figure 4(B,C)); phialides ampulliform to acerose, tapering into very thin neck, 10.0–11.5 × 3.1–3.7 µm (Figure 4(B,C)); conidia narrow ellipsoidal, smooth walled, 2.9–3.7 × 2.0–2.7 µm (Figure 4(D)).

Habitat: Filtered freshwater.

Specimen examined: Singung-ri, Naebuk-myeon, Boeun-gun, Chungbuk, South Korea; March 9 2018; NNIBRFG5602 (ITS = PQ771863; BenA = PQ772845; CaM = PQ772867; RPB2 = PQ772856).

Note: Penicillium xuanhanense was initially reported in 2023, isolated from the soil in China [23]. Comparing the Korean P. xuanhanense NNIBRFG5602 and CS31-04^T, the cultural characteristics from PDA, MEA, CYA, and YES media are similar, but the strain NNIBRFG5602 P. xuanhanense shows slower growth on PDA (22–25 vs. 27–28 mm), YES (29–33 vs. 35–36 mm), and CYA (19–21 vs. 30–31 mm) (Figure 4(A)) [23]. For the morphological characteristics, both strains exhibit long flask-shaped phialides at the ends of monoverticillate conidiophores (Figure 4(B,C)), producing narrow ellipsoidal of conidia (Figure 4(D)) [23].

Penicillium soppii K. Zaleski, Bull. Int. Acad. Polon. Sci., Cl. Sci. Math., Sér. B., Sci. Nat. 1927: 476 (1927) [MB#121424]

Cultural characteristics: On PDA at 25 °C for seven days: flat, and radially sulcate with irregular colonies having white mycelium; dull green conidia; margins undulate; texture is velutinous to floccose; no exudate present; no soluble pigments detected; coloration of reverse is light brown to brown (Figure 5(A)). On MEA at 25 °C for seven days: flat, and radially sulcate with irregular colonies having white mycelium; dull green conidia; margins undulate; texture is velutinous; no exudate present; no soluble pigments detected; coloration of reverse is light brown (Figure 5(A)). On CYA at 25 °C for seven days: flat, and radially sulcate with irregular colonies having white mycelium; margins undulate; dull green conidia; texture is velutinous; no exudate present; no soluble pigments detected; coloration of reverse is light brown (Figure 5(A)). On YES at 25 °C for seven days: flat with irregular colonies having white mycelium; margins undulate; dull green conidia; texture is floccose; no exudate present; no soluble pigments detected; coloration of reverse is light brown to brown (Figure 5(A)). Colony diameters after seven days at 25 °C are as follows: PDA 20–25; MEA 17–21; CYA 27–31; YES 22–26 (Figure 5).

Figure 5.

Morphological characteristics of Penicillium soppii NNIBRFG4602. (A) Colonies after seven days at 25 °C, from left to right (top row) PDA obverse, MEA obverse, CYA obverse, YES obverse; (bottom row) PDA reverse, MEA reverse, CYA reverse, YES reverse; (B, C) Conidiophore consists of the stipe (black arrows), metula (red arrows head), and phialide (red arrows); (D) Conidia; scale bars = 10 µm.

Morphological characteristics: Conidiophores biverticillate (Figure 5(B,C)); stipes smooth walled 320.0–450.0 × 2.5–4.0 µm (Figure 5(B,C)); metula 10.8–13.4 × 2.8–4.4 µm, cylindrical (Figure 5(B,C)); phialides parallel in the cluster, short tapered necks, 8–10 × 2.3–3 µm (Figure 5(B,C)); conidia globose to subglobose, smooth walled, 2.0–2.8 µm (Figure 5(D)).

Habitat: Endophyte from Cypripedium macranthum.

Specimen examined: Gohan-ri, Gohan-eup, Jeongseon-gun, Gangwon, South Korea; October 23 2015; NNIBRFG4602 (ITS = PQ771865; BenA = PQ772847; CaM = PQ772869; RPB2 = PQ772858).

Note: The species Penicillium soppii was first reported in 1927 [24]. However, in Korea, P. soppii was first reported under the synonym P. meleagrinum var. viridiflavum [11]. Furthermore, according to the Mycobank database (http://www.mycobank.org/), it is revealed that P. sumatraense is the current name of P. meleagrinum var. viridiflavum. Hence, the discrepancy supports that P. soppii has not been reported in Korea yet. Comparing the Korean P. soppii NNIBRFG4602 and CBS 226.28^T, the cultural characteristics from MEA, CYA, and YES are similar (Figure 5(A)). For the morphological characteristics, both strains exhibit long flask-shaped phialides at the ends of cylindrical metula (Figure 5(B,C)). Metula are formed on the branching point of biverticillate conidiophores (Figure 5(B,C)), producing globes to subglobose of conidia (Figure 5(D)) [24].

4. Discussion

The morphological and phylogenetic analyses conducted on the four previously unrecorded Penicillium strains in this study revealed significant diversity within the genus, underscoring the potential for discovering unreported species in Korea. Phylogenetic analyses based on the ITS regions, BenA, CaM, and RPB2 sequences successfully classified the strains, placing them into distinct subgenera and sections within Penicillium. In this study, all isolates were obtained from freshwater sources and their nearby substrates, suggesting that these environments may serve as significant habitats for Penicillium species. The isolation of these fungi from aquatic habitats indicates a potential adaptation to the unique conditions. Ongoing taxonomic investigations have further expanded the known diversity of Penicillium, with novel and previously unreported species continuously being identified across various ecological niches, including freshwater ecosystems [25–34]. Recently, a lot of studies have been conducted on the species of Penicillium, and many new species that have not been recorded in aquatic and terrestrial environments in Korea have been continuously reported [25–34]. Among these, P. annulatum, P. camponotum, P. echinulonalgiovense, P. globosum, P. limosum, P. onobense, and P. yunnanense have been identified from freshwater and soil samples in 2021, contributing to the expanding diversity of Penicillium in Korea. Similarly, P. aquadulcis, P. flavigenum, and P. lenticrescens have been isolated from freshwater samples, further enhancing the understanding of their ecological roles in aquatic environments [26]. Throughout this study, these finding adds four new Penicillium species to the current list of native Penicillium species in Korea, and suggests that Korea’s unique environment may harbor more unexplored fungal diversity, further offering insights into regional biodiversity. Consequently, the compiling of data on the genus Penicillium in Korea is essential for advancing our understanding of its adaptations and the potential applications of its bioactive properties [35]. More than 20 species of Penicillium such as P. expansum, P. citrinum, and P. digitatum were reported as the cause of post-harvest disease on crops in Korea [36]. Moreover, recent study indicated that P. labradorum were agents of disseminated fungal disease in a dog [37]. However, the species isolated from this study have not been studied for any pathogenicity tests. Furthermore, the identification of these unreported strains has implications for potential industrial applications, since Penicillium species can be sources of new bioactive compounds. P. rubens was first studied for its production of the antibacterial antibiotic penicillin [38], and the antibiotic production is being continuously studied with other various Penicillium species, such as P. griseofulvin and P. brasiliensis, which produced anti-inflammatory activities, antibiotic enzymes, and other pharmaceutical metabolites [39,40]. Furthermore, some of the Penicillium species such as P. roqueforti and P. camemberti are used in the production of many varieties of blue cheese [38]. Thus, future studies on these strains should focus on the pathogenicity, and biochemical properties to fully assess their potential applications. To the best of our knowledge, this is the first report of P. contaminatum, P. jinfoshanicum, P. xuanhanense, and P. soppii identified in Korea.

Funding Statement

This work was supported by a grant from the Nakdonggang National Institute of Biological Resources (NNIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NNIBR20251105).

Disclosure statement

No potential conflict of interest was reported by the author(s).