Identification and Characterization of Five Previously Unrecorded Penicillium Species of Subgenus Aspergilloides Isolated in Korea

Abstract

In a survey of freshwater and near-freshwater soils in Chungbuk, Gyeonggi, Gangwon, Gyeongbuk, and Gyeongnam provinces in Korea, seven fungal strains were isolated and identified as members of the genus Penicillium based on the internal transcribed spacer (ITS) regions sequence analyses. Identification was performed through observing morphological characteristics and conducting phylogenetic analyses based on concatenated partial ITS, β-tubulin, calmodulin, and RNA polymerase II subunit gene sequences. The phylogenetic analyses revealed that strain NNIBRFG6577 was distinct from known Penicillium species, and based on morphological comparisons with the closest related species, P. scruposum CGMCC 3.25167^T and P. subasperum CGMCC 3.25173^T, it was proposed as a novel species. Strains NNIBRFG4823, NNIBRFG4610, and NNIBRFG4600 were closely related to P. ellipsoideum CGMCC 3.25156^T, P. skrjabinii CBS 439.75^T, and P. yezoense CBS 350.59^T, respectively, while strains NNIBRFG1486, NNIBRFG48324, and NNIBRFG49919 clustered with P. pancosmium CBS 276.75^T, and all strains showed morphological characteristics similar to those of their respective reference strains. These findings enrich Korea’s fungus inventory, providing a basis for valuable future resource development.

1. Introduction

The genus Penicillium was first described by Link with P. candidum, P. expansum, and P. glaucum, the latter of which, designated as the type species, is notable for its production of the antibiotic penicillin [1–3]. These species were characterized by their distinctive brush-like conidiophores, a key feature used for species identification within the genus [1]. Initially, species classification within this genus was based on cultural and morphological characteristics [1,4,5]. Subgenus Aspergilloides is one of the main phylogenetic lineages of genus Penicillium, originally classified by Pitt based on morphology of monoverticillate conidiophores [4]. However, with the development of molecular phylogenetic techniques, particularly analyses incorporating multiple marker loci, such as the ITS regions and the β-tubulin (BenA), calmodulin (CaM), and RNA polymerase II subunit (RPB2) genes, species delineation within Penicillium has become more precise [6–8]. With the adoption of polyphasic classification, the genus has been divided into two subgenus, 32 sections, 89 series, and 535 accepted species [7]. Thus, modern classification of subgenus Aspergilloides encompasses species having monoverticillate, biverticillate, and terverticillate conidiophores, but is distinguishable from subgenus Penicillium by its simpler and less organized branching patterns, typically lacking the complex, symmetric terverticillate arrangements that characterize the subgenus Penicillium [4,5,7]. In addition, Penicillium species have been isolated from a wide range of substrates, including soil, aquatic habitats, air, indoor environments, and food products [9–11]. Notably, 160 Penicillium species have been reported in Korea, highlighting the rich fungal diversity of the region and the need for the continued exploration of uncharacterized native species [12]. Beyond their ecological diversity, many species within this genus have economic significance [13–15]. While some are recognized as pathogens, others play important roles in fermentation and antibiotic production [13–15]. Penicillium digitatum, P. crustosum, P. expansum, and P. solitum are primary postharvest pathogens responsible for fruit decay [13]. In contrast, P. camemberti and P. roqueforti are widely employed in cheese production, while P. rubens and P. griseofulvum remain key penicillin-producing species [2,14,15]. Given the wide range of habitats from which Penicillium can be isolated, there is significant potential for the discovery of previously unrecorded or novel species exhibiting unique metabolic capabilities with biotechnological applications.

To secure domestic fungal resources for applications in agriculture and biotechnology, ongoing research in Korea is focused on discovering unreported and novel Penicillium species in air, soil, freshwater, and other environments [16–22]. Such research is expected to not only advance fungal taxonomy but also lead to the discovery of industrially valuable enzymes and bioactive compounds. This study aimed to expand the catalog of Penicillium species in Korea’s soils, establishing a valuable resource for future mycological research and biotechnological applications. Isolated strains were identified based on molecular, cultural, and morphological characteristics.

2. Materials and methods

2.1. Sample collection and fungal isolation

Fungal strains used in this study were isolated from freshwater, stream sediments and Cypripedium guttatum-associated sediments collected from Chungbuk (36° 59′ 38′′ N 128° 13′ 45′′ E), Gyeonggi (37° 59′ 13.3′′ N 127° 27′ 5.7′′ E), Gangwon (37° 8′ 57.4′′ N 128° 54′ 10.8′′ E), Gyeongbuk (36° 17′ 36.2′′ N 128° 50′49.6′′ E, and 36° 47′7.87′’′N 128° 9′16.63′′ E), and Gyeongnam (35° 8′ 54.1′′ N 127° 51′ 13.8′′ E) provinces, Korea. Isolation was performed using methods appropriate for each sample type (freshwater or Cypripedium guttatum-associated), as described in a previous study [23]. Among the isolated fungal strains, seven were chosen for cultural, morphological, and phylogenetic analyses. The strains used in this study were deposited at the Nakdonggang National Institute of Biological Resources under the accession numbers NNIBRFG6577, NNIBRFG4823, NNIBRFG4610, NNIBRFG4600, NNIBRFG1486, NNIBRFG48324, and NNIBRFG49919.

2.2. Cultural and morphological characterization

Isolated strains were cultured on various media to observe their cultural characteristics. All strains were cultured at three points on Czapek yeast extract agar (CYA; MB Cell, Seoul, Korea), yeast extract sucrose agar (YES; 4 g of yeast extract, 20 g of sucrose, 1 g of KH₂PO₄, 0.5 g of MgSO₄, and 15 g of agar in distilled water to 1000 mL), and creatine sucrose agar (CREA; 30 g of sucrose, 3 g of creatine, 1.6 g of K₃PO₄·7H₂O, 0.5 g of MgSO₄·7H₂O, 0.5 g of KCl, 0.01 g of FeSO₄·7H₂O, 1 mL of trace elements stock solution, 0.05 g of bromocresol purple, and 20 g of agar in distilled water to 1000 mL) [6]. Additional cultural media were used for five strains. Strains NNIBRFG6577 and NNIBRFG4823 were also cultured on potato dextrose agar (PDA; Difco, Detroit, MI), malt extract agar (MEA; Difco, Detroit, MI), and dichloran 18% glycerol agar (DG18; 31.5 g of dichloran glycerol agar base, 220 g of glycerol, 1 mL of trace element stock solution, 0.05 g of chloramphenicol, and 20 g of agar in distilled water to 1000 mL) [6], while strains NNIBRFG4610 and NNIBRFG4600 were cultured on MEA and strain NNIBRFG1486 was cultured on PDA. All cultures were incubated at 25 °C for 7 d in darkness [6]. Cultural characteristics, including the size and color of the colonies, were observed. A light microscope (BX-50; Olympus, Tokyo, Japan) was used to study the morphological characteristics of diagnostic fungal structures, including conidiophores, stipes, metulae, phialides, and conidia.

2.3. Genomic DNA extraction, PCR amplification, and sequencing

Using tissue from strains cultured on PDA, DNA was extracted with the HiGene^™ Genomic DNA Prep Kit for fungi (Biofact, Daejeon, Korea) following the manufacturer’s instructions. The primer pairs ITS1F/ITS4, Bt2a/Bt2b, CMD5/CMD6, and RPB2-5f/RPB2-7cR were used to amplify the internal transcribed spacer (ITS) regions, β-tubulin (BenA), calmodulin (CaM), and RNA polymerase II subunit (RPB2) genes, respectively [24–27]. To confirm successful amplification, electrophoresis was performed using 1.0% HP Agarose (BIOPURE, Cambridge, MA) gels. ExoSAP-IT (Thermo Fisher Scientific, Waltham, MA) was used to purify amplified products, and sequencing services were provided by Macrogen (Seoul, Korea).

2.4. Phylogenetic analyses

The isolated strains were preliminarily identified by identifying related species in the NCBI GenBank database using the Basic Local Alignment Search Tool (BLAST). For phylogenetic analyses, closely related fungal strains of seven isolated strains were obtained from the NCBI GenBank database (Table 1). The sequences were aligned using the ClustalW alignment tool in Mega11 and gaps were removed [28]. Then, phylogenetic analyses based on the maximum likelihood (ML) method were conducted in MEGA11 and Bayesian inference (BI) with MrBayes version 3.2.7 based on the concatenated sequences of the four loci (ITS, BenA, CaM, and RPB2) [28,29]. The ML phylogenetic tree was constructed using the general time reversible model in MEGA11, and bootstrap values were generated based on 1000 replicates [28]. BL analysis was performed with four Markov Chain Monte Carlo (MCMC) using the GTR + I + G model. Four MCMC were run for 10 million generations, sampling every 1000 generations. Posterior probabilities were calculated after discarding the first 25% of samples as burn-in. The consensus tree was viewed in FigTree version 1.4.4 [30].

Table 1.

Strain and GenBank accession numbers of the sequences used for the phylogenetic analyses in this study

Species	Strain ID	GenBank accession number
		ITS	BenA	CaM	RPB2
Penicillium additum	CGMCC 3.25145T	OQ870831	OR051180	OR051355	OR062046
Penicillium alagoense	URM 93058T	MK804503	MK802333	MK802336	MK802338
Penicillium araracuaraense	CBS 113149T	GU981597	GU981642	MN969237	KF296414
Penicillium atrofulvum	CBS 109.66T	JN617663	JN606677	JN606387	JN606620
Penicillium aurantiacobrunneum	CBS 126228T	JN617670	JN606702	MN969238	MN969106
Penicillium aurantioviolaceum	CBS 137777T	KM189756	KM089005	KM089392	KM089779
Penicillium austroafricanum	CBS 137773T	KM189610	KM088854	KM089241	KM089628
Penicillium brachycaulis	CGMCC 3.25148T	OQ870832	OR051181	OR051356	OR062047
Penicillium brasilianum	CBS 253.55T	GU981577	GU981629	MN969239	KF296420
Penicillium cairnsense	CBS 124325T	JN617669	JN606693	MN969240	MN969108
Penicillium cartierense	CBS 137956T	KM189564	KM088804	KM089189	KM089576
Penicillium cataractarum	CBS 140974T	KT887847	KT887808	KT887769	MN969180
Penicillium celere	CGMCC 3.25172T	OQ870848	OR051197	OR051372	OR062062
Penicillium choerospondiatis	CGMCC 3.18411T	KX885063	KX885043	KX885053	KX885034
Penicillium christenseniae	CBS 126236T	JN617674	JN606680	MN969243	JN606624
Penicillium chrzaszczii	CBS 217.28T	GU944603	JN606758	MN969244	JN606628
Penicillium contaminatum	CBS 345.52T	KM189554	KM088793	KM089178	KM089565
Penicillium cosmopolitanum	CBS 126995T	JN617691	JN606733	MN969249	MN969113
Penicillium creberum	CGMCC 3.25153T	OQ870833	OR051182	OR051357	OR062048
Penicillium crocicola	CBS 745.70T	KM189581	KJ834445	KM089210	JN406535
Penicillium decaturense	CBS 117509T	GU944604	JN606685	MN969252	JN606621
Penicillium echinulonalgiovense	CBS H-23172T	GU981587	GU981631	KX961269	KX961301
Penicillium ellipsoideum	CGMCC 3.25156T	OQ870835	OR051184	OR051359	OR062050
Penicillium ellipsoideum	CS28-04	OQ870837	OR051186	OR051361	OR062051
Penicillium ellipsoideum	CS29-01	OQ870838	OR051187	OR051362	OR062052
Penicillium ellipsoideum	NNIBRFG4823	PV702369	PV741155	PV741148	PV741141
Penicillium flosculum	CGMCC 3.25159T	OQ870839	OR051188	OR051363	OR062053
Penicillium fusisporum	CBS 137463T	KF769424	KF769400	KF769413	MN969117
Penicillium globosum	CBS 144639T	KY495014	KY495123	MN969330	KY495067
Penicillium godlewskii	CBS 215.28T	JN617692	JN606768	MN969258	JN606626
Penicillium grevilleicola	CBS 137775T	KM189630	KM088874	KM089261	KM089648
Penicillium griseoflavum	HMAS 247729T	KY495011	KY495120	MN969331	KY495064
Penicillium guangxiense	CBS 144526T	KY494986	KY495095	MN969332	KY495045
Penicillium herquei	CBS 336.48T	JN626101	JN625970	JN626013	JN121494
Penicillium infrabuccalum	CBS 140983T	KT887856	KT887817	KT887778	MN969181
Penicillium jejuense	CBS 138646T	KF818464	KF818461	KF818470	KF818467
Penicillium jiangjinense	CGMCC 3.25160T	OQ870840	OR051189	OR051364	OR062054
Penicillium laevigatum	HMAS 247728T	KY495015	KY495124	MN969335	KY495068
Penicillium malachiteum	CBS 647.95T	KC773838	KC773794	KC773820	MN969125
Penicillium manginii	CBS 253.31T	GU944599	JN606651	MN969274	JN606618
Penicillium mariae-crucis	CBS 270.83T	GU981593	GU981630	MN969275	KF296439
Penicillium miczynskii	CBS 220.28T	GU944600	JN606706	MN969277	JN606623
Penicillium neoherquei	CBS 148692T	MW341222	OL840853	OL840855	MW349119
Penicillium neomiczynskii	CBS 126231T	JN617671	JN606705	MN969278	MN969128
Penicillium nothofagi	CBS 130383T	JN617712	JN606732	JN606507	MN969129
Penicillium onobense	CBS 174.81T	GU981575	GU981627	MN969281	KF296447
Penicillium pancosmium	CBS 276.75T	JN617660	JN606790	MN969284	MN969130
Penicillium pancosmium	NNIBRFG1486	PV702372	PV741158	PV741151	PV741144
Penicillium pancosmium	NNIBRFG48324	PV702373	PV741159	PV741152	PV741145
Penicillium pancosmium	NNIBRFG49919	PV702374	PV741160	PV741153	PV741146
Penicillium panissanguineum	CBS 140989T	KT887862	KT887823	KT887784	MN969182
Penicillium paraherquei	CBS 338.59T	AF178511	KF296465	MN969285	KF296449
Penicillium pasqualense	CBS 126330T	JN617676	JN606673	MN969286	JN606617
Penicillium pedernalense	CBS 140770T	KU255398	KU255396	MN969322	MN969184
Penicillium quebecense	CBS 101623T	JN617661	JN606700	JN606509	JN606622
Penicillium raphiae	CBS 126234T	JN617673	JN606657	MN969292	JN606619
Penicillium retrofuscum sp. nov.	NNIBRFG6577 T	PV702368	PV741154	PV741147	PV741140
Penicillium roseoviride	CBS 267.35T	KM189549	KM088787	KM089172	KM089559
Penicillium scruposum	CGMCC 3.25167T	OQ870841	OR051190	OR051365	OR062055
Penicillium simplicissimum	CBS 372.48T	GU981588	GU981632	MN969297	JN121507
Penicillium skrjabinii	CBS 439.75T	GU981576	GU981626	MN969299	EU427252
Penicillium skrjabinii	NNIBRFG4610	PV702370	PV741156	PV741149	PV741142
Penicillium sphaerioides	CGMCC 3.25175T	OQ870850	OR051199	OR051374	OR062064
Penicillium spinuliferum	CBS 144483T	KY495040	KY495149	MN969338	KY495090
Penicillium subasperum	CGMCC 3.25173T	OQ870849	OR051198	OR051373	OR062063
Penicillium subglobosum	CGMCC 3.25171T	OQ870844	OR051193	OR051368	OR062058
Penicillium sucrivorum	CBS 135116T	JX140872	JX141015	JX141506	MN969140
Penicillium tanzanicum	CBS 140968T	KT887841	KT887802	KT887763	MN969183
Penicillium tardicrescens	CGMCC 3.25178T	OQ870853	OR051202	OR051377	OR062067
Penicillium thomii	CBS 225.81T	KM189560	KM088799	KM089184	KM089571
Penicillium ubiquetum	CBS 126437T	JN617680	JN606800	MN969306	MN969142
Penicillium umkhoba	CBS 147457T	MT949912	MT957417	MT957459	MT957485
Penicillium valentinum	CBS 172.81T	KM189550	KM088788	KM089173	KM089560
Penicillium vancouverense	CBS 126323T	JN617675	JN606663	MN969307	MN969143
Penicillium verrucisporum	CGMCC 3.18415T	KX885069	KX885049	KX885059	KX885040
Penicillium waksmanii	CBS 230.28T	GU944602	JN606779	MN969310	JN606627
Penicillium wanyuanense	CGMCC 3.25182T	OQ870854	OR051203	OR051378	OR062068
Penicillium wellingtonense	CBS 130375T	JN617713	JN606670	MN969311	JN606616
Penicillium westlingii	CBS 231.28T	GU944601	JN606718	MN969312	JN606625
Penicillium wotroi	CBS 118171T	GU981591	GU981637	MN969313	KF296460
Penicillium wulientehii	CGMCC 3.25183T	OQ870856	OR051205	OR051380	OR062070
Penicillium yezoense	CBS 350.59T	KM189553	KM088792	KM089177	KM089564
Penicillium yezoense	NNIBRFG4600	PV702371	PV741157	PV741150	PV741143
Hamigera avellanea	CBS 295.48T	AF454075	EU021664	EU021682	EU021627

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

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

3. Results

3.1. Phylogenetic analyses

A BLASTn search was performed using the sequences of the ITS regions, BenA, CaM, and RPB2 to determine the phylogenetic affiliations of strains NNIBRFG6577, NNIBRFG4823, NNIBRFG4610, NNIBRFG4600, NNIBRFG1486, NNIBRFG48324, and NNIBRFG49919. The results indicated that strain NNIBRFG6577 belongs to the genus Penicillium, being closely related to P. scruposum CGMCC 3.25167^T (with sequence similarities of 99.80%, 97.20%, 95.33%, and 98.73% for the ITS, BenA, CaM, and RPB2 sequences, respectively) and P. subasperum CGMCC 3.25173^T (with sequence similarities of 100%, 96.97%, 97.76%, and 98.73% for the ITS, BenA, CaM, and RPB2 sequences, respectively). Similarly, strain NNIBRFG4832 was found to be most closely related to P. ellipsoideum CGMCC 3.25156^T (with sequence similarities of 99.81%, 99.78%, 100%, and 99.61% for the ITS, BenA, CaM, and RPB2 sequences, respectively), and strain NNIBRFG4610 was most closely related to P. skrjabinii CBS 439.75^T (with sequence similarities of 99.62%, 99.14%, 99.42%, and 99.47% for the ITS, BenA, CaM, and RPB2 sequences, respectively). Sequences from strain NNIBRFG4600 were identical to those of P. yezoense CBS 350.59^T (with sequence similarities of 100% for all four loci). Lastly, strains NNIBRFG1486, NNIBRFG48324, and NNIBRFG49919 were closely related to P. pancosmium CBS 276.75^T (with sequence similarities of 99.83–100%, 99.76–100%, 99.39–99.82%, and 99.10–99.18% for the ITS, BenA, CaM, and RPB2 sequences, respectively). ML phylogenetic trees were constructed using the concatenated sequences of the four loci to better characterize the taxonomic affiliations of strains. Strain NNIBRFG6577 formed a distinct cluster with multiple strains within ser. Herqueorum, suggesting that it represents a novel species within the genus Penicillium (Figure 1). In contrast, strain NNIBRFG4832 was clustered within a monophyletic group with various P. ellipsoideum strains, including the type strain CGMCC 3.25156^T (Figure 1); strain NNIBRFG4610 was closely paired with P. skrjabinii CBS 439.75^T (Figure 2); strain NNIBRFG4600 was closely paired with P. yezoense CBS 350.59^T (Figure 3); and strains NNIBRFG1486, NNIBRFG48324, and NNIBRFG49919 were clustered within a monophyletic group with P. pancosmium CBS 276.75^T (Figure 4).

Figure 1.

Maximum likelihood phylogenetic tree of Penicillium series Herqueorum based on a combined dataset of partial ITS, BenA, CaM, and RPB2 sequences. Hamigera avellanea CBS 295.48^T was used as an outgroup. Bootstrap values (bs) ≥70% (left) and posterior probability values (pp) ≥ 0.90 (right) are indicated at nodes. Values below 70 bs and 0.90 pp are not shown or indicated with a hyphen. The Penicillium species isolated in this study are highlighted in bold and red. The scale bar represents 0.050 substitutions per nucleotide position.

Figure 2.

Maximum likelihood phylogenetic tree of Penicillium series simplicissima based on a combined dataset of partial ITS, BenA, CaM, and RPB2 sequences. Hamigera avellanea CBS 295.48^T was used as an outgroup. Bootstrap values (bs) ≥70% (left) and posterior probability values (pp) ≥0.90 (right) are indicated at nodes. Values below 70 bs and 0.90 pp are not shown or indicated with a hyphen. The Penicillium species isolated in this study is highlighted in bold and red. The scale bar represents 0.050 substitutions per nucleotide position.

Figure 3.

Maximum likelihood phylogenetic tree of Penicillium series thomiorum based on a combined dataset of partial ITS, BenA, CaM, and RPB2 sequences. Hamigera avellanea CBS 295.48^T was used as an outgroup. Bootstrap values (bs) ≥70% (left) and posterior probability values (pp) ≥0.90 (right) are indicated at nodes. Values below 70 bs and 0.90 pp are not shown or indicated with a hyphen. The Penicillium species isolated in this study is highlighted in bold and red. The scale bar represents 0.050 substitutions per nucleotide position.

Figure 4.

Maximum likelihood phylogenetic tree of Penicillium series westlingiorum based on a combined dataset of partial ITS, BenA, CaM, and RPB2 sequences. Hamigera avellanea CBS 295.48^T was used as an outgroup. Bootstrap values (bs) ≥70% (left) and posterior probability values (pp) ≥0.90 (right) are indicated at nodes. Values below 70 bs and 0.90 pp are not shown or indicated with a hyphen. The Penicillium species isolated in this study are highlighted in bold and red. The scale bar represents 0.050 substitutions per nucleotide position.

3.2. Taxonomy

Penicillium retrofuscum M.G. Kim, S.Y. Lee and H.Y. Jung sp. nov. (Figure 5)

Figure 5.

Cultural and morphological characteristics of Penicillium retrofuscum sp. nov. NNIBRFG6577^T. (A) Colonies after 7 d at 25 °C, from left to right (top row) PDA obverse, MEA obverse, CYA obverse, YES obverse, DG18 observer, CREA observe; (bottom row) PDA reverse, MEA reverse, CYA reverse, YES reverse, DG18 reverse, CREA reverse;

(B–D) Conidiophores; (E) Conidia. Scale bars = 10 µm.

Mycobank: 859413

Etymology: The specific epithet refers to the brown color on the underside of the colony on PDA, CYA, and YES media. In Penicillium subgenus Aspergilloides section Sclerotiorum series Herqueorum.

Typus: Freshwater sediment from Neungganggyegok Valley, Neunggang-ri, Susan-myeon, Jecheon-si, Chungbuk, South Korea (36° 59′ 38″ N 128° 13′ 45″ E) in 2018.

Cultural characteristics: On PDA at 25 °C for 7 d: Colonies sulcate, circular, margin entire with velvety texture; mycelium white and conidia dull green; exudates and soluble pigments not found; coloration of reverse orange to brownish orange (Figure 5(A)). On MEA at 25 °C for 7 d: Colonies sulcate, circular, margin entire with velutinous texture; mycelium white and conidia dull green; exudates and soluble pigments not found; coloration of reverse brownish to orange (Figure 5(A)). On CYA at 25 °C for 7 d: Colonies sulcate, circular, margin entire with velvety to floccose texture; mycelium white and conidia dull green; exudates and soluble pigments not found; coloration of reverse light brown to beige (Figure 5(A)). On YES at 25 °C for 7 d: Colonies sulcate, circular, margin entire with velvety to floccose texture; mycelium white and conidia dull green; exudates and soluble pigments not found; coloration of reverse orange to brownish orange (Figure 5(A)). On DG18 at 25 °C for 7 d: Colonies circular, margin entire with floccose texture; mycelium white and conidia dull green; exudates and soluble pigments not found; coloration of reverse light brown to brown (Figure 5(A)). On CREA at 25 °C for 7 d: Colonies circular with diffuse and sparse aerial mycelium; acid production absent (Figure 5(A)). Colony diameters (mm) after 7 d at 25 °C: 19–22 on PDA; 24–27 on MEA, 20–24 on CYA, 28–31 on YES, 23–24 on DG18, and 15–19 on CREA (Figure 5(A)).

Morphological characteristics: Conidiophores symmetrically biverticillate (Figure 5(B–D)); stipes smooth walled, 186–493 × 4.0–6.3 µm (Figure 5(B–D)); metulae cylindrical, 9.0–12.0 × 3.0–4.0 µm (Figure 5(B–D)); phialides ampulliform, tapering to a very thin neck, 7.0–8.5 × 2.5–3.5 µm (Figure 5(B–D)); conidia smooth-walled, ellipsoidal, 2.5–3.4 × 2.3–3.0 µm (Figure 5(E)).

Habitat: Freshwater soil

Specimen examined: Neungganggyegok Valley, Neunggang-ri, Susan-myeon, Jecheon-si, Chungbuk, South Korea; July, 2018; NNIBRFG6577 (ITS = PV702368; BenA=PV741154; CaM=PV741147; RPB2 = PV741140)

Note: Based on comparisons of cultural and morphological characteristics along with its placement in the phylogenetic tree, NNIBRFG6577 is closely related to P. scruposum CGMCC 3.25167^T and P. subasperum CGMCC 3.25173^T (Figure 1). To evaluate the characteristics of the newly discovered Penicillium species, strain NNIBRFG6577 was compared to P. scruposum CGMCC 3.25167^T and P. subasperum CGMCC 3.25173^T. Compared to P. scruposum CGMCC 3.25167^T, NNIBRFG6577 showed slower growth on PDA (19–22 vs. 25–26 mm), MEA (24–27 vs. 29–31 mm), CYA (20–24 vs. 26–27 mm), and YES (28–31 vs. 39–40 mm) [31]. Similarly, NNIBRFG6577 exhibited slower growth than P. subasperum CGMCC 3.25173^T on PDA (19–22 vs. 24–25 mm), MEA (24–27 vs. 29–31 mm), CYA (20–24 vs. 30–32 mm), and YES (28–31 vs. 37–39 mm) [31]. Morphologically, strain NNIBRFG6577 produces smaller metulae than both P. scruposum CGMCC 3.25167^T (9.0–12.0 × 3.0–4.0 vs. 9.0–23.0 × 2.5–6.5 µm) and P. subasperum CGMCC 3.25173^T (9.0–12.0 × 3.0–4.0 vs. 8.5–13.5 × 3.0–4.5 µm) [31], and smaller conidia than P. scruposum CGMCC 3.25167^T (2.5–3.4 × 2.3–3.0 vs. 3.5–4.0 × 3.0–3.5 µm) and P. subasperum CGMCC 3.25173^T (2.5–3.4 × 2.3–3.0 vs. 3.0–4.0 × 2.5–3.5 µm) [31]. Thus, since strain NNIBRFG6577 exhibited significant differences from both previously described closely related Penicillium strains, it was considered a novel species within subgenus Aspergilloides, section Sclerotiorum, and series Herqueorum of the genus Penicillium (Table 2).

Table 2.

Comparisons of morphological characteristics of penicillium retrofuscum sp. nov. NNIBRFG6577^T with related species.

Characteristics	Penicillium retrofuscum sp. nov. NNIBRFG6577Ta	Penicillium subasperum CGMCC 3.25173Tb	Penicillium scruposum CGMCC 3.25167Tb
Conidiophores	Symmetrically biverticillate	Biverticillate, terverticillate	Biverticillate
Stipes	Smooth-walled, 186–493 × 4.0–6.3 µm	Smooth-walled, 125–315 × 2.5–4.0 µm	Rough-walled, 185–435 × 3.0–3.5 µm
Metulae	9.0–12.0 × 3.0–4.0 µm	8.5–13.5 × 3.0–4.5 µm	9.0–23.0 × 2.5–6.5 µm
Phialides	Ampulliform, tapering into very thin neck 7.0–8.5 × 2.5–3.5 µm	Ampulliform, tapering into very thin neck 7.5–9.0 × 2.5–3.5 µm	Ampulliform, tapering into very thin neck 8.5–11.5 × 2.5–4.0 µm
Conidia	Rough-walled, ellipsoidal 2.5–3.4 × 2.3–3.0 µm	Rough-walled, ellipsoidal 3.0–4.0 × 2.5–3.5 µm	Rough-walled, ellipsoidal 3.5–4.0 × 3.0–3.5 µm

^aFungal strain used in this study, ^bSource of description [28]; ^TType strain.

Penicillium ellipsoideum X.C. Wang & W.Y. Zhuang, J. Fungi 9 (12, no. 1150): 69 (2023) [MB#571544]

Cultural characteristics: On PDA at 25 °C for 7 d: Colonies flat, margin entire with velvety texture; mycelium white to light green and conidia dull green; exudates and soluble pigments not found; coloration of reverse light brown to beige (Figure 6(A)). On MEA at 25 °C for 7 d: Colonies flat, margin entire with velvety texture; mycelium white to yellow and conidia dull green; exudates and soluble pigments not found; coloration of reverse yellow (Figure 6(A)). On CYA at 25 °C for 7 d: Colonies flat, margin entire with velutinous texture; mycelium white to yellow and conidia dull green; exudates hyaline at center; soluble pigments not found; coloration of reverse light brown to beige (Figure 6(A)). On YES at 25 °C for 7 d: Colonies flat, margin entire with velvety texture; mycelium white to yellow and conidia dull green; exudates and soluble pigments not found; coloration of reverse white to yellow (Figure 6(A)). On DG18 at 25 °C for 7 d: Colonies circular to slightly irregular, margin entire with velvety texture; mycelium white and conidia dull green; exudates and soluble pigments not found; coloration of reverse light brown (Figure 6(A)). On CREA at 25 °C for 7 d: Colonies circular, sparse, compact with limited radial expansion; acid production absent (Figure 6(A)). Colony diameters (mm) after 7 d at 25 °C: 21–23 on PDA, 32–34 on MEA, 24–27 on CYA, 32–34 on YES, 14–17 on DG 18, and 8–10 on CREA (Figure 6(A)).

Figure 6.

Cultural and morphological characteristics of Penicillium ellipsoideum NNIBRFG4823. (A) Colonies after 7 d at 25 °C, from left to right (top row) PDA obverse, MEA obverse, CYA obverse, YES obverse, DG18 observe, CREA observe; (bottom row) PDA reverse, MEA reverse, CYA reverse, YES reverse, DG18 reverse, CREA reverse; (B–D) Conidiophores; (E) Conidia. Scale bars = 10 µm.

Morphological characteristics: Conidiophores biverticillate or terverticillate (Figure 6(B–D)); stipes smooth walled, 206–270 × 3.5–4.0 µm (Figure 6(B–D)); metulae cylindrical, 8.0–14.5 × 3.6–5.2 µm (Figure 6(B–D)); phialides ampulliform, 7–12 × 2.7–3.5 µm (Figure 6(B–D)); conidia smooth-walled, ellipsoidal, and slightly ovoid, 3.0–4.1 × 2.2–2.8 µm (Figure 6(E)).

Habitat: Freshwater sediment

Specimen examined: Domacheon Stream, Jeongmok-ri, Buk-myeon, Gapyeong-gun, Gyeonggi, South Korea; September, 2017; NNIBRFG4823 (ITS = PV702369; BenA = PV741155; CaM = PV741148; RPB2 = PV741141)

Note: Penicillium ellipsoideum was first reported in 2023, isolated from soil in China [31]. To evaluate the characteristics of Korean P. ellipsoideum, strain NNIBRFG4823 was compared to P. ellipsoideum CGMCC 3.25156^T. Regarding cultural characteristics, NNIBRFG4823 showed faster growth than P. ellipsoideum CGMCC 3.25156^T on MEA (32–34 vs. 28–32 mm) but slower growth on CYA (24–27 vs. 34–35 mm) and YES (32–34 vs. 35–37 mm) [31]. For morphological characteristics, NNIBRFG4823 and P. ellipsoideum CGMCC 3.25156^T both produce ampulliform phialides tapering into a very thin neck, producing smooth-walled, ellipsoidal conidia. Metulae form at the branching point of biverticillate conidiophores [31].

Penicillium skrjabinii Schmotina & Golovleva, Mikol. Fitopatol.: 530 (1974) [MB#319296]

Cultural characteristics: On MEA at 25 °C for 7 d: Colonies circular, raised, and dense; margin diffusely radiating and slightly uneven; texture floccose, with a matted center; mycelium dark olive-green; exudates and soluble pigments not found; coloration of reverse white to pale olive-green (Figure 7(A)). On CYA at 25 °C for 7 d: Colonies finely deep and radially sulcate with symmetrical radial growth, whit to yellow mycelium; texture velvety to floccose, appearing denser in the center and softer at the margin; margins entire and slightly fimbriate; exudates and soluble pigments not found; coloration of reverse cream to mustard-yellow (Figure 7(A)). On YES at 25 °C for 7 d: Colonies circular, finely deep and radially sulcate with circular colonies having white to yellowish mycelium; margins regular, slightly undulating; texture is velvety to slightly floccose; exudates and soluble pigments were not found; coloration of reverse is yellow (Figure 7(A)). On CREA at 25 °C for 7 d: Diffuse and sparse aerial with circular colonies having white to grayish mycelium; margins entire, feathered to slightly diffuse; texture is floccose to granular; mycelium white to grayish; no acid production (Figure 7(A)). Colony diameters (mm) after 7 d at 25 °C: 18–20 on MEA, 28–30 on CYA, 33–35 on YES, and 8–10 on CREA (Figure 7(A)).

Figure 7.

Cultural and morphological characteristics of Penicillium skrjabinii NNIBRFG4610. (A) Colonies after 7 d at 25 °C, from left to right (top row) MEA obverse, CYA obverse, YES obverse, CREA obverse; (bottom row) MEA reverse, CYA reverse, YES reverse, CREA reverse; (B,C) Conidiophores; (D) Conidia. Scale bars = 10 µm.

Morphological characteristics: Conidiophores biverticillate (Figure 7(B,C)); stipes long, rough-walled (Figure 7(B,C)); metulae 10–13 × 3–4 µm (Figure 7(B,C)); phialides flask-shaped, 9–12 × 2.5–3.5 µm (Figure 7(B,C)); conidia globose to subglobose, smooth-walled, 2–3 µm (Figure 7(D)).

Habitat: Soils associated with Cypripedium guttatum roots

Specimen examined: Mt. Hambaek, Gohan-ri, Gohan-eup, Jeongseon, Taebaek-si, Gangwon, South Korea; July, 2017; NNIBRFG4610 (ITS = PV702370; BenA=PV741156; CaM=PV741149; RPB2 = PV741142).

Note: Penicillium skrjabinii was originally isolated from soil near Blagoveshchensk in the Amur region of Siberia [32]. To evaluate the characteristics of Korean P. skrjabinii, strain NNIBRFG4610 was compared to P. skrjabinii CBS 439.75^T. In our phylogenetic tree, strain NNIBRFG4610 formed a distinct cluster with P. skrjabinii CBS 439.75^T (Figure 2). Regarding morphological characteristics, strain NNIBRFG4610 and P. skrjabinii CBS 439.75^T both produce globose to subglobose conidia from flask-shaped phialides borne on the tips of metulae, which form the branching point of biverticillate conidiophores (Figure 7(B–D)) [32,33]. Thus, the morphological characteristics of the two strains appear to be identical.

Penicillium yezoense Hanzawa, J. Agric. Chem. Soc. Japan: 774 (1943) [MB#335775]

Cultural characteristics: On MEA at 25 °C for 7 d: Colonies circular, radially floccose to slightly granular, fine, margins entire and tight; mycelium pale to white; exudates and soluble pigments not found; coloration of reverse pale to white (Figure 8(A)). On CYA at 25 °C for 7 d: Colonies strongly radial sulcate, with prominent concentric rings alternating in tone and texture; texture velvety to floccose, appearing denser in the center; margins entire; grayish-green to olive in the center, transitioning to white toward the margins; exudates and soluble pigments not found; coloration of reverse bright yellow to pale golden. On YES at 25 °C for 7 d: Colonies with radiating wrinkles and concentric zones; margins well-defined, smooth, slightly undulating; texture velvety to floccose, denser in the center; mycelium white to gray-greenish white; exudates and soluble pigments not found; coloration of reverse bright yellow. On CREA at 25 °C for 7 d: Colony small, sparse, and compact, with limited radial expansion; margins entire, feathered to slightly diffuse; texture floccose to powdery in the center; mycelium white and low-growing; exudates and soluble pigments not found; acid production absent (Figure 8(A)). Colony diameters (mm) after 7 d at 25 °C: 32–35 on MEA, 34–36 on CYA, 30–33 on YES, and 9–11 on CREA (Figure 8(A)).

Figure 8.

Cultural and morphological characteristics of Penicillium yezoense NNIBRFG4600. (A) Colonies after 7 d at 25 °C, from left to right (top row) MEA obverse, CYA obverse, YES obverse, CREA obverse; (bottom row) MEA reverse, CYA reverse, YES reverse, CREA reverse; (B,C) Conidiophores; (D) Conidia. Scale bars = 10 µm.

Morphological characteristics: Conidiophores monoverticillate (Figure 8(B,C)); stipes smooth-walled (Figure 8(B,C)); phialides flask-shaped, 7–10 × 2–3 µm (Figure 8(B,C)); conidia globose to subglobose, smooth-walled to finely ornamented, 2.5–3.5 µm (Figure 8(D)).

Habitat: Soils associated with Cypripedium guttatum roots

Specimen examined: Mt. Hambaek, Gohan-ri, Gohan-eup, Jeongseon, Taebaek-si, Gangwon, South Korea; July, 2017; NNIBRFG4600 (ITS = PV702371; BenA=PV741157; CaM=PV741150; RPB2 = PV741143).

Note: Penicillium yezoense was first described in 1943 and was subsequently treated as a synonym of P. thomii by Pitt [4]. However, a more recent study, in 2014, provided additional morphological and phylogenetic evidence, recognizing it as a distinct, phylogenetically unique species [34]. To evaluate the characteristics of Korean P. yezoense, strain NNIBRFG4600 was compared with the type strain, P. yezoense CBS 350.59^T [34]. In the phylogenetic tree, P. yezoense NNIBRFG4600 was closely paired with P. yezoense CBS 350.59^T (Figure 3). Morphologically, P. yezoense NNIBRFG4600 was similar to P. yezoense CBS 350.59^T, with phialides at the end of the monoverticillate conidiophores producing ellipsoidal conidia (Figure 9(B,C)) [34].

Figure 9.

Cultural and morphological characteristics of Penicillium pancosmium NNIBRFG1486. (A) Colonies after 7 d at 25 °C, from left to right (top row) PDA obverse, CYA obverse, YES obverse, CREA obverse; (bottom row) PDA reverse, CYA reverse, YES reverse, CREA reverse; (B,C) Conidiophores; (D) Conidia. Scale bars = 10 µm.

Penicillium pancosmium Houbraken, Frisvad & Samson, Stud. Mycol. 70: 108 (2011) [MB#563191]

Strains NNIBRFG1486, NNIBRFG48324, and NNIBRFG49919 were found to be morphologically identical, and they clustered together with P. pancosmium CBS 276.75^T in the ML phylogenetic tree. Thus, in this study, only the cultural and morphological characteristics of strain NNIBRFG1486 are described.

Cultural characteristics: On PDA at 25 °C for 7 d: Colonies with distinct radial sulcation and occasional sectoring or cracking, slightly raised, center cottony with faint radial striations; margins entire to slightly diffuse; mycelium white to pale gray; exudates and soluble pigments not found; coloration of reverse yellow to golden yellow (Figure 9(A)). On CYA at 25 °C for 7 d: Colonies dense, with floccose white center and light gray periphery, weak radial sulcation; margins circular and well-defined; texture velvety to floccose, appearing denser in the center; exudates and soluble pigments not found; coloration of reverse pale-yellow margin and golden center with pronounced radial grooves (Figure 9(A)). On YES at 25 °C for 7 d: Colonies circular, with prominent concentric zonation and deep radial sulcation; margins well-defined; texture velvety to floccose, with a dense and raised center; mycelium white in the center, grayish-white toward the margins; exudates and soluble pigments not found; coloration of reverse pale yellow to golden yellow (Figure 9(A)). On CREA at 25 °C for 7 d: Colonies circular and small, compact; margins entire, feathered to slightly diffuse; texture floccose to powdery; mycelium white and low-growing; exudates and soluble pigments not found; reverse showing faint pink halos, indicating weak acid production (Figure 9(A)). Colony diameters (mm) after 7 d at 25 °C: 32–35 on PDA, 30–33 on CYA, 30–35 on YES, and 10–12 on CREA (Figure 9(A)).

Morphological characteristics: Conidiophores symmetrically biverticillate (Figure 9(B,C)); stipes smooth-walled (Figure 9(B,C)); metulae 9–13 × 4–6 µm (Figure 9(B,C)); phialides flask-shaped, tapering toward the apex, 7–9 × 3–4 µm (Figure 9(B,C)); conidia globose to subglobose, rough-walled and hyaline, 2.5–4.0 µm (Figure 9(D)).

Habitat: Freshwater soil

Specimen examined: Namdae-cheon stream, Uiseong-gun, Gyeongbuk, South Korea; March, 2016; NNIBRFG1486 (ITS = PV702372; BenA = PV741158; CaM = PV741151; RPB2 = PV741144).

Other species examined: Hwajeong-ri, Bukcheon-myeon, Hadong-gun, Gyeongnam, Korea (35° 8′ 54.1″ N 127° 51′ 13.8″ E), freshwater, Mar 2023, NNIBRFG48324; Galpyeong-ri, Mungyeong-eup, Mungyeong, Gyeongbuk, South Korea (36° 47′ 7.87″ N 128° 9′ 16.63″ E), soil in freshwater, Jul 2023, NNIBRFG49919.

Note: Penicillium pancosmium was first described in 2011, being placed within section Citrina [35]. To evaluate the characteristics of Korean P. pancosmium, strain NNIBRFG1486 was compared to P. pancosmium CBS 276.75^T. Regarding cultural characteristics, strain NNIBRFG1486 showed slower growth than P. pancosmium CBS 276.75^T on YES (30–35 vs. 30–40 mm) and CREA (10–12 vs. 15–20 mm) [35]. Regarding morphological characteristics, NNIBRFG4823 and P. pancosmium CBS 276.75^T both produce roughened, globose to subglobose conidia on ampulliform phialides, with metulae forming at the branching point of biverticillate conidiophores (Figure 9(B–D)) [35].

4. Discussion

In this study, one novel Penicillium species and four Penicillium species newly reported in Korea were isolated from freshwater and near-freshwater substrates, identified, and classified within the infrageneric taxonomy based on cultural and morphological characteristics and phylogenetic analyses. Strains NNIBRFG6577 and NNIBRFG4823, assigned to subgen. Aspergilloides, sect. Sclerotiorum, and ser. Herqueorum, were identified as the novel species Penicillium retrofuscum sp. nov. and P. ellipsoideum, respectively. The series Herqueorum, comprising 22 species reported globally, including two in Korea, is characterized by monoverticillate, biverticillate, or terverticillate conidiophores; ellipsoidal conidia with grey-green or dull green coloration; and the ability to grow at 37 °C [7,8,12,31]. Strain NNIBRFG4610 was identified as P. skrjabinii, within subgen. Aspergilloides, sect. Lanata-Divaricata, and ser. Simplicissima. This series includes 21 species worldwide, with seven recorded in Korea, and is distinguished by biverticillate or divaricate conidiophores, grey-green or dull green conidia that vary in size and shape, and colony growth at 37 °C [7,8,12,31]. Strain NNIBRFG4600 was assigned to the species P. yezoense of subgen. Aspergilloides, sect. Aspergilloides, and ser. Thomiorum. The series Thomiorum, including 12 species reported globally, with three in Korea, is characterized by monoverticillate conidiophores; dull green, ellipsoidal or fusiform conidia; and colony growth at 37 °C [7,8,12,31]. Lastly, strains NNIBRFG1486, NNIBRFG48324, and NNIBRFG49919 were identified as P. pancosmium, which is placed in subgen. Aspergilloides, sect. Citrina, and ser. Westlingiorum. The series Westlingiorum, encompassing 22 species worldwide, eight of which are known to be in Korea, is characterized by biverticillate or divaricate conidiophores; blue-green to grayish conidia that are globose, subglobose, or ellipsoidal in shape; and colony growth at 37 °C [7,8,12,31].

Most species belonging to the Penicillium series Herqueorum, Simplicissima, Thomiorum, and Westlingiorum were initially isolated from a variety of terrestrial and aerial environments, including soil, plant debris, fruits, air, and insects, as well as marine habitats [31]. Notably, among the 77 species classified within these four series, only two, P. aquadulcis and P. wandoense, both reported from Korea, have been documented in freshwater environments [31,36,37]. The limited number of freshwater-associated species in these series suggests that fungal diversity in freshwater ecosystems has been insufficiently investigated, highlighting the need for further research in these underrepresented environments.

Currently, in Korea, taxonomic studies on the genus Penicillium are actively being conducted under the “one fungus one name”, which has led to the re-identification of existing species and the discovery of novel species or species previously unreported in the region [38,39]. Additionally, these new Penicillium species are being identified across various habitats, including soil and aquatic environments, underscoring the success of efforts to catalog and secure fungal biodiversity as a domestic resource [16–22]. Given these ongoing efforts, it is highly probable that additional Penicillium species, including those belonging to new taxonomic groups, will continue to be discovered in Korea. The identification of Penicillium retrofuscum sp. nov., P. ellipsoideum, P. skrjabinii, P. yezoense, and P. pancosmium from freshwater and near-freshwater substrates enhances the current understanding of fungal diversity and distributions in Korea. These findings enrich the national fungus inventory, providing a basis for valuable future resource development.

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 authors.