Three Unrecorded Species Belonging to Penicillium Section Sclerotiora from Marine Environments in Korea.

Species that belong to Penicillium section Sclerotiora are commonly found in various terrestrial environments, but only a few have been reported in marine environments. Because the number of Penicillium species reported in marine environments is increasing, we investigated the diversity of Penicillium section Sclerotiora in marine environments in Korea. Based on sequence analyses of β-tubulin and calmodulin loci, 21 strains of section Sclerotiora were identified as P. bilaiae, P. daejeonium, P. exsudans, P. herquei, P. cf. guanacastense, P. mallochii, P. maximae, and P. viticola. Three of them were confirmed as new to Korea: P. exsudans, P. mallochii, and P. maximae. Here, we have provided detailed morphological descriptions of these unrecorded species.

Introduction

Penicillium is one of the most common genera found in various terrestrial and marine environments [1-4], and many species in this genus play important ecological roles as decomposers and plant pathogens [3,5]. Penicillium species produce a variety of bioactive compounds, such as mycotoxins, antibiotic compounds, and enzymes [6], therefore, have been industrially exploited for potential applications of the bioactive compounds [7-9]. Recently, reports on these species have increased in marine environments, such as sand, seawater, and macroalgae [4,10-13]. In Korea, more than 130 Penicillium species have been introduced, and most of them originated from terrestrial environments [14-18].

Since the launch of the Marine Fungal Resource Bank (MFRB) by the Ministry of Maritime Affairs and Fisheries, we have been studying a number of Penicillium species from marine environments of the Korean peninsula [4]. Because of plasticity of the distinguishing morphological features [6], it is very difficult to identify Penicillium members to the species level. Therefore, we used a reverse taxonomic approach, in which the species was first identified using molecular markers and then confirmed morphologically [19]. The nuclear rDNA internal transcribed spacer (ITS), β-tubulin (BenA), calmodulin (CaM), and RNA polymerase II second largest subunit (RPB2) have been introduced as standard molecular markers for the identification and phylogenetic study of Penicillium [6].

Penicillium section Sclerotiora is characterized by yellow and/or orange mycelia, orange or reddish colony reverses, and bright-colored sclerotia and cleistothecia if produced and commonly isolated from the soil, plants, and insects [20]. The species in section Sclerotiora can be distinguished by combinations of morphological features such as colony characters, conidiophore branching pattern and stipe roughening, and sclerotia production [21]. BenA and CaM were successfully used for accurately identifying the species in section Sclerotiora in previous study [22]. Currently, 30 species have been described in this section [21-24]. In Korea, seven species have been reported: five species from terrestrial environments [17,18,25,26] and two from marine environments [12,13].

Because the number of Penicillium species reported in marine environments is increasing, we hypothesized that there are more species in the section Sclerotiora. To investigate the diversity of Penicillium section Sclerotiora in marine environments, we re-identified previously isolated Penicillium species by using sequence analysis of BenA and CaM. In this process, a total of eight species were detected in this section and three species– P. exsudans, P. mallochii, and P. maximae–were confirmed as new to Korea. Their detailed descriptions have been provided in this study.

Materials and methods

Materials

A total of 21 Penicillium strains were identified in this study. They were isolated from egg masses of Arctoscopus japonicus, mudflats, sea sand, and seaweeds by using previously described methods [10,13,27]. All plates were incubated at 25 °C for 7–15 days, and each Penicillium strain was transferred to a new PDA plate. The isolated strains are stored in 20% glycerol and −80 °C at the Seoul National University Fungus Collection (SFC; Table 1).

Table 1.

Summary and GenBank accession numbers for Penicillium strains isolated from marine environments.

Species	Strain No.	Substrate	GPS coordinates	Accession No.
				BenA	CaM
P. bilaiae	SFC20151118-M26	Agarum clathratum	37°57'14.8″N 128°46'24.0″E	KX712485	KX712499
P. daejeonium	SFC102721	Sea sand	35°03'44.4″N 126°20'17.0″E	MK134649	MK134665
	SFC100824	Sea sand	36°09'51.2″N 126°31'18.9″E	MK134646	MK134662
	SFC20151014-M05	Sea sand	34°20'59.5″N 126°31'15.7″E	MK134657	MK134675
	SFC20160805-M22	Agarum clathratum	38°07'05.0″N 128°38'02.1″E	KX712486	KX712500
	SFC20160805-M23	Agarum clathratum	38°07'05.0″N 128°38'02.1″E	KX712487	KX712501
P. exsudans*	SFC100716	Mudflat	34°59'50.8″N 128°41'36.9″E	MK134645	MK134661
	SFC101878	Egg masses of Arctoscopus japonicas	38°29'26.4″N 128°25'49.4″E	MK134647	MK134663
	SFC102481	Mudflat	37°36'33.8″N 126°31'16.7″E	MK134648	MK134664
	SFC102936	Seasand	34°50'30.2″N 127°29'09.7″E	MK134650	MK134666
P. cf. guanacastense	SFC100711	Mudflat	34°57'29.4″N 127°50'44.6″E	MK134644	MK134660
	SFC106100	Sea water	33°29'51.6″N 126°27'09.7″E	MK134652	MK134668
	SFC106870	Chondria crassicaulis	33°57'12.9″N 126°18'08.3″E	MK134653	MK134669
	SFC20150402-M23	Colpomenia sp.	33°30'57.4″N 126°30'44.7″E	KU600433	MK134672
P. herquei	SFC20180817-M07	Mudflat	37°36'33.8″N 126°31'16.7″E	MK134658	MK134676
P. mallochii*	SFC20150915-M03	Seasand	36°09'51.2″N 126°31'18.9″E	MK134655	MK134673
P. maximae*	SFC103236	Mudflat	35°01'38.7″N 126°25'15.7″E	MK134651	MK134667
	SFC20150303-M16	Mudflat	34°49'59.6″N 127°23'13.7″E	MK134654	MK134670
	SL-CL7	Shell	36°01'22.6″N 126°39'49.2″E	MK134659	MK134677
P. viticola	SFC20150915-M05	Sea sand	37°35'32.0″N 126°27'27.5″E	MK134656	MK134674
	SFC20150402-M20	Ulva sp.	33°30'57.4″N 126°30'44.7″E	KU600432	MK134671

*New to Korea found in this study.

DNA extraction, amplification, and sequencing

Genomic DNA was extracted using a modified cetyltrimethylammonium bromide extraction protocol [28]. BenA and CaM were amplified using the primer sets Bt2a/Bt2b [29] and CF1/CF4 [30], respectively. Each PCR was performed in a C1000 thermal cycler (Bio-Rad, Richmond, CA) by using previously described methods [12]. The PCR products were purified using the ExpinTM PCR Purification Kit (GeneAll Biotechnology, Seoul, Korea), according to the manufacturer’s instructions. DNA sequencing was performed in both forward and reverse directions by using the corresponding PCR primers at Macrogen (Seoul, Korea) and ABI Prism 3700 genetic analyzer (Life Technologies, Gaithersburg, MD).

Phylogenetic analysis

The sequences were assembled, proofread, and aligned using MEGA6 [31]. The resulting consensus sequences were deposited in GenBank (accession Nos. in Table 1). Phylogenetic analyses were performed in two steps. First, we identified strains belonging to section Sclerotiora by analyzing BenA sequences with 432 type strains. Next, each strain was identified to the species level by analyzing the combined dataset (BenA + CaM) with 42 GenBank sequences (32 type strains) belonging to section Sclerotiora. Penicillium levitum CBS 345.48 was used as the outgroup [19]. The sequence similarities were calculated for each gene by using MEGA6 [31]. The sequences were aligned using the default settings of MAFFT v7 [32], and ambiguously aligned positions were adjusted manually. Maximum likelihood phylogenetic analyses were performed with RAxML [33], using the GTR + G model of evolution and 1000 bootstrap replicates.

Morphological analysis

The morphology of the three unrecorded species was observed using previously described methods [6]. Five different culture media were used: creatine sucrose agar, Czapek’s agar, Czapek yeast autolysate agar (CYA; Difco, Sparks, MD), malt extract agar (MEA; Oxoid, Hampshire, UK), and yeast extract sucrose agar (YES; Difco). Culture inoculation and incubation for each strain were conducted according to previously described methods [6]. To observe microscopic characters using a light microscope (Eclipse 80i, Nikon, Tokyo, Japan), mounts of the strains were made in lactic acid from colonies grown on MEA, and conidiophores were washed with a drop of ethanol to remove excess spores.

Results and discussion

Except for BenA and CaM sequences of P. bilaiae and BenA sequence of P. viticola (SFC20150402-M20), BenA and CaM of the other strains were sequenced successfully. All sequences were deposited in GenBank (Table 1). The two-step phylogenetic analysis was used to identify 21 Penicillium strains as eight species: P. bilaiae, P. daejeonium, P. exsudans, P. herquei, P. cf. guanacastense, P. mallochii, P. maximae, and P. viticola. The strains formed strongly supported monophyletic groups with each type strain, except P. herquei (Figure 1). Penicillium daejeonium was the dominant species, followed by P. exsudans and P. cf. guanacastense (Table 1).

Figure 1.

Maximum-likelihood phylogenetic analysis of the combined data set of BenA and CaM used to identify strains to the species level in Penicillium section Sclerotiora. Bootstrap scores of >70 are presented at the nodes. The scale bar indicates the number of nucleotide substitutions per site. “T” indicates the extype strains, and * indicates the new records found in this study.

Strains SFC100716, SFC101878, SFC102481, and SFC102936 grouped with the type strain (HMAS248735) of P. exsudans (sequence similarity for BenA = 99.5–99.7% and CaM = 98.1–99.3%; bootstrap support = 100%). SFC20150915-M03 formed a monophyletic group with P. mallochii DAOM 239917 (type strain), DAOM 239922, and DAOM 239926 (sequence similarity for BenA = 98.4–100% and CaM = 99.5–99.8%; bootstrap support = 100%). SFC103236, SFC20150303-M16, and SL-CL7 grouped with the type strain (NRRL2060) of P. maximae (sequence similarity for BenA = 100% and CaM = 99.8–100%; bootstrap support = 98%). SFC100711, SFC106100, and SFC106870 formed a monophyletic group with the type strain (DAOM239912) of P. guanacastense and SFC20150402-M23, which previously was designated Penicillium sp. 1 [13]. However, Korean strains were clearly separated from the reference sequences and showed morphological differences such as growth morphology and rate. Therefore, we designated them as P. cf. guanacastense in this study (Figure 1).

To date, seven species in section Sclerotiora have been reported in Korea: P. adametzioides, P. cainii, P. daejeonium, P. herquei, and P. sclerotiorum from terrestrial environments [17,18,25,26] and P. bilaiae and P. viticola from marine environments [12,13]. Through this study, we found eight species from marine environments. Four of them (P. herquei, P. bilaiae, P. daejeonium, and P. viticola) match previously reported species, whereas the other four have been newly identified in this study. As a result, there are eight species in section Sclerotiora in marine environments. To the best of our knowledge, this is the first report of P. exsudans, P. mallochii, and P. maximae in Korea. In case of P. cf. guanacastense, further studies are required to determine whether it is a new species. We have presented detailed taxonomic information of the three species below.

Taxonomy

4.1. Penicillium exsudans X.C. Wang & W.Y. Zhuang (2017)

Description: Colony diam, 7 d, in mm: CYA 33–40; CYA 5 °C no growth; CYA 37 °C no growth; MEA 31–36; YES 34–40 (Figure 2).

Figure 2.

Penicillium exsudans SFC101878 in 7-day-old cultures at 25 °C. (A–C) Colonies grown on Czapek yeast autolysate agar (CYA), malt extract agar (MEA), and yeast extract sucrose agar (YES) from left to right (top = obverse, bottom = reverse); (D–F) Conidiophores; (G) Conidia (scale bar: D–G = 10 μm).

CYA, 25 °C: conidia dull green (28E3); colony texture velvety; sporulation strong, absent towards the margins; nonsporulating margins 2 mm; exudates yellow droplets in central areas; soluble pigments absent; reverse color deep yellow (4A8). MEA, 25 °C: conidia greenish grey (28D2) to dull green (28D3); colony texture velvety; sporulation strong, absent toward the margins; nonsporulating edges 2 mm; exudates hyaline to yellow droplets in central areas; soluble pigments absent; reverse color reddish orange (7B8) to yellowish red (8B8) in center but light yellow (4A5) at margins. YES, 25 °C: conidia greenish grey (28D2) to dull green (28D3); colony texture velvety; sporulation strong; nonsporulating margins 1 mm; exudates absent; soluble pigments absent; reverse color light yellow (4A5) to orange (6A6) at center in SFC101878, light brown (6D6) in SFC102936 at center, but pale yellow (2A3) at margins (Figure 2(A–C)).

Conidiophores monoverticillate, smooth walls, 2.3–3.0 µm wide, phialides ampulliform, 8.0–9.0 × 2.7–3.4 µm (Figure 2(D–F)). Conidia subglobose to broadly ellipsoidal, 2.4–2.8 × 2.1–2.8 µm, with finely roughened walls (Figure 2(G)). Sclerotia absent. Asci and ascospores not observed.

Strain examined: SFC101878 and SFC102936

Note: Penicillium exsudans is phylogenetically similar to P. mallochii. The former species can be distinguished from the latter by the shape of the conidia. P. exsudans is characterized by subglobose to broadly ellipsoidal spores, whereas P. mallochii has globose to subglobose conidia (see below). When compared with the type strain of P. exsudans (HMAS 248735), P. exsudans in Korea grows slower on YES at 25 °C (34–40 vs. 39–42) [23].

4.2. Penicillium mallochii K.G. Rivera, Urb & Seifert (2012)

Description: Colony diam, 7 d, in mm: CYA 39–42; CYA 5 °C no growth; CYA 37 °C no growth; MEA 33–37; YES 34–37 (Figure 3).

Figure 3.

Penicillium mallochii SFC20150915-M03 in 7-day-old cultures at 25 °C. (A–C) Colonies grown on Czapek yeast autolysate agar (CYA), malt extract agar (MEA), and yeast extract sucrose agar (YES) from left to right (top = obverse, bottom = reverse); (D–F) Conidiophores; (G) Conidia (scale bar: D–G = 10 μm).

CYA, 25 °C: conidia greenish grey (28D2); colony texture velvety with pale yellow (4A3) mycelium in the center; sporulation strong, absent towards the margins; non-sporulating margins 3–4 mm; exudates hyaline; soluble pigments absent; reverse color deep yellow (4A8). MEA, 25 °C: conidia dull green (27E3); colony texture velvety; sporulation strong, pale yellow (3A3) and white towards the margins; nonsporulating edges 1–2 mm; exudates absent; soluble pigments absent; reverse color orange (6B7). YES, 25 °C: conidia dull green (27E3); colony texture velvety; sporulation strong; nonsporulating margins 3–4 mm; exudates absent; soluble pigments absent; reverse color light (5A5) (Figure 3(A–C)).

Conidiophores monoverticillate, smooth or finely roughened walls, 2.4–3.2 µm wide; phialides ampulliform, 8.0–10.0 × 2.5–3.3 µm (Figure 3(D–F)). Conidia globose to subglobose, 2.4–2.7 × 2.2–2.5 µm, with finely roughened walls (Figure 3(G)). Sclerotia absent. Asci and ascospores not observed.

Strain examined: SFC20150915-M03

Note: When compared with the type strain of P. mallochii (DAOM 239917), P. mallochii in Korea grows faster on CYA at 25 °C (39–42 vs. 29–39 mm) [34].

4.3. Penicillium maximae Visagie, Houbraken & Samson 2013

Description: Colony diam, 7 d, in mm: CYA 35–40; CYA 5 °C no growth; CYA 37 °C no growth; MEA 33–38; YES 39–45 (Figure 4).

Figure 4.

Penicillium maximae SFC20150303-M16 in 7-day-old cultures at 25 °C. (A–C) Colonies grown on Czapek yeast autolysate agar (CYA), malt extract agar (MEA), and yeast extract sucrose agar (YES) from left to right (top = obverse, bottom = reverse); (D–F) Conidiophores; (G) Conidia (scale bar: D–G = 10 μm).

CYA, 25 °C: conidia greyish green (27C3); colony texture floccose; sporulation moderate; angular in the margins, moderately raised, with white and light orange (5A4) mycelia; pale yellow (3A3) toward the margins; exudates hyaline to orange droplets; soluble pigments brownish orange (6C4); reverse color brownish red (8C8). MEA, 25 °C: conidia dull green (27D3); colony texture floccose; sporulation strong at center; light orange (5A4) dominates margins; mycelia white and light orange (5A4); exudates hyaline; soluble pigments absent; reverse color reddish brown (8D7). YES, 25 °C: conidia unclear; colony texture floccose; sporulation moderate; mycelia white and pastel red (7A5); exudates hyaline droplets; soluble pigments greyish yellow (4B4); reverse color reddish brown (8E7) (Figure 4(A–C)).

Conidiophores monoverticillate, smooth walls, 2.0–2.5 µm wide; phialides ampulliform, 7.0–11.0 × 2.3–3.0 µm (Figure 4(D–F)). Conidia broadly ellipsoidal to ellipsoidal, 3.2–3.5 × 2.4–2.8 µm, with smooth walls (Figure 4(G)). Sclerotia absent. Asci and ascospores not observed.

Strain examined: SFC103236 and SFC20150303-M16

Note: Penicillium maximae is closely related to P. austrosinicum and P. sclerotiorum. This species can be distinguished from P. austrosinicum by slower growth on YES at 25 °C and from P. sclerotiorum by the absence of sclerotia [22].