Abstract

1. Introduction

Freshwater fungi have an important role in the decomposition of organic matter in ecological systems, by virtue of producing enzymes that break down wood and producing bioactive compounds against pathogenic bacteria, fungi, and nematodes [1–4]. There are more than 622 species (170 genera) of Ascomycetes; more than 226 species of Hyphomycetes (55 genera); and 183 species of Trichomycetes (3 orders), while little is known about the Basidiomycetes and Zygomycetes [3].

Sordariomycetes is one of the largest classes of Ascomycetes, with six subclasses, 32 orders, 105 families, and 1331 genera [5]. Its species are characterized by nonlichenized, flask-shaped fruiting bodies (perithecia) and unitunicate asci [6]. They can be found in soil, dung, leaf litter, fresh water, plants, arthropods, and mammals [3,5,7,8].

The genus Acrostalagmus, which belongs to the class Sordariomycetes, order Hypocreales, and family Hypocreaceae, was established by Corda (1838) [9], with the type species A. cinnabarinus Corda. The species belonging to this genus are characterized as verticillate conidiophores, with hyaline, egg-shaped conidia formed singly [10]. Members of Acrostalagmus are found in saffron soil, needle mushroom, vermicompost, and branches of cacao [11–14]. They are also known for their ability to produce a variety of secondary metabolites. To date, 27 species belonging to this genus are known, according to the Index Fungorum (www.indexfungorum.org).

The genus Bartalinia, which belongs to the class Sordariomycetes, order Amphisphaeriales, and family Bartaliniaceae, was established by Tassi (1900) [15], with the type species Bartalinia robillardoides. It is characterized by the production of fusiform conidia with an acute or blunt apex and having three to four septate. Members of the genus have frequently been isolated from the leaves, stems of medicinal plants, or dead aerial spines of Rosa canina L. [16–19]. B. robillardoides has been reported to produce taxol, an anticancer drug [20]. Several types of compounds have been investigated from B. robillardoides strain LF550, such as three new chloroazaphilones named helicusin E, isochromophilone X, and isochromophilone XI, which expressed antimicrobial activity towards Bacillus subtilis, Staphylococcus lentus, Candida albicans, Trichophyton rubrum, and Septoria tritici, and also inhibited PDE4 and AChE [21]. Currently, there are 17 accepted species in this genus [22].

The genus Collariella, which belongs to the class Sordariomycetes, order Sordariales, and family Chaetomiaceae, was introduced by Wang et al. [23]. Based on phylogenies derived from ITS, LSU rDNA, rpb2, and tub2 sequence data combined with morphological observations, seven species have been recognized [23]. Currently, nine species have been registered in Index Fungorum. The species belonging to this genus are characterized by the production of a dark collar-like apex around the ostiolar pore of the ascomata [23]. They are found in soil, dust, and air [23–25]. Some of them have been reported to produce a variety of metabolites, including chaetoquadrin E, cochliodinol B, prenisatin, and SB236049/SB236050/SB238569 [23].

In Korea, many studies on the fungal diversity of various habitats have been conducted, although the occurrence of freshwater fungi remains poorly understood. Until now, only five new species and eight new records from freshwater habitat have been reported in Korea [19,26–30].

During our collection from freshwater samples, three rare species were found in Korea: A. luteoalbus, B. robillardoides, and C. carteri.

2. Materials and methods

3. Results

3.1. Phylogenetic analysis

A BLAST search of ITS sequences via the NCBI database indicated that the isolates CNUFC-YR537-1, CNUFC-CNUP1-1, and CNUFC-NDR3-1 most closely resembled A. luteoalbus (GenBank accession no. KP050692), B. robillardoides (GenBank accession no. KJ710460), and C. carteri (GenBank accession no. KX976747), with 99.6% (542/544bp), 99.6% (539/541bp), and 99.8% (499/500bp) homology, respectively. The 28S sequences of A. luteoalbus (GenBank accession no. KJ443145), B. robillardoides (GenBank accession no. KJ710438), and C. carteri (GenBank accession no. KX976742) showed 100% (850/850bp), 99.8% (871/873bp), and 100% (487/487bp) homology with the 28S sequence of the isolates CNUFC-YR537-1, CNUFC-CNUP1-1, and CNUFC-NDR3-1, respectively.

On the basis of the ITS and 28S sequence analysis, the isolates CNUFC-YR537-1, CNUFC-CNUP1-1, and CNUFC-NDR3-1 were identical to A. luteoalbus, B. robillardoides, and C. carteri, respectively (Figures 1–3).

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Figure 1.

Phylogenetic tree based on neighbor-joining analysis of internal transcribed spacers (ITS rDNA) and 28S rDNA sequences of A. luteoalbus CNUFC-YR537-1 and A. luteoalbus CNUFC-YR537-2. Colletotrichum boninense and Glomerella cingulata were used as outgroups. Bootstrap support values of ≥50% are indicated at the nodes. The bar indicates the number of substitutions per position.

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Figure 2.

Phylogenetic tree based on neighbor-joining analysis of internal transcribed spacers (ITS rDNA) and 28S rDNA sequences of B. robillardoides CNUFC-CNUP1-1 and B. robillardoides CNUFC-CNUP1-2. Pestalotiopsis malayana and Phlogicylindrium uniforme were used as outgroups. Bootstrap support values of ≥50% are indicated at the nodes. The bar indicates the number of substitutions per position.

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Figure 3.

Phylogenetic tree based on neighbor-joining analysis of internal transcribed spacers (ITS rDNA) and 28S rDNA sequences of C. carteri CNUFC-NDR3-1 and C. carteri CNUFC-NDR3-2. Microascus trigonosporus was used as an outgroup. Bootstrap support values of ≥50% are indicated at the nodes. The bar indicates the number of substitutions per position.

3.2. Taxonomy

3.2.1. Taxonomy of CNUFC-YR537-1

A. luteoalbus (Link) Zare, W. Gams & Schroers, Mycological Research 108 (5): 581 (2004) (Table 2, Figure 4).

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Figure 4.

Morphology of A. luteoalbus CNUFC-YR537-1. (A), Colonies in corn meal agar (CMA). (B), Colonies in potato dextrose agar (PDA). (C–E), Conidiophores bearing conidia. (F,G), Conidiophores and phialides. (H), Conidia (scale bars D–H=20µm).

Table 2.

Morphological characteristics of CNUFC-YR537-1 compared to A. luteoalbus reference strain.

Characteristics	Present isolate	A. luteoalbusa
Colony	Slow-growing, attaining a diameter of 24–26mm after 7 d, dull orange to orange-brown; reverse of colonies were orange, becoming light orange at the center	Slow-growing, attaining a diameter of 35–10mm after 10 d, dull orange to orange-brown
Phialide	Flask-shaped, hyaline, measured 10–29×2.0–3.5 µm	Flask-shaped, hyaline, 12–23×2–4 µm
Conidia	Conidia were oval, pale reddish, measured 3.5–6.2×2.2–2.9 µm	Conidia oval, pale reddish brown in mass, 3.5–5.0×2.0–2.5 µm

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^aFrom the description by Zare et al. [10].

=Sporotrichum luteoalbum (Link), Magazin der Gesellschaft Naturforschenden Freunde Berlin 3 (1): 13 (1809).

=Verticillium luteoalbum (Link) Subram., Hyphomycetes: an account of Indian species, except Cercosporae: 649 (1971).

=Verticillium tenerum Nees, System der Pilze und Schwämme: 57, t. 4:55 (1817).

=Sporotrichum lateritium Ehrenb., Sylvae mycologicae Berolinenses: 22 (1818).

=Sporotrichum hippocastani Corda, Icones fungorum hucusque cognitorum 1: 10, t. 2:159 (1837).

=Acrostalagmus cinnabarinus Corda, Icones fungorum hucusque cognitorum 2: 15, t. 10:66 (1838).

=Sporotrichum luteoalbum Thüm., Fungi pomicoli. Monographische Beschreibung der auf den Obstfrüchten der gemässigten Klimate vorkommenden Pilze: 21 (1879).

=Sporotrichum vile P. Karst., Hedwigia 30: 303 (1891).

Description: Colonies of the strain grew slowly on PDA, appearing dull orange to orange-brown, and reaching 24–26mm in diameter after 7days of incubation at 25°C. The reverse of the colonies were orange, appearing a lighter orange in the centers. Conidiophores were erect, pale orange-brown, measured 33–121×2.0–4.0µm, and repeatedly verticillately branched in one to several orders. Phialides were flask-shaped, hyaline, and measured 10–29×2.0–3.5µm. Conidia were oval, pale reddish, and measured 3.5–6.2×2.2–2.9µm.

3.2.2. Taxonomy of CNUFC-CNUP1-1

B. robillardoides Tassi, Bollettino del Laboratorio de Orto Botanico Reale Universita Siena 3: 5 (1900) (Table 3, Figure 5).

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Figure 5.

Morphology of B. robillardoides CNUFC-CNUP1-1. (A), Colonies in potato dextrose agar (PDA). (B), Colonies in malt extract agar (MEA). (C,E), Conidiogenous cells with developing conidia. (D,F), Conidia. (C, D: observed under light microscope; E, F: observed under SEM) (scale bars C, D=20µm; E=15µm, F=10µm).

Table 3.

Morphological characteristics of CNUFC-CNUP1-1 compared to B. robillardoides reference strain.

Characteristics	Present isolate	B. robillardoidesa
Conidiogenous cell	Cylindrical to subcylindrical, ampuliform, measured 8.4–11.3×2.5–3.8μm	Ampulliform, hyaline, thin-walled, smooth, measured 4–8×3–4.5μm
Conidia	Subcylindrical to slightly curved fusoid, 4-septate, measured 18.3–24.2×3.0–3.8μm	Subcylindrical, 4-septate, smooth, slightly constricted at the septa, measured (19–) 21–24(–27)×3–4μm
Apical appendage	Three-branched, 12.4–18.2μm long	Three-branched, measured (15–)16–20(–22) μm long
Basal appendage	Single, unbranched, filiform, excentric, 3.8–7.3μm long	Single, unbranched, filiform, flexuous, excentric, 4–7μm long

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^aFrom the description by Crous et al. [16].

≡Seimatosporium robillardoides (Tassi) Arx, The genera of fungi sporulating in pure culture: 224 (1981).

Description: Colonies of the strain grew rapidly on PDA, were greyish, and reached 80–83mm in diameter after 14d culture at 25°C. Conidiomata were globose or subglobose, appearing black to brownish black, and measured 276.7–482.2×228.1–364.8μm. Conidiophores were reduced to conidiogenous cells, which were hyaline, cylindrical to subcylindrical, ampuliform, measured 8.4–11.3×2.5–3.8μm, and were formed from the inner cells of the peridial wall. Conidia were subcylindrical to slightly curved fusoid, basal cell obconic with a truncate base, 4-septate, and measured 18.3–24.2×3.0–3.8μm. The apical appendage was 12.4–18.2μm long and three-branched. The basal appendage was 3.8–7.3μm long, single, unbranched, filiform, and excentric.

3.2.3. Taxonomy of CNUFC-NDR3-1

C. carteri X. Wei Wang, Houbraken & Samson, Studies in Mycology 84: 179 (2016) (Table 4, Figure 6).

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Figure 6.

Morphology of C. carteri CNUFC-NDR3-1. (A), Colonies in potato dextrose agar (PDA). (B), Colonies in malt extract agar (MEA). (C), Ascomata mounted in lactophenol. (D), Tip of terminal hairs. (E,F), Asci containing ascospores. (G,H), Ascospores. (C– F: observed under light microscope; G, H: observed under SEM) (scale bars E, F=20µm; G, H=5µm).

Table 4.

Morphological characteristics of CNUFC-NDR3-1 compared to C. carteri reference strain.

Characteristics	Present isolate	C. carteria
Ascomata	Globose to subglobose, 81–364.5×78–247.9 µm	Globose to subglobose, 175–320×110–220 µm
Ascomatal hair	3–5 µm diameter at the base, tapering and fading towards the tips	4–8.5μm near the base, tapering and fading towards the nearly hyaline tips
Asci	Fasciculate, clavate or slightly fusiform, contained seven to eight ascospores, measured 16.5–31.5×7.8–10.8 µm	Fasciculate, clavate or slightly fusiform, eight ascospores, measured 17–26×8.5–11.5μm
Ascospores	Brown, limoniform, usually biapiculate at both ends, measured 5.0–6.7×4.5–5.5 µm	Olivaceous when mature, limoniform, usually biapiculate at both ends, bilaterally flattened, measured (4.5–)5–6(–7.5)×4.4–5μm

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^aFrom the description by Wang et al. [23].

Description: Colonies of this strain grew moderately slowly on PDA, reaching 35–37mm in diameter after 7d incubation at 25°C. The colony color was pale yellowish and then became greenish/olivaceous with age. Ascomata were globose to subglobose, and measured 81–364.5×78–247.9µm. Ascomatal hair were 3–5µm diameter at the base, tapering and fading towards the tip. Asci were fasciculate, clavate, or slightly fusiform, contained seven to eight ascospores, and measured 16.5–31.5×7.8–10.8µm. Ascospores were brown, limoniform, usually biapiculate at both ends, and measured 5.0–6.7×4.5–5.5µm.

4. Discussion

The use of DNA sequences has dramatically increased the number of fungal species being recognized and identified [38,39]. Especially the ITS-5.8S rDNA sequences have become important features for the rapid identification of fungi [40,41]. The LSU region on its own or in combination with the ITS region is also valuable in identifying fungi at the intermediate taxonomic level [41,42].

Phylogenetic analysis based on ITS and 28S sequences showed that CNUFC-YR357-1 and CNUFC-YR357-2 strains were clustered within the same clade as A. luteoalbus from NCBI (Figure 1). The results of our molecular data analysis were consistent with the phylogeny presented by Grum-Grzhimaylo et al. [43]. The morphological characteristics of the A. luteoalbus isolate studied were generally similar to those previously described by Zare et al. [10], who transferred this species to genus Acrostalagmus from genus Verticillium, based on their molecular studies. Species of A. luteoalbus have been reported to produce two new indole diketopiperazines, namely luteoalbusins 1 and 2, along with eight previously known ones (3–10). These compounds were evaluated for their cytotoxic activities against SF-268, MCF-7, NCI-H460, and HepG-2 cell lines, and compounds 1–5 showed significant cytotoxicity against all four cancer cell lines [44]. Two new epipolythiodioxopiperazines, named chetracins E and F (1 and 2), along with the previously known chetracin C, exhibited cytotoxicity against the five tested cancer cell lines at the low-micromolar or nanomolar IC50 values isolated from A. luteoalbus [45]. A novel alkali-tolerant rhamnosidase was isolated from the soil fungus A. luteoalbus from Argentina [46].

Our analyses of ITS and 28S sequences showed that the strains CNUFC-CNUP1-1 and CNUFC-CNUP1-2 clustered with B. robillardoides CBS 122705 (type species), with well-supported branches, in full agreement with previous studies by Crous et al. [16] and Wanasinghe et al. [19] (Figure 2). The CNUFC-CNUP1-1 isolate was morphologically most similar to B. robillardoides, as described by Crous et al. [16]. B. robillardoides, B. laurina, and B. rosicola have 4-septate conidia. However, there is significant morphological variation in the conidial measurement; B. robillardoides has a conidia length/width ratio of 6.4 [16]; B. laurina has a conidia length/width ratio of 7.4 [47]; B. rosicola has a conidia length/width ratio of 3.8 [19]. Some Bartalinia species have been recorded as plant pathogens that cause diseases on economically important crops [22]; while some species, such as B. robillardoides, are well known for their secondary metabolites used in pharmaceutical industry. Hence, it is necessary to explore the biodiversity of Bartalinia species in Korea.

Phylogenetic analysis based on ITS and 28S sequences showed that CNUFC-NDR3-1 clustered within the same clade as C. carteri CBS 128.85 (type species) (Figure 3). The morphological features of our isolates corresponded well with the description of C. carteri by Wang et al. [23]. However, our observations showed that the CNUFC-NDR3-1 isolate produces seven to eight ascospores per ascus, whereas the ascus of C. carteri CBS 128.85 produces eight ascospores, as described by Wang et al. [23]. Our results suggest that different isolates of the same species of C. carteri have different number of ascospores. Eight ascospores in each ascus are frequently occurring in comparison to seven ascospores (rare). This species is also showed to produce metabolite such as cochliodinol B and prenisatin [23].

This study significantly improved our understanding of the rare ascomycete genera Acrostalagmus, Bartalinia, and Collariella in freshwater from Korea. There are numerous unknown freshwater derived species still awaiting description. In addition, three species obtained from this study may potentially be highly valuable. Thus, the potential biological activities of A. luteoalbus, B. robillardoides, and C. carteri should be further studied.

Funding Statement

This work was in part supported by the Graduate Program for the Undiscovered Taxa of Korea, and in part by the Project on Survey and Discovery of Indigenous Fungal Species of Korea funded by NIBR and Project on Discovery of Fungi from Freshwater and Collection of Fungarium funded by NNIBR of the Ministry of Environment (MOE), and in part carried out with the support of Cooperative Research Program for Agriculture Science and Technology Development (PJ013744), Rural Development Administration, Republic of Korea. This work was in part supported by the BK21 plus program through the National Research Foundation (NRF) funded by the Ministry of Education of Korea.

References