Diversity of Marine-Derived Aspergillus from Tidal Mudflats and Sea Sand in Korea.

Aspergillus (Trichocomaceae, Eurotiales, and Ascomycota) is a genus of well-defined asexual spore-forming fungi that produce valuable compounds such as secondary metabolites and enzymes; however, some species are also responsible for diseases in plants and animals, including humans. To date, 26 Aspergillus species have been reported in Korea, with most species located in terrestrial environments. In our study, Aspergillus species were isolated from mudflats and sea sand along the western and southern coasts of Korea. A total of 84 strains were isolated and identified as 17 Aspergillus species in 11 sections on the basis of both morphological characteristics and sequence analysis of the calmodulin gene (CaM) locus. Commonly isolated species were A. fumigatus (26 strains), A. sydowii (14 strains), and A. terreus (10 strains). The diversity of Aspergillus species isolated from mudflats (13 species) was higher than the diversity of those from sea sand (five species). Four identified species-A. caesiellus, A. montenegroi, A. rhizopodus, and A. tabacinus-are in the first records in Korea. Here, we provide detailed descriptions of the morphological characteristics of these four species.

Genus Aspergillus consists of well-defined asexual sporeforming fungi and is classified as Trichocomaceae, Eurotiales, Eurotiomycetes, and Ascomycota. Approximately 64% of the 250 described species of aspergilli have not been studied regarding their sexual state [1]. Historically, species of Aspergillus were called as several names: Chaetosartorya, Cristaspora, Dichotomomyces, Emericella, Eurotium, Fennellia, Neocarpenteles, Neopetromyces, Neosartorya, and Petromyces [2]. However, genus Aspergillus has been recently unified by mycologists in an effort to simplify the taxonomy by using a single genus name following ICN guidelines (International Code of Nomenclature for algae, fungi, and plants) [3].

Since Aspergillus was first described in the 18th century [4], 339 species have been reported worldwide and have been categorized into four subgenera (Aspergillus, Circumdati, Fumigati, and Nidulantes) and 20 sections [2]. Morphological features of Aspergillus are crucial for initial identification [2]; however, morphology alone has proven unreliable because several morphological features are influenced by environmental conditions such as composition of the medium, pH, additives, or temperature [5]. To overcome these limitations, mycologists recently proposed standardized methods involving polyphasic analysis including morphological and molecular analyses and extrolite profiling [2]. Molecular identification methods were first introduced into fungal taxonomy as sequencing of the internal transcribed spacer (ITS) region, and this locus has been proposed as the universal DNA barcode marker for all fungi. More recently, additional DNA markers for Aspergillus such as calmodulin (CaM), β-tubulin (BenA), and the RNA polymerase II second largest subunit (RPB2) have been used in Aspergillus taxonomy [2]. In particular, CaM has proven a useful marker for Aspergillus identification because the CaM database is well established, the locus is relatively easy to amplify, and adequate polymorphism allows for accurate identification of Aspergillus species [2].

Aspergillus is known for plant pathogen and cause of respiratory diseases in humans, and produces various mycotoxins such as aflatoxin and ochratoxin A [6], however, Aspergillus plays important ecological roles by degrading starches, hemicelluloses, celluloses, and other polysaccharides [7]. Therefore, Aspergillus species have been industrially exploited for potential applications of their enzymes [68]. Aspergillus species are isolated from various environments [2]. In addition, many Aspergillus species have been reported from marine environments including sponges and corals, but most Aspergillus studies have been focused on their bioactive compounds rather than their diversity [910].

There are various types of the intertidal zone depending on various geographical factors [1112]. The intertidal zone in Korea usually appears as mudflats or sea sand. Intertidal zones are ecologically valuable and provide habitats and feeding areas for diverse flora and fauna populations [13]. In addition, many microbes live in intertidal zones and perform critical ecological roles such as degradation of organic materials and synthesis of various bioactive compounds [1415]. On the other hand, most researches about intertidal zones have been focused on bacterial communities [1617] rather than fungal communities.

To date, 26 species of Aspergillus have been reported in Korea [18]. Although some Aspergillus species were reported from soil and inside buildings, most of them were isolated from fermented foods and studied as a potential fermentation initiator for production of foods and drinks such as meju, soy sauce, and traditional alcohol [19]. On the other hand, diversity of marine-derived Aspergillus has been poorly studied. Since the Ministry of Ocean and Fisheries established the Marine Fungal Resource Bank, marine-derived fungi have been studied to promote exploration of marine biodiversity and biological resources. We studied Aspergillus from intertidal mudflats and sea sand on both the western and southern coasts of Korea from 2014 to 2015. Based on sequence analysis of the CaM locus, several Aspergillus strains were identified at the species level. During the study, we found four previously unrecorded species in Korea (A. caesiellus, A. montenegroi, A. rhizopodus, and A. tabacinus). In this study, we describe macromorphological and micromorphological characteristics of these four species.

MATERIALS AND METHODS

Collection and isolation

Samples were collected from both the western and southern coasts of Korea. Collection was carried out during summer and winter from 2014 to 2015. Each collection was repeated five times after removal of the top 0.5 to 1.0 cm of soil, and each collection site was randomly chosen within a 10 m distance. After collection, the samples were stored at 4℃ prior to isolation. Each sample was diluted 10-fold with artificial sea water [20]. Next, 100 µL of each dilution was spread on three different media plates, which contained artificial sea water: potato dextrose agar (PDA; Difco, Becton Dickinson, Sparks, MD, USA), glucose yeast extract agar (1 g/L glucose, 0.1 g/L yeast extract, 0.5 g/L peptone, and 15 g/L agar), and dichloran rose bengal chloramphenicol agar (Difco, Becton Dickinson). All plates were incubated at 25℃ for 1 wk to confirm distinguishable morphological features. Each Aspergillus strain was transferred to a new PDA plate. Each strain was then stored in 20% glycerol at −80℃ at the Seoul National University Fungus Collection (SFC).

Molecular procedures and phylogenetic analysis

DNA was extracted using a modified cetyltrimethylammonium bromide protocol [21]. The PCR amplification of the CaM locus was conducted as described by Park et al. [22]. Gel electrophoresis was carried out for each amplicon on a 1% agarose gel, and these PCR products were purified using the Expin PCR Purification Kit (Geneall Biotechnology, Seoul, Korea) following the manufacture's instruction. The purified amplicons were sequenced using corresponding PCR primers by Macrogen (Seoul, Korea) in both forward and reverse directions using an ABI Prism 3700 genetic analyzer (Life Technologies, Gaithersburg, MD, USA).

Sequences were assembled, proofread, edited, and aligned using MEGA v.5 [23] and were deposited in GenBank (Table 1). For multiple sequence alignments, MAFFT v.7 [24] was used, and each sequence was checked and adjusted by eye. After alignment, maximum likelihood phylogenetic trees were constructed. The phylogenetic trees were generated using RAxML 8.0.2 [25] and the GTR-GAMMA model of evolution with 1,000 bootstrap replicates.

Table 1

Information of isolated Aspergillus strains from mudflat and sea sand in different seasons

aSFC stands for abbreviation of ‘Seoul National University Fungus Collection’.

Morphological analysis

To examine both macro- and micromorphological characteristics of the unrecorded species, each strain was inoculated as a spore suspension at three points on plates with one of three different media: Czapek yeast autolysate agar (CYA, yeast extract; Difco), malt extract agar (MEA, Oxoid), and yeast extract sucrose agar (YES, yeast extract; Difco). All CYA, MEA, and YES plates were incubated at 25℃ for 7 days, and CYA plates were incubated at 30℃, 37℃, and 50℃ for additional 7 days. All plates with media were prepared as described by Samson et al. [2]. The color names and alphanumeric codes for macromorphological characteristics were assigned using the criteria of Kornerup and Wanscher [26]. Analysis of micromorphological characteristics was performed following the methods of Samson et al. [2].

RESULTS

Totally, 84 Aspergillus strains were isolated from two intertidal substrates (mudflats and sea sand) at 30 sites from the western and southern coasts of Korea. More Aspergillus strains were isolated (74 strains) from mudflats rather than from sea sand (10 strains). Furthermore, higher diversity of Aspergillus was observed in mudflats (16 species) compared to sea sand (six species). Five species (A. fumigatus, A. terreus, A. welwitschiae, A. venenatus, and Aspergillus sp. 1) were commonly found from both substrates, and A. sydowii was found only from mudflats. More Aspergillus strains were isolated during winter (52 strains) than summer (32 strains). Diversity of Aspergillus species was higher in winter (14 species) than in summer (10 species), and six species (A. clavatus, A. fumigatus, A. rhizopodus, A. ruber, A. sydowii, A. terreus, and A. welwitschiae) were commonly found during both seasons (Table 1). On the basis of CaM sequences, 84 isolates were identified as 17 species in 11 sections, and three strains were identified as Aspergillus sp. due to ambiguous phylogenetic relationship (Table 1). For convenience, sections were grouped into four groups regardless of phylogenetic relationship.

Three sections were included in group 1: Versicolores, Nidulantes, and Flavi. Three species (A. sydowii, A. tabacinus, and A. venenatus) were confirmed to belong to section Versicolores. Seven representative strains (SFC20160407-M09, SFC20160805-M44, SFC20160805-M46, SFC20160805-M47, SFC20160805-M48, SFC20160805-49, and SFC20160805-M51) were identified as A. sydowii (sequence similarity, 99%; bootstrap support, 99%) (Fig. 1). Although one isolated strain (SFC20160407-M11) formed a monophyletic group with low bootstrap support, its morphology and CaM sequence similarity were very similar to those of strains of A. tabacinus (NRRL 4791T, NRRL5031, S823, and NRRL A-23173); thus, it was identified as A. tabacinus (sequence similarity, 99.8~100%; bootstrap support, 78%) (Fig. 1). One strain (SFC20160407-M10) was identified as A. venenatus (sequence similarity, 99.8%; bootstrap support, 99%) (Fig. 1). In section Nidulantes, two strains (SFC20150812-M13 and SFC20160805-M50) were isolated from mudflats (Table 1), and each strain was identified as either A. nidulans (sequence similarity, 99.8%; bootstrap support, 100%) or A. montenegroi (sequence similarity, 99.3%; bootstrap support, 100%), respectively. Two strains (SFC20160805-M30 and SFC20160805-M31) in section Flavi were identical to type strains of A. flavus (NRRL 1957T) and A. oryzae (NRRL 447T), with 100% sequence similarity (Fig. 1). However, we identified these specimens as Aspergillus sp. 1 because it was difficult to distinguish these two species by CaM sequences.

Fig. 1

A maximum likelihood phylogenetic tree of Aspergillus on the basis of sequences of the calmodulin (CaM) locus. Bootstrap scores > 70 are presented at the nodes. The scale bar indicates the number of nucleotide substitutions per site. “T” indicates ex-type strains. Each group was designed depending on the arrangement of sections in the phylogenetic tree.

Group 2 included sections Terrei and Flavipedes and showed high bootstrap support. Two strains (SFC20160805-M43 and SFC20160805-M45) were identified as A. terreus in section Terrei (sequence similarity, 99.6~100%; bootstrap support, 76%) (Fig. 1). In section Flavipedes, SFC20160805-M32 formed a group with type strains of A. mangaliensis (CCF4698T: sequence similarity, 96.5%) and A. templicola (DTO270C6T: sequence similarity, 97.1%), but we identified it as Aspergillus sp. 2 because of unclear phylogenetic relationship (Fig. 1).

Group 3 included three sections (Clavati, Fumigati, and Nigri). Six strains (SFC20160805-M33, SFC20160805-M34, SFC20160805-M35, SFC20160805-M36, SFC20160805-M37, and SFC20160805-M38) were confirmed to belong to section Fumigati and were identified as A. fumigatus (sequence similarity, 99.6~100%; bootstrap support, 96%) (Fig. 1). SFC20160805-M39 was found to be closely related to A. papuensis (CBS 841.96T), A. coversis (CCF4190T), and A. brevistipitatus (CCF419T) (sequence similarity, 93.1~95.3%), but it was named Aspergillus sp. 3 due to unresolved phylogeny. In section Clavati, the strains (SFC20150303-M01 and SFC20160407-M08) were identified as A. clavatus (sequence similarity, 99.8%; bootstrap support, 100%) and A. rhizopodus (sequence similarity, 99.9%; bootstrap support, 100%), respectively (Fig. 1). In section Nigri, four strains (SFC20160317-M20, SFC20160805-M40, SFC20160805-M41, and SFC20160805M-42) were identified as A. welwitschiae (sequence similarity, 100%; bootstrap support, 97%) (Fig. 1).

Group 4 included three distantly related sections (Aspergillus, Restricta, and Circumdati). In section Aspergillus, two strains (SFC20160317-M25 and SFC20160805-M29) were identified as A. ruber (sequence similarity, 99.8~100%; bootstrap support, 99%), and the other strain (SFC20160317-M27) was identified as A. pseudoglaucus (sequence similarity, 99.6%; bootstrap support, 94%) (Fig. 1). Strain SFC201601112-M06 was identified as A. caesiellus in section Restricta (sequence similarity, 100%; bootstrap support, 100%). One strain (SFC20160112-M05) was identified as A. westerdijkiae in section Circumdati (sequence similarity, 99.83%; bootstrap support, 100%).

Taxonomy

Aspergillus caesiellus

Saito, J. Coll. (1904) (Fig. 2A)

Fig. 2

Morphology of Aspergillus caesiellus (SFC201601112-M06) (A), A. montenegroi (SFC20160805-M50) (B), A. rhizopodus (SFC20160407-M08) (C), and A. tabacinus (SFC20160407-M11) (D). a~c, incubation for 7 days at 25℃. Upper: a, Czapek yeast autolysate agar (CYA); b, malt extract agar (MEA); c, yeast extract sucrose agar (YES). Lower: a, CYA reverse; b, MEA reverse; c, YES reverse. d, e, conidiophores; f, conidia (scale bars = 10 µm).

Description

Colony diameter, at 25℃ for 7 days, CYA 8~13 mm; CYA 30℃: 14~15 mm; no growth at 37℃ and 50℃; MEA 13~14 mm; YES 29~31 mm.

Colonies on CYA, lightly sulcate, heavy sporulation, velutinous, central part gray (26C1) to grayish green (25C3) with 3-mm gray margin (26C1), no exudates, no pigmentation, reverse central part light brown (6D4) to dull green (28E4).

Colonies on MEA, sporulation, floccus, central part dull green (25D3) to dark turquoise (24F5) with 1-mm white margin, no exudates, no pigmentation, reverse central part dull green (27D3) to dull green (30D4).

Colonies on YES, sporulation, central floccus, velutinous, central part greenish gray (25D2) to dark green (25F4) with 1 mm white margin, no exudates, no pigmentation, reverse grayish yellow (4C4).

Conidiophore smooth with slightly green wall (61~) 82~ 95 (~114) × 4.6~5.2 (~5.5) µm wide, vesicles spatulate (10.7~) 11.1 × 13.5 (~15.4), conidial head uniseriate, metulae covering half of upper surface, (4.4~) 4.5~5.6 (~5.8) × (2.6~) 2.8~3.4 (~3.9) µm, phialide (2.4~) 2.5~2.6 × 7.0~8.2 (~8.6) µm, conidia ellipse and pyriform with greenish gray (4.4~) 4.5~5.5 (~6) × (2.4~) 2.6~2.8 µm diam.

Strain examined

SFC20160112-M06.

Remarks

A. caesiellus is similar to A. penicillioides but is distinguished by more vigorous growth and formation of conidia on CYA and MEA media. A. caesiellus grows more slowly and its cylinder becomes more round.

Aspergillus montenegroi

Y. Horie, Miyaji & Nishim (1996)(Fig. 2B)

Colony diameter, at 25℃ for 7 days, CYA 52~58 mm; CYA 30℃ 64~70 mm; CYA 37℃ 66~72 mm; no growth at 50℃; MEA 64~72 mm; YES 66~70 mm.

Colonies on CYA, not sulcate, sporulation, floccus, and velutinous, central part yellowish gray (3C2) to olive (3D3) and grayish green (28E5) with 3 mm white margin, no exudate, pigmentation grayish red (7B3), reverse central part reddish gray (7B2) to brownish orange (7C4) with reddish gray (8B2) margin.

Colonies on MEA, not sulcate, sporulation velutinous, dull green (27E3) with 1-mm white margin, no exudate, no pigmentation, reverse central part brown (6E4) to light brown (6D4).

Colonies on YES, sulcate, sporulation, velutinous, central part purplish gray (13B2) to greenish yellow (1B8) and grayish green (27E6) with 2 mm white margin, no exudate, no pigmentation, reverse yellowish brown (5D5).

Conidiophore smooth and thick-walled with orange-brown (63.9~) 65~88.8 (~110.8) × (3.5~) 4.3~5.3 (~5.5) µm, vesicles hemispherical with orange-brown to dark brown 9.7~9.9 × (10.8~) 11.8~13.5 (~13.8) µm, conidial heads biseriate, metulae covering over half of upper surface (4.4~) 4.5~5.6 (~5.8) × (2.6~) 2.8~3.4 (~3.9) µm, phialide (4.7~) 5.2~5.6 (~6.1) × (2~) 2.1~2.6 (~2.7) µm, conidia hyaline with grayish green to green (2.8~) 2.9~4.3 (~4.7) × (2.1~) 2.9~3.4 (~3.6) µm.

SFC20160805-M50.

The spore of Aspergillus montenegroi has similar morphology to that of the Aspergillus nidulans. However, the spore of A. montenegroi has reticulate ornamentation on the convex wall, whereas the spore of A. nidulans has a smooth wall.

Aspergillus rhizopodus

J. N. Rai, Wadhwani & S. C. Agarwal (1975) (Fig. 2C)

Colony diameter, at 25℃ for 7 days, CYA 58~62 mm; CYA 30℃ 65~68 mm; CYA 37℃ 19~22 mm; no growth at 50℃; MEA 52~53 mm; YES 74~76 mm.

Colonies on CYA, weakly sulcate, heavy sporulation, velutinous with a floccus central part, dull green (25D3) with 2mm white margin, no exudate, no pigmentation, reverse central part light brown (5D4) to olive brown (4D4).

Colonies on MEA, weakly sulcate, heavy sporulation floccus, dull green (25D3) with 7-mm white margin, slight exudates, no pigmentation, reverse central part brownish orange (5C6) to grayish orange (5B4).

Colonies on YES, weakly sulcate, heavy sporulation, floccus, central part grey (24C1) to grayish turquoise (24D3) with 2 mm white margin, no exudates, no pigmentation, reverse grayish yellow (4C5).

Conidiophore smooth-walled with light brown (210~) 340~502 (~550) × (5.9~) 6.3~8.2 µm, vesicles clavate form 14.9~15.9 (~16.4) × 19~20.8 µm, and conidial heads biseriate, metulae covering entire vesicle surface, (4~) 4.9~5.5 (~5.8) × (2.4~) 2.5~3.1 (~3.2) µm, phialide (5.2~) 5.6~5.9 (~6) × (1.4~) 1.6~2 (~2.1) µm, conidia spherical or subspherical with a light green smooth wall (1.9~) 2.1~2.7 µm.

SFC20160407-M08.

A. rhizopodus is similar to A. giganteus and A. longivesica but is distinguished by variously shaped foot cells with fingerlike projections and large conidial heads in the presence of light.

Aspergillus tabacinus

Nakaz., Y. Takeda, Simo & A. Watan (1934) (Fig. 2D)

Colony diameter, at 25℃ for 7 days, CYA 25~26 mm; CYA 30℃ 22~24 mm; no growth at 37℃ and 50℃; MEA 18~19 mm; YES 31~33 mm.

Colonies on CYA, sulcate, sporulation in central area, central part grayish beige (4C2) to greenish grey (27D2), brownish orange (5C4) with 2-mm white margin, no exudate, no pigmentation, reverse central part grayish orange (5B3) to yellowish white (4A2). No growth at 37℃ and 50℃.

Colonies on MEA, sulcate, sporulation moderate, dull green (26E2) with 2-mm white margin, no exudates, no pigmentation, reverse brownish orange (5C5).

Colonies on YES, lightly sulcate, rings in central area, central part yellowish white (2A2) to grayish green (27C3) with 3-mm white margin, no exudates, no pigmentation, reverse central part deep yellow (4A8) to pale yellow (4A3).

Conidiophore smooth-walled, light brown (353~) 368~ 448 (~514) × (5.2~) 5.4~6.3 µm wide, vesicles spatulate form 14.9~15.9 (~16.4) × 19~20.8 µm, conidial heads biseriate, metulae covering entire vesicle surface (4~) 4.9~5.5 (~5.8) × (2.4~) 2.5~3.1 (~3.2) µm, phialide (5.2~) 5.6~5.9 (~6) × (1.4~) 1.6~2 (~2.1) µm, conidia spherical or subspherical with light green smooth wall (1.9~) 2.1~2.7 µm.

SFC20160407-M08.

A. tabacinus has smooth-walled conidia similar to those of A. amoenus and A. austroafricanus, but A. tabacinus produces no soluble pigment, whereas A. austroafricanus produces a brown soluble pigment on the CYA medium.

DISCUSSION

In this study, we explored the diversity of Aspergillus derived from marine environments in Korea and identified each Aspergillus strain using CaM sequences, a locus that has previously been shown to be an effective DNA marker for Aspergillus identification [2]. A total of 17 species in 11 sections were identified, while three species were left unidentified (Aspergillus sp. 1~3) due to their ambiguity in morphological and phylogenetic analyses. Several Aspergillus species (A. fumigatus, A. nidulans, A. sydowii, A. terreus, A. versicolor, and A. westerdijkiae) have been reported from marine substrates such as deep sea sediments, marine sponges, corals, and mudflat [272829], and particularly A. fumigatus and A. sydowii were dominant in mud substrates [30]. Similarly, in our study, higher diversity of Aspergillus was detected in mudflats, and both A. fumigatus and A. sydowii were the most abundant species, as another study has shown [30]. The number of isolated strains and diversity of Aspergillus were similar in winter and summer, and we could not find a significant difference between the seasons. A. montenegroi, A. venenatus, and A. tabacinus are reported for the first time in marine environments.

Group 1: sections Versicolores, Nidulantes, and Flavi.

Section Versicolores was first introduced by Thom and Church [31], and 13 species are included in this section [32]. Pathogenic species in section Versicolores are generally isolated from various substrates including hypersaline water [33]. In our study, A. sydowii, A. tabacinus, and A. venenatus were isolated from a marine environment. A. sydowii is commonly isolated from marine environments as well and is known as a serious pathogen of marine organisms [34]. A. venenatus and A. tabacinus have previously been isolated from plant materials [32], but our study is the first report of these species in a marine environment. This study is the first report of A. tabacinus in Korea.

Species in section Nidulantes are usually found in soil, indoor environments, and foods, and are characterized by red or purple pseudoparenchymatous cleistothecium surrounded by hülle cells and purple ascospores [35]. In the present study, we identified two strains as A. nidulans and A. montenegroi. A. nidulans is characterized by a “nestlike” fruiting body [36] and is known for producing xylanase [37]. A. montenegroi was first isolated from soil by Horie et al. [38] (as Emericella montenegroi). A. montenegroi is characterized by ascospores with reticulate and convex walls [38]. This is the first report of this species in a marine environment and in Korea.

Species in section Flavi have a relatively thin and rough cell wall as compared to species in other sections [39]. Species in this section are found in soil, air, organic materials, and plants [40]. In our study, five isolated strains were confirmed within section Flavi and form a monophyletic group with type strains of A. flavus and A. oryzae. Both species are generally found in terrestrial environments such as soil and plant products, but are also found in marine environments [4142]. A. flavus is a representative species in that section and is known as an opportunistic pathogen of plants and animals due to aflatoxin production, whereas A. oryzae is used in food fermentation [4243]. However, it is difficult to distinguish A. flavus and A. oryzae because of their similar morphological characteristics and ambiguous phylogenetic relationship [44]. Therefore, we denoted five strains as Aspergillus sp. 1, which showed high sequence similarity to both A. flavus and A. oryzae; thus, further research is necessary to unambiguously identify these strains.

Group 2: sections Terrei and Flavipedes.

Species in section Terrei were first described by Gams et al. [45] and commonly have brown columnar conidial heads. They are ubiquitous and considered economically important, especially in the fermentation industry [46]. In section Terrei, 10 strains were identified as A. terreus. This species is commonly isolated from terrestrial habitats such as deserts, grassland soils, and plant products but has also been isolated from a soft coral [28]. A. terreus is used for food fermentation because of its production of itaconic or itatartaric acid [4748]; however, A. terreus is also a known human pathogen that induces an inflammatory response [49]. A. terreus produces aspernolides A and B as well as butenolides [28]. In section Flavipedes, only one strain, Aspergillus sp. 2, is present, and it could not be clearly distinguished from either A. mangaliensis or A. templicola due to weak phylogenetic support and ambiguous morphological features.

Group 3: sections Fumigati, Clavati, and Nigri.

Section Fumigati was first categorized as an A. fumigatus group by Raper and Fennel [40]. This identification was based on morphology and has remained a controversial issue due to the similarity of these morphological features among other taxa [5051]. Most species in this section are pathogenic or allergenic to humans owing to mycotoxins [5253]. In our study, two Aspergillus species (A. fumigatus and Aspergillus sp. 3) were found in this section. A total of 26 strains were identified as A. fumigatus in section Fumigati. A. fumigatus is the most abundant species isolated in this study. A. fumigatus is generally found in forest soils and air. A. fumigatus is often the dominant fungal species in mudflats [30, 54,55]. Like other species in section Fumigati, A. fumigatus is generally characterized by green conidia and phialides [5657]. A. fumigatus is known for causing a pulmonary disease and an allergic reaction in the respiratory tract via mycelia colonization [57]. In addition, one strain, Aspergillus sp. 3 (SFC20160805-M39), was confirmed to belong to section Fumigati; however, it could not be identified at the species level because it is phylogenetically and morphologically different from reported type species such as A. papuensis (CBS 841.96T), A. coversis (CCF4190T), and A. brevistipitatus (CCF419T).

Section Clavati was initially classified as a part of the Aspergillus clavatus group along with A. giganteus by Thom and Church [31]. Section Clavati is considered a sibling group of section Fumigati and includes six Aspergillus species that show alkali tolerance and produce various mycotoxins such as patulin and tryptoquivalones [585960]. In the present study, two species (A. clavatus and A. rhizopodus) were found in section Clavati. A. clavatus is generally found in soil, dung, and stored foods [61] and produces various compounds such as hydroxylamine [59]. A. rhizopodus was first reported by Rai et al. [62] and was considered a synonym of A. giganteus [63] because of their similar macromorphology. In contrast, the former species is characterized by distinct, long conidiophores and is an alkalophilic fungus [6062]. A. rhizopodus has never been recorded in Korea.

Section Nigri, which is generally called black molds, currently consists of 26 species [64]. Prior to the introduction of molecular markers, this section was difficult to identify to the species level because of very similar morphology within the section. In our study, six strains were isolated and identified as A. welwitschiae. The latter was first introduced by Wehmer [65] and is sometimes confused with A. niger owing to similar morphological characteristics, but it can be distinguished by the ability to produce ochratoxin A [66].

Group 4: sections Circumdati, Aspergillus, and Restricta.

Section Circumdati was initially classified as a part of the A. ochraceus group by Raper and Fennell [40] but later categorized into section Circumdati [67]. Twenty-seven species are currently recognized in this section and are characterized by yellow conidia [68]. Species in section Circumdati are economically important due to their production of various bioactive compounds such as ochratoxin [68]. In the present study, one isolated strain of A. westerdijkiae was identified as belonging to section Circumdati. A. westerdijkiae is commonly isolated from fruits or grains but has been isolated from deep-sea environments as well [69]. A. westerdijkiae produces various alkaloid compounds [69].

Section Aspergillus was first reported by Raper and Fennell [40] and is considered one of the main sections in subgenus Aspergillus [2]. Most species in section Aspergillus were formerly classified within Eurotium [7]. Species in section Aspergillus are generally distinguished from other sections by their distinct morphological characteristics such as yellowish hyphae, greenish yellow conidial heads, and red hyphae [7]. A. pseudoglaucus and A. ruber were isolated from sea sand and mudflats, respectively. These species are generally isolated from fermented foods [70, 71]. A. ruber is recognized as a highly valuable species via its production of tannase and various secondary metabolites including aflatoxin B [7072]. A. pseudoglaucus was first named Eurotium repens by de Bary [73] but was recently renamed A. pseudoglaucus by Hubka et al. [7]. A. pseudoglaucus is known for cytotoxic activity and production of echinulin [74].

Section Restricta was first classified within the A. glaucus group by Thom and Raper [75], but later renamed as section Restricta by Gams et al. [45]. In our study, A. caesiellus was isolated from mudflats, and this is the first report of this species in Korea. A. caesiellus has been generally found in plants [76]; several enzymes, including keratinase, cellulases and xylanases, have been isolated from this species [7677].

In conclusion, in this study, we explored the diversity of Aspergillus in marine environments (mudflats and sea sand) during both summer and winter in Korea. We identified 17 species in 11 sections based on CaM sequences, whereas three specimens were identified to the genus level. This study shows four previously unrecorded Aspergillus species in Korea: A. caesiellus, A. montenegroi, A. rhizopodus, and A. tabacinus. Collectively, marine-derived Aspergillus species have been shown to produce over 120 secondary metabolites that have cytotoxic and antimicrobial properties and are therefore potentially important pharmacologically [7879]. Despite the high known diversity within the Aspergillus genus and the remarkable potential for production of useful industrial and medical compounds that have been found in this group, most of the recorded taxa within this genus remain unidentified to the species level. Therefore, we believe that this study will spur future research into the diversity of marine Aspergillus and will form the basis for the discovery of new bioresources through accurate identification of species in this genus.