Investigation of Marine-Derived Fungal Diversity and Their Exploitable Biological Activities

Marine fungi are potential producers of bioactive compounds that may have pharmacological and medicinal applications. Fungi were cultured from marine brown algae and identified using multiple target genes to confirm phylogenetic placement. These target genes included the internal transcribed spacer (ITS), the nuclear large subunit (LSU), and the β-tubulin region. Various biological activities of marine-derived fungi were evaluated, including their antifungal, antioxidant and cellulolytic enzyme activities. As a result, a total of 50 fungi was isolated from the brown algae Sargassum sp. Among the 50 isolated fungi, Corollospora angusta was the dominant species in this study. The genus Arthrinium showed a relatively strong antifungal activity to all of the target plant pathogenic fungi. In particular, Arthrinium saccharicola KUC21221 showed high radical scavenging activity and the highest activities in terms of filter paper units (0.39 U/mL), endoglucanase activity (0.38 U/mL), and β-glucosidase activity (1.04 U/mL).


Introduction
The marine environment is extremely complex and contains a broad spectrum of fungal diversity. Although a variety of new marine-derived fungal genera have been identified and evaluated [1], numerous marine fungi are identical to terrestrial fungi, e.g., Aspergillus spp., Cephalosporium spp.,

Fungal Phylogeny and Identification
A total of 50 fungal strains were isolated and investigated from the brown algae (Table 1). Two were identified as zygomycetes: Mucor circinelloides. The other 48 were identified as ascomycetes. Among isolated fungi, six strains were not identified at the species or genus level because their best matched references from BLAST search were lower than 95% of similarities.
In the phylogenetic tree based on ITS (Figure 1), most species were grouped in their own clade. However, five species, i.e., Acremonium sp. 1, Chaetomium sp., Colletotrichum sp., Eutypella sp., and Paradendryphiella sp., were identified at the genera level because they were grouped in their respective species complex. Six species, i.e., Acremonium sp. 2, Arthrinium sp. 1, Arthrinium sp. 2, Pleosporaceae sp. 1, Pleosporaceae sp. 2, and Valsaria sp., were identified at genera or family level due to their best matched sequences from BLAST search had low similarities. They were placed in their own clade without close reference species. Four species, i.e., Aspergillus sp., Sordariomycetes sp. 2, Sordariomytcetes sp. 3, and Xylaliales sp., were not identified at species level, despite their own clades with references, because two of the other analyzed regions, LSU and β-tubulin, were not supported by the ITS-based identifications.
The phylogenetic relationship of the ITS-based tree was not clear because some nodes had low posterior probabilities. Thus, phylogenetic tree based on combined dataset from ITS, LSU and β-tubulin sequences was constructed to confirm simultaneous relationship of the isolates (Figure 2). The outline of this tree was similar to the ITS-based tree. Especially, branching pattern of early diverging nodes that classified taxa into class level was agreed with the ITS-based tree. Therefore, the phylogenetic tree based on ITS region could represent their phylogenetic relationships.
The most abundant species was Corollospora angusta (26%). C. angusta has a broad distribution in marine habitats, such as algae and mangroves [26][27][28], but it has not been reported in Korea. It has been suggested that this species is highly associated with its substrate, Sargassum sp. In both MEA and PDA media, this species showed a very slow growth rate (less than 1 mm per day). The most diverse genus was Arthrinium (12%), which contained four species (Figures 2 and 3). Arthrinium is a commonly isolated genus from not only plants and soil but also marine environments [7,11]. In Korea, two Arthrinium species, A. arundinis and A. phaeospermum, have been reported [29,30] but have not been fully described. A. arundinis is also known as a plant pathogen of barley [31]. In Korea, A. arundinis was isolated from bamboo [30], but marine A. arundinis has not been reported. The first A. saccharicola was isolated from Saccharum officinarum [32]. Although A. saccharicola has been reported from various habitats, such as plants, air [11], and seawater [33], it has not been reported in Korea.      In our phylogenetic tree of Arthrinium spp. (Figure 3), each Arthrinium species was classified at the species level with high posterior probability (p.p.). A. arundinis KUC21261 and KUC21229 were placed in a monophyletic clade of A. arundinis with a high p.p. (1.0). A. saccharicola KUC21221 was grouped with reference A. saccharicola with a high p.p. (1.0). Conversely, Arthrinium sp. 1 KUC21228, KUC21232, and Arthrinium sp. 2 KUC21220 did not join the clade with reference Arthrinium but instead had their own clades. They also demonstrated low similarities with best matched references from BLAST search. We suggest that these unidentified Arthrinium are new species candidates. Further studies with detailed morphological analyses are required to prove the novelty of these species.
Acremonium was the third largest genus in this study (8%). It is a cosmopolitan genus that is commonly isolated from soil, wood [34,35], and seaweed [7,36]. Aspergillus spp. is a common saprophyte of various substrates [35], and two species were isolated. Some Aspergillus species are halophilic and are commonly observed in marine environments [7,37]. A. niger is common in various environments, including seawater [38,39]. In addition, Fusarium oxysporum, a well-known plant pathogen [40], is a commonly isolated soil fungus worldwide [35], and has been found in marine environments [41]. Mucor circinelloides was the solitary zygomycetous species found in this study. The genus, Mucor has been reported to occur in a variety of seaweed [7]. This saprophytic species is commonly isolated from various environments [35], including seawater [42], and has been utilized in the production of fermented food [43,44] and in biotechnology applications to produce lipids [45] and ethanol [46].
For marine fungi which were not identifiable at the species or genus level, further studies with morphological approaches are required. Certainly, identified fungal diversity data can help further studies on marine fungal diversities.

Antifungal Activity and Antioxidant Activity
The antifungal activity of extracts from each strain were examined against three species, A. saccharicola KUC21221, Arthrinium sp. 1 KUC21228, Arthrinium sp. KUC21232 and Arthrinium sp. 2 KUC21220. In contrast, C. gloeosporioides was weakly inhibited by marine-derived fungal extracts in this study. Plant pathogenic fungi are the main infection sources of damage during pre-or post-harvest in the agricultural industry.
There are several studies on the biological control of marine-derived compounds against phytopathogenic fungi [14]. Aqueous and ethanolic extracts from marine algae were shown to inhibit B. cinerea and Phytophthora cinnamon [47].
In this study, the genus Arthrinium showed potential antifungal activity to target fungi. To date, over 20 compounds have been isolated and studied from the genus Arthrinium. In particular, the origins of some compounds were marine habitats. Four new compounds, arthrinins A-C and arthrinins D were isolated from the sponge-derived Arthrinium sp. [48]. Additionally, (−)-hexylitaconic acid was isolated from sponge-derived Arthrinium sp. [49]. Three new chemical compounds, i.e., myrocin D, libertellenone E, and libertellenone F, were isolated from the marine fungus A. sacchari [50]. Plant-derived Arthrinium phaeospermum produced the antifungal compound arthrinic acid [51]. Biotic pesticide antifungal compounds can be useful in the control of infections caused by phytopathogenic fungi. The results of this study demonstrate the possibility of biological control of marine-derived fungal extracts.

Antioxidant Activity
In this study, 50 marine-derived fungal extracts were screened for their antioxidant capacity. The antioxidant activity was measured by the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. Among the fungal extracts, Arthrinium saccharicola KUC21221 and Aspergillus niger KUC21224 showed strong ABTS radical scavenging. A. saccharicola KUC21220 exhibited the highest DPPH radical scavenging activity, although A. niger KUC21224, Arthrinium sp. 2 KUC21220 and Xylariales sp. KUC21219 also exhibited activities. To the best of our knowledge, this is the first report of A. saccharicola activity on radical scavenging. Additionally, Diaporthe arecae, which is generally considered a plant pathogenic fungus, exhibited an antioxidant effect. Numerous marine filamentous fungi can produce antioxidant compounds and play an important role in the capture of free radicals. Excess free radicals or reactive oxygen species are harmful to human life [52]. Other studies have shown that various marine-derived fungal extracts are potent radical scavengers [53,54]. These efficient antioxidant compounds can be applied to the cosmetic industry. Kojic acid, a tyrosinase inhibitor, was screened through 600 marine-derived fungi and has been used as a cosmetic agent [21]. As an additional experiment, the radical scavenging activities of the extracts of cellulase-inducing media were investigated. However, there were no positive results among the extracts, although many fungal strains, such as A. saccharicola, showed high radical scavenging activity when cultivated on PDA (Table 1). It is believed that fungi are poor producers of antioxidants in cellulase-inducing media. This influence of culture media, such as the carbon source, on the secretion of secondary metabolites by fungi has been previously identified [55,56].

Cellulolytic Enzymes
To investigate the specific cellulolytic enzyme activities of the isolated fungi, the filter paper unit (FPU), endoglucanase (EG), β-glucosidase (BGL) and β-xylosidase (BXL) activities of all fungal strains were measured. FPU indicates the total cellulolytic enzyme activity. EG creates a free chain-end by attacking the non-crystalline regions of the cellulose fiber. BGL acts on β-1,4 bonds linking two glucose or glucose-substituted molecules, and BXL catalyzes the hydrolysis of 1,4-β-D-xylans [57]. Among the fungal isolates, Arthrinium saccharicola KUC21221 showed the highest activities in FPU (0.39 U/mL), EG (0.38 U/mL), and BGL (1.04 U/mL), and it showed a remarkable radical scavenging activity in this study (Table 1). These activities were comparable to the reported values of other wooddecaying fungi ( Table 2). The FPU activity of A. saccharicola KUC21221 was also higher than that of Penicillium echinulatum (0.27 U/mL) [58]. It was expected that A. saccharicola KUC21221 would have a great saccharification ability because the FPU is closely related to the saccharification yield. According to a previous study, Hypoxylon oceanicum showed the highest EG activity among the five fungi isolated from mangroves [59]. However, A. saccharicola KUC21221 showed much higher activity in EG than did H. oceanicum (0.04 U/mL), and it exhibited higher BGL activity than did Aspergillus niger (1.02 U/mL) [60]. High BGL activity is important because BGL minimizes product inhibition and transglycosylation and promotes the production of cellulase inducer compounds [61]. Considering that A. saccharicola KUC21221 was derived from the marine environment, it was expected that it may secrete more enzyme when cultured in media with a proper amount of salinity [62]. Conversely, no strain showed a highly elevated BXL activity. Because all of the fungal strains were isolated from brown algae, low BXL activities could occur because the hemicellulose content is low in brown algae [63]. Our results support these fungal characteristics and further demonstrate the untapped potential of novel fungal lineages and their potentially useful metabolites in marine systems.

Sampling and Isolation of Fungi
Marine brown algae, Sargassum sp., were collected from Hyeopjae beach in Jeju, Korea. The samples were washed thoroughly with distilled water two times and cut into approximately 0.5 cm × 0.5 cm squares using a sterile surgical blade. Small segments were treated with 70% ethanol for 60 s and then washed in sterile distilled water for 10 s [5,64]. The segments were placed on malt extract agar (MEA) with 0.01% streptomycin and ampicillin to prevent bacterial growth. The grown fungi from the segments were transferred to MEA periodically and incubated at 25 °C.

Phylogenetic Analysis
The obtained sequences were aligned using MAFFT 7.130 [69] and modified manually using MacClade 4.08 [70]. The ITS, LSU and β-tubulin datasets contained 50 taxa and 778, 1005 and 657 nucleotide characters, respectively. They were respectively tested by MrModeltest 2.3 with default options using the AIC criteria [71]. The GTR+I+G model was chosen under the AIC criteria for all datasets. Three datasets were combined and the selected model was applied. Bayesian analysis was performed using MrBayes 3.2.1 [72]. Two runs with 1,000,000 generations were performed, and every 100th generation was sampled. Among them, the first 25% of the trees was eliminated, and the last 75% was used. A 50% majority-rule consensus tree was constructed, and tree reliability was confirmed by posterior probability.
The phylogenetic tree based on ITS was constructed to prove the identity of each fungus. The ITS sequences were aligned using MAFFT 7.130 with reference sequences obtained from GenBank using a BLAST search. The dataset has 95 taxa and 810 nucleotide characters. Phylogenetic analysis was performed using the described method. The GTR+I+G model was selected under the AIC criteria.
To confirm the identification of Arthrinium spp., which shows strong bioactivity, an additional phylogenetic tree was constructed with reference sequences from Crous and Groenewald [11]. Phylogenetic analysis was performed as described in this study. The ITS, LSU and β-tubulin datasets contained 38 taxa and 643, 893 and 560 nucleotide characters, respectively. The SYM+I+G model was selected for the ITS dataset, and the GTR+I+G model was chosen for the last two datasets.

Preparation of Extracts
A total of 50 fungal species were cultivated on 50 mL of potato dextrose agar (PDA; Difco, Detroit, MI, USA) at 25 °C for 7 days in the dark. After the incubation periods, the solid culture was extracted with 200 mL of methanol (SK chemicals, Ulsan, Korea) for a day. The harvested MeOH solution was filtered through Whatman No. 1 filter paper. The solvents were evaporated to dryness under a vacuum at 37 °C, and 4 °C was used during the cooling circulation to obtain effective yields. The condensed residues were re-dissolved in ethyl acetate and distilled water (1:1). Then, the partitioned ethyl acetate fraction was evaporated. The extracts were solubilized in dimethyl sulfoxide (DMSO; Junsei, Tokyo, Japan) to a final concentration of 10 mg/mL and stored at 4 °C before testing [2].

Antifungal Activity
The capability of marine-derived fungi to control three plant pathogenic fungi was evaluated.
Fusarium oxysporum KUC20267, Botrytis cinerea KUC21265, and Collectotrichum gloeosporioides KUC21266 were tested as the target fungi. Each well of a 96-well microplate contained 2 μL of fungal extracts, 20 μL of spore suspension, and 178 μL of media [73]. The plant pathogenic fungi spore suspension was adjusted to 4 × 10 6 per mL. The absorbance was measured every 24 h at 595 nm.

ABTS Radical-Scavenging Assay
The 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (Sigma-Aldrich Inc., St. Louis, MO, USA) radical-scavenging activity was measured as described by Roberta et al. (1999) [47]. ABTS was dissolved in phosphate-buffered saline (PBS, pH 7.4) to 7 mM, and the ABTS radical cation was produced by adding potassium persulfate at a final concentration of 2.45 mM. The mixture was incubated for 24 h at RT in the dark, and the resultant ABTS radical solution was diluted to an absorbance of 0.70 (±0.02) at 734 nm. A 990 μL aliquot of the prepared ABTS radical solution was reacted with 10 μL of the extracts (10 mg/mL) in a cuvette. After 6 min, the absorbance at 734 nm was measured using a spectrophotometer. Trolox was used as the antioxidant positive control solution.

DPPH Radical-Scavenging Assay
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Sigma-Aldrich Inc., St. Louis, MO, USA) radical scavenging activity of the samples was analyzed according to Fukumoto et al. [4]. The DPPH solution was prepared in methanol (80%) at 150 μM. For analysis in the 96-well plate, we added 22 μL of the extracts of the sample (10 mg/mL) and 200 μL of the DPPH solution to each well and then incubated the plate at room temperature for 30 min. The absorbance at 520 nm was measured using a microplate reader. A control consisted of 22 μL of methanol instead of the sample extracts. Trolox was used as the antioxidant standard.

Cellulolytic Enzyme Enzyme Preparation
All fungal isolates were inoculated on solid medium containing 2% (w/v) malt extract (Bacto, Sparks, MD, USA) and 1.5% agar powder (Showa, Tokyo, Japan). The strains were grown for seven days, and two agar plugs of each strain were used for the inoculums. To prepare the fungal enzyme, the fungi were cultivated in 50 mL conical tubes that contained 10 mL Mandels' medium (0.3 g of urea, 1.4 g of KH2PO4, 2.0 g of (NH4)2SO4, 0.3 g of CaCl2, 0.3 g of MgSO4, 0.25 g of yeast extract, 0.75 g of peptone, 5 mg of FeSO4·7H2O, 36 mg of COCl2·6H2O, 1.8 mg of MnSO4·H2O, and 2.5 mg of ZnSO4·7H2O per liter of distilled water) with 1% cellulose as the sole carbon source [74]. Fungal cultures were incubated aerobically for 1 week at 25 °C on a rotary shaker at 150 rpm in the dark. The cultures were prepared in triplicate, and the mean values are presented. After cultivation, the samples were centrifuged at 4000 rpm for 25 min at 4 °C, and the supernatant was extracted and filtered through a 0.45 μm filter (Minisart, Sartorius, Göttingen, Germany) to determine the enzymatic activities of the crude enzyme solutions.

Enzyme Assays
The FPU activities were measured using a 60 μL filter paper assay (FPA) method [75]. Briefly, the reaction mixtures were prepared in a total volume of 60 μL consisting of 20 μL of the enzyme solutions and 40 μL of 50 mM citrate buffer (pH 4.8) in a 0.2 mL PCR tube (Axygen, cat. No. 321-10-051); the substrate was a 7 mm diameter Whatman No. 1 filter paper disk. After one hour of the reaction at 50 °C, 120 μL of dinitrosalicylic acid (DNS) was added. The tubes were boiled and cooled for 5 min each. Thirty-six microliters of the reaction mixtures were transferred to a 96-well tissue microplate (Falcon, cat. No. 353072) with 160 μL of distilled water, and the absorbance was measured at 540 nm. The absorbance was used to calculate the FPU using glucose as the standard. One FPU was defined as 1 mM of glucose equivalent released per minute. The EG activities were assayed according to standard procedures [76,77]. The procedure was similar to the above FPA method. Briefly, the reaction mixtures were prepared in a total volume of 50 μL consisting of 25 μL of enzyme solutions and 25 μL of 2% CMC in 50 mM citrate buffer (pH 4.8) in a 0.2 mL PCR tube. After one hour of the reaction at 50 °C, 150 μL of the DNS reagent was added. The following procedures were the same as the above FPA method. The tubes were boiled and cooled for 5 min each. Thirty-six microliters of the reaction mixtures was transferred to flat-bottom 96-well tissue culture plates with 160 μL of distilled water, and the absorbance was measured at 540 nm. The absorbance was used to calculate the EG activity using glucose as the standard. One unit of EG activity was defined as 1 mM of glucose equivalent released per minute. The activities of BGL and BXL were determined according to the method of Valásková and Baldrian [78]. These activities were determined by measuring the concentration of p-nitrophenyl (pNP) released by p-nitrophenyl glucosidase (pNPG) or p-nitrophenyl xylosidase (pNPX). Briefly, reaction mixtures consisting of 20 μL of the enzyme solutions, 20 μL of 10 mM pNPG or pNPX, and 20 μL of 1 M sodium acetate buffer (pH 5.0) were incubated for 5 and 10 min, respectively, at 50 °C. The reaction was stopped by adding 20 μL of 2 M Na2CO3, and the absorbance was measured at 405 nm. One unit of BGL or BXL activity was defined as the amount of enzyme that released 1 mM of pNP per minute [79].

Statistical Analysis
Inhibitory concentration (IC50) values were calculated from nonlinear regression analysis using the SAS software (version 9.2 by SAS Institute Inc., Cary, NC, USA).

Conclusions
In conclusion, this study provided reliable DNA information for marine-derived fungi and their biological activities. A total of 50 fungal strains were obtained from the brown algae Sargassum sp. Based on the BLAST search with ITS, LSU, and the β-tubulin region, a phylogenetic analysis was performed to verify the identification. Among the collected fungi, Corollospora angusta was the dominant species, and the most diverse genus was Arthrinium. In addition, Arthrinium spp. showed relatively strong biological activities for antifungal, antioxidant, and cellulolytic activity. The potential bioactive compounds will be isolated and purified in the near future.