Diversity of Trichoderma spp. in Marine Environments and Their Biological Potential for Sustainable Industrial Applications

: Microorganisms are regarded as a sustainable source of biologically active molecules. Among them, Trichoderma spp. have been an attractive source of biological compounds. However, the study of marine-derived Trichoderma has developed slowly because of the difficulty in isolating the fungi. In our study, 30 strains of marine-derived Trichoderma were identified through the translation elongation factor 1-alpha (EF1α) sequences, and their biological activities, such as antioxidant activity by ABTS and DPPH assays, antifungal activity against Asteromyces cruciatus and Lindra thalassiae , and tyrosinase inhibition activity, were investigated. As a result, the 30 marine Trichoderma species were classified into 21 taxa, including three new species candidates. Three strains of T. asperellum showed the highest ABTS radical scavenging activity and antifungal activity. T. bissettii SFC20170821-M05 and T. guizhouense SFC20180619-M23 showed notable DPPH radical scavenging activity and tyrosinase inhibition activity, respectively. This study showed the potential of marine-derived Trichoderma as a source of bioactive compounds.


Introduction
In a specific environment, biodiversity is the most essential information for sustainable development and is important in discovering biological resources [1][2][3][4][5][6]. In particular, microorganisms have been considered as a sustainable source of various bioactive compounds as well as useful enzymes [7][8][9][10][11][12][13][14]. Traditional sources of natural products have been terrestrial plants, fungi, and bacteria. The idea of natural products from the ocean has recently been of interest, but the need for additional efforts, such as scuba diving or instruments for collecting or culture, have made the development of marine natural compounds slow in comparison with its terrestrial counterpart [15,16]. Nevertheless, the structural uniqueness and profound effect of marine-derived compounds have been of interest to the pharmaceutical and cosmetic industry [17,18]. A total of 200 million microorganisms in the ocean covering 71% of the surface of the earth have been presumed, although the exact number of microorganisms is still open to debate [19,20]. For this reason, marine organisms, especially microorganisms, have attracted the attention of the pharmaceutical and cosmetic industries as a new reservoir of novel natural compounds [17,20].
The genus Trichoderma is well known for its ability to produce antibiotic compounds and parasitize other fungi [21][22][23]. The actions of antifungal secondary metabolites (i.e., 6-pentyl-αpyrone and trichodermaketones) and cell wall hydrolytic enzymes (i.e., β-1,3,-glucanases and β-1,6,glucanases) secreted by Trichoderma spp. can cause the death of prey [21,[24][25][26]. Several Trichoderma provide a beneficial effect to host plants by activating plant defense mechanisms, preventing pathogen attacks and promoting plant growth [21,27]. To date, more than 250 species of the genus Trichoderma have been reported [28]. Approximately 78 metabolites have been described from marine-derived Trichoderma so far, and most of them showed a variety of industrially useful biological activities, such as antifungal, antibacterial, and antioxidant activity [29,30]. Reliable phylogenetic information is important to discover the diversity of secondary metabolites of microorganisms, and it is difficult to distinguish Trichoderma spp. by morphology alone, because they share many morphological features [31][32][33]. Thus, a molecular biological analysis is essential for the accurate identification of Trichoderma [34]. The internal described spacer (ITS) is the most universal fungal molecular barcode [35,36]. However, the ITS has low species resolution in the genus Trichoderma [32]. Instead of ITS, translation elongation factor 1-alpha (EF1α) sequences were recommended for phylogenetic analysis of this genus [32].
The aim of this study was to investigate the diversity of Trichoderma spp. in marine environment in South Korea using phylogenetic analysis and evaluate the marine Trichoderma spp. as a source of bioactive secondary metabolites by investigating the antifungal, antioxidant, and tyrosinase activities of the fungal extracts.

Preparation of Trichoderma Cultures
A total of 30 marine Trichoderma cultures were obtained from the Marine Fungal Resource Bank (http://mfrb.snu.ac.kr; Seoul, Korea). A list of the fungal species with their general information is shown in Table 1. Their sampling sites are indicated in Figure 1. They were subcultured on potato dextrose agar (PDA) media in room temperature: 20−25°C.  * The sampling sites in Figure 1 were indicated by A-L.

Phylogenetic Analysis
EF1α sequences of marine Trichoderma were obtained from Marine Fungal Resource Bank. The closely related sequences for references were downloaded from GenBank using nucleotide BLAST. Type specimens were chosen for reference sequences except for Trichoderma virens. EF1α sequence data of the CBS 249.59, type specimen of T. virens has only 200 bp in GenBank. Protocrea illinoensis GJS 94-54 (EU703904) was downloaded as outgroup of the genus Trichoderma. The obtained sequences were aligned using MAFFT 7.388 [37]. The aligned dataset was proofread and modified manually using MacClade 4.08 [38]. The aligned dataset contained 113 taxa and 752 characters. A neighbor joining tree was constructed with PAUP 4.0b10 [39]. The Kimura 2-parameter model was applied [40]. To indicate branch stability, 1000 replications of bootstrap analysis were carried out.

Preparation of Fungal Extracts
All of the fungal species were cultivated on 50 mL of PDA at 25 °C for seven days in darkness. After 7 days of cultivation, the solid media were extracted with 200 mL of methanol for 24 h. the methanol solution was filtrated with Whatman No. 1 filter paper. The filtrated solutions were evaporated at 37 °C under a vacuum. The condensed residues were dissolved in 20 mL of ethyl acetate and 20 mL of distilled water. After 6 h, the supernatant, the partitioned ethyl acetate fraction, was evaporated. The extracts were stored at 4 °C. The fungal cultures were incubated in triplicate, and all the subsequent biological assays were performed in triplicate.

Measurement of Antioxidant Activity by ABTS Scavenging Ability
The 2.2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS, Sigma-Aldrich, Inc., St. Louis, MO) solution dissolved in PBS (7 mM) was oxidized with potassium persulfate (2.45 mM) for 24 h in darkness at room temperature. The ABTS •+ solution was diluted with PBS to an absorbance of 0.70 (± 0.02) at a wavelength of 734 nm. Then, 990 µL of the ABTS •+ solution and 10 µL of each fungal extract sample (10 mg/mL in DMSO) were mixed in the cuvette and measured at 734 nm after 6 min.

Measurement of Antifungal Activities
Asteromyces cruciatus SFC20161110-M19 was obtained from Marine Fungal Resource Bank (MFRB) at Seoul National University as a marine bioresource bank of Korea by the Ministry of Oceans and Fisheries, and Lindra thalassiae NBRC106646 was purchased from Biological Resource Center under National Institute of Technology and Evaluation. Asteromyces cruciatus SFC20161110-M19 and Lindra thalassiae NBRC106646 were tested as the target fungi and antifungal activity was determined in a 96-well plate. The 25 µL of spore suspensions (4 × 10 5 conidia/mL) of the target fungi was added to each well containing 49 µL of distilled water and 25 µL of potato dextrose broth. The 2 µL of Trichoderma extracts solubilized in dimethyl sulfoxide (DMSO) to a final concentration of 100 µg/mL was added to each well at the last. The 96-well plates were incubated at 25 °C for 3 days and the fungal growth was detected by measuring the absorbance at the wavelength of 595 nm [41]. The minimum inhibitory concentration (MIC) values were determined in the concentration range of 6.25-50 µg/mL.

Tyrosinase Inhibition Activity
The assay was modified from the method described by Lai et al. [42]. An amount of 40 µL of each fungal extract samples dissolved in 50% DMSO at the same concentration (2.5 mg/mL), 70 µL of a 0.1 M potassium phosphate buffer (pH 6.8), and 30 µL of 0.02 mg/mL of tyrosinase were mixed in 96-well plate. The mixture was heated to 30 °C for 5 min and mixed with 100 µL of 2.5 mM Ldihydroxyphenylalanine (L-DOPA). After 30 min, to terminate the reaction put the plate in the ice and the absorbance was measured at 492 nm. Kojic acid was used as a positive control and all mixtures without L-DOPA was used as a blank.

Antioxidant Activity
ABTS and DPPH radical scavenging assays were used to evaluate the antioxidant activity of 30 methanol crude fungal extracts of marine Trichoderma, and many of the marine Trichoderma extracts contained antioxidative compounds. Trichoderma asperellum SFC20160907-M21, SFC20180619-M22, and SFC20180619-M24 showed over 70% ABTS radical scavenging activity, and T. bissettii SFC20170821-M05 and T. longibrachiatum SFC20171019-M03 exhibited over 70% DPPH radical scavenging activity (Table 2). Other extracts showed lower inhibition rates from 2.3% to 69%. Only four strains, T. bissettii SFC20170821-M05, Trichoderma sp. 1 SFC20190312-M13, T. longibrachiatum SFC20171019-M03, and T. subviride SFC20170919-M07, exhibited over 50% antioxidant activities in both the ABTS and DPPH radical scavenging tests. Numerous studies have shown that various marine-derived natural compounds have radical scavenging properties that could be used as raw materials in the cosmetic and pharmaceutical industries [18,49,50]. For example, the major antioxidant in the food supplement Seanol is phlorotannins extracted from Ecklonia Cava [51]. Several natural compounds that have antioxidant ability, such as polyphenols and vitamins, also have other biological activities, such as antiaging and skin-whitening [52,53]. Some antioxidants have the ability to inhibit tyrosinase by eliminating reactive quinone products [52]. In this study, the extracts of T. atroviride SFC20161110-M05, T. gamsii SFC20160907-M22, T. guizhouense SFC20180619-M23, and T. songyi SFC20171120-M04, which showed a remarkable ability to inhibit tyrosinase (IC50 < 100 µg/mL), exhibited low radical scavenging activity (<50%), indicating that they have other mechanisms for inhibiting tyrosinase rather than scavenging reactive quinone products. For examples, they may be the competitive inhibitors such as copper chelators that inhibit this metalloenzyme or suicide inhibitors that inactivate tyrosinase by changing the tertiary and quaternary structures of the enzyme [54].

Antifungal Activity
The antifungal activity was determined using the fungal extracts of marine Trichoderma on Asteromyces cruciatus and Lindra thalassiae as target fungi. A. cruciatus is a ubiquitous marine fungus and has the ability to degrade alginate, which is the major material of the brown algal construct; therefore, it is regarded as a potentially harmful fungus to brown algae [55]. Lindra thalassiae is a wellknown pathogen of brown algae and seagrasses and causes raisin disease [56]. In this study, eight strains showed remarkable growth inhibitory ability against both A. cruciatus and L. thalassiae: T. afroharzianum SFC20180619-M25, T. asperelloides SFC20180619-M20, T. asperellum SFC20160907-M21, SFC20180619-M22, and SFC20180619-M24, T. capillare SFC20160907-M2190, T. citrinoviride SFC20180510-M16 and T. virens SFC20180817-M24. T. asperellum SFC20160907-M21, SFC20180619-M22, and SFC20180619-M24 showed a high inhibitory effect against both target fungi ( Table 2). In particular, T. asperellum SFC20160907-M21 and SFC20180619-M24 showed the highest inhibitory effect, as they could inhibit L. thalassiae at 6.25 µg/mL. Since Trichoderma spp. are well known producers of antifungal compounds, such as 6-pentyl-α-pyrone and trichodermaketones, it was expected that Trichoderma species could exhibit antagonistic ability against A. cruciatus and L. thalassiae, which are regarded as pathogenic fungi to algae [25,26]. Similarly, T. asperellum has already been investigated for its antagonistic ability against pathogenic fungi in several previous studies [57][58][59]. In addition, the synergistic effects of antifungal secondary metabolites and enzymes from Trichoderma could provide support to the plant rhizosphere attacked by other pathogenic fungi [21,27]. The results of this antifungal activity suggested that the antifungal compounds from Trichoderma could also support marine algae defense systems that are related to symbiosis, similar to the action of the plant rhizosphere.

Conclusions
We investigated the diversity of marine-derived Trichoderma spp. and provided reliable DNA information of 30 marine Trichoderma species isolated in South Korea as well as their exploitable biological activities. Based on the phylogenetic analysis, the 30 marine Trichoderma species were classified into 21 taxa, including three new species candidates. Among them, three species-Trichoderma sp. 1, T. asperellum, and T. longibrachiatum-showed remarkable abilities in most of the biological activities investigated in this study. The potent bioactive compounds of these species will be studied in the near future. This study proved that marine-derived Trichoderma can be a useful source of bioactive compounds.