Classification and Molecular Insights of Undiscovered Marine Fungi PDF

Article Classification and Molecular Insights of Undiscovered Marine Fungi with Bioeconomy Prospects By Kamollanat Chaiyabut, Esra Shaiban, Fahad Jamil, Saranya Yapa Pathirannehelage, Durotun Ainiyah Abstract Marine fungi, a largely overlooked group, represent a significant ye...

Article Classification and Molecular Insights of Undiscovered Marine Fungi with Bioeconomy Prospects By Kamollanat Chaiyabut, Esra Shaiban, Fahad Jamil, Saranya Yapa Pathirannehelage, Durotun Ainiyah Abstract Marine fungi, a largely overlooked group, represent a significant yet understudied component of marine biodiversity. Despite their potential for biotechnological applications, their diversity and bioactivity remain poorly characterized. This study aimed to identify and characterize marine fungi through a combination of morphological and molecular techniques. Twenty fungal strains were successfully isolated and cultivated, with most exhibiting growth on WSP30 media. Morphological variations were observed among strains grown on different media, highlighting the influence of culture conditions on fungal development. Molecular identification based on the Internal Transcribed Spacer (ITS) region revealed that the majority of isolates belonged to the genus Penicillium. One strain, Corollospora sp. (MF59), was identified as a potential new species, as its ITS sequence exhibited low similarity to known taxa. Antimicrobial activity assays indicated that certain strains, particularly Penicillium sp. (MF9) and Fusarium sp. (MF40), showed significant inhibitory effects against both Escherichia coli as well as Gram-positive bacteria. These findings underscore the potential of marine fungi as a valuable source of bioactive compounds and highlight the need for further exploration of their diversity and biotechnological applications. Keywords: fungal diversity, molecular identification, Penicillium, ITS region, bioactive compounds Introduction Fungi, an important element in terrestrial as well as marine habitats, recording a significant amount of microbial diversity on Earth. Marine-derived fungi constitute a diverse and largely understudied group of microorganisms inhabiting the vast expanse of the marine environment. While the phyla Ascomycota and Basidiomycota represent the dominant lineages within this domain, encompassing major clades such as Pezizomycotina, Saccharomyces, Agaricomycotina, and others [2, 3, 4], our comprehension of their overall diversity remains incomplete [2, 5, 6]. These marine-derived fungi possess a remarkable capacity to synthesize a wide array of bioactive compounds. These metabolites exhibit a diverse spectrum of biological activities, encompassing antimicrobial, anticancer, antiviral, antioxidant, and anti-inflammatory properties [6, 7], demonstrating significant promise for drug discovery and development across various sectors, including pharmaceuticals, medicine, and agriculture. Beyond their capacity to produce bioactive compounds, marine-derived fungi play crucial roles in the intricate workings of marine ecosystems. They significantly contribute to nutrient cycling by decomposing organic matter and facilitating nutrient turnover. Moreover, they form intricate symbiotic associations with other marine organisms, such as corals and algae [1, 8]. Furthermore, they exhibit bioremediation capabilities, effectively degrading pollutants, including petroleum hydrocarbons and plastics [9, 10]. The internal transcribed spacer (ITS) region within nuclear ribosomal DNA (nrDNA) has emerged as the preferred DNA barcode for fungal identification. This marker effectively identifies both individual fungal taxa and mixed fungal communities within environmental samples ('environmental DNA barcoding'). The ITS region has been formally recognized as the primary DNA barcode for fungi by a consensus of mycologists. Currently, over 100,000 fungal ITS sequences, generated primarily through Sanger sequencing, are publicly available in international nucleotide sequence databases. Recent advancements in molecular biology, genomics, and bioinformatics have revolutionized our ability to explore the hidden diversity of marine-derived fungi [12, 13]. These technological advancements have enabled the discovery of novel fungal species, the identification of novel enzymes, and the characterization of bioactive compounds with potential applications across diverse fields [12, 13]. This has spurred a growing interest in harnessing the biotechnological potential of marine-derived fungi, particularly in the development of novel pharmaceuticals, bioremediation strategies, and sustainable biofuels [14, 15, 16, 17, 18, 19]. However, significant challenges persist in fully exploiting the biotechnological potential of these organisms. Cultivating and isolating marine-derived fungi can be challenging due to their specific growth requirements and sensitivities. Furthermore, their complex genomes and the limited availability of reference sequences can hinder genome sequencing and annotation efforts [21, 22]. The aim of this study was to expedite fungal species identification through the utilization of the Internal Transcribed Spacer (ITS) region in conjunction with morphological characteristics, for that we used to select the marine fungi based on pigmentation. Difficulty in differentiation between closely related marine fungi based solely on appearance and have morphological limitations.. We had face challenges during culturing marine fungi according to , marine fungi often required specific nutrient, temperature, salinity and have slow growth rate. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (rDNA) has been established as the primary fungal barcode, enabling accurate and reliable identification of fungal species , This approach typically involves amplifying the ITS region using specific primers, such as ITS1 and ITS4, followed by DNA sequencing, often using Sanger sequencing technology. To effectively assess antimicrobial activity, the approach is often employed. Initial screening typically involves the agar diffusion assay, a rapid method for identifying compounds with inhibitory effects against target microorganisms This approach, as outlined by , provides a robust framework for evaluating antimicrobial potential. Materials and methods Sample Collection and Inoculation Marine fungal strains were obtained from the Flensburg Strain Collection of Marine Fungi (mFSC), focusing on samples with unique morphological traits such as pigmentation, which could indicate the presence of secondary metabolites. Sixty strains were randomly selected and carefully inoculated onto WSP30 and GPY agar plates using sterile techniques [48,49]. The media used in this study were modified to better simulate specific nutrient conditions. Modified Wickerham media (WSP30) was prepared by dissolving 10.0 g of glucose H₂O, 5.0 g of peptone from soymeal, 3.0 g of malt extract, 3.0 g of yeast extract, 30.0 g of NaCl, and 18.0 g of agar in 1 L of distilled water. The pH was used as is without adjustment. The mixture was then autoclaved. GPY agar was prepared by dissolving 1.0 g of glucose·H₂O, 0.5 g of peptone from soymeal, 0.1 g of yeast extract, 18.0 g of agar, and 30.0 g of artificial seawater in 1 L of distilled water. The pH was adjusted to 7.2–7.4 before autoclaving.. Each fungal strain was inoculated onto the media using a sterile inoculation loop or pipette tip under aseptic conditions in a laminar flow hood to prevent contamination. Plates were incubated at 25°C, an optimal temperature for marine fungal growth, for seven days. Colony morphology, pigmentation, and growth patterns were observed and documented weekly using ShpereFlash device and the microscope. This allowed for continuous monitoring of colony variation and ensured proper growth before further analyses. DNA Extraction and PCR Amplification Genomic DNA was extracted from fungal mycelia using the DNeasy® Plant Pro Kit (Qiagen). Fresh fungal mycelia were scraped from agar plates and transferred to sterile 2 mL bead tubes. Approximately 5-100 mg of fungal biomass was subjected to mechanical disruption using the kit’s bead beating step with the addition of each 500 µL of the proprietary lysis buffer CD1 and CD2. The mixture was undergone cell lysis using a Tissuelyser for 5 minutes to ensure the complete cell lysis. The lysate was then centrifuged at 12,000 rpm for 2 minutes to remove debris, and the supernatant was transferred to a new tube. DNA was purified using the Qiagen spin-column system. The supernatant was mixed with the binding buffer APP and transferred to the spin column, where DNA was bound to the silica membrane. Following sequential washes with 650µL AW1 and 650µL AW2 wash buffer to remove contaminants respectively, following centrifugation of 12,000 rpm for 1 minute in between each washing step. Then all the accumulated liquid was discarded and the remains were centrifuged at 16,000 rpm for 2 minutes. After the final centrifugation, the liquid remains were discarded if there were any, and the filter was transferred to a new Eppendorf tube. DNA was eluted with 50 µL of EB buffer following a final centrifugation step of 12,000 rpm for 1 minute. The concentration and purity of the extracted DNA were determined using a NanoDrop spectrophotometer. The absorbance at 260/280 nm was measured to assess protein contamination, with acceptable ratios ranging from 1.8 to 2.0. DNA integrity was confirmed via agarose gel electrophoresis using a 1% (W/V) agarose gel stained with ethidium bromide [50,6]. PCR amplification of the ITS regions was performed using the ITS1F (5’-CTTGGTCATTTAGAGGAAGTAA-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) primers, targeting the preserved flanking regions of fungal ITS sequences. Each PCR reaction (50 µL) contained 25 µL of 2X OneTaq® Hot Start Quick-Load Master Mix, 1 µL of each primer (10 µM), 1 µL of DNA template, and nuclease-free water. Thermal cycling conditions included an initial denaturation at 94°C for 30 seconds, preceded by 35 iterations of 94°C for 30 seconds, 55°C for 30 seconds, and 68°C for 1 minute, with a final extension at 68°C for 5 minutes. PCR samples were confirmed by running 10 µL of each reaction on a 1% agarose gel stained with RotiSafe® Gel Stain and visualized under UV light [50,53]. Sequencing and Data Analysis The PCR products were purified using a commercial PCR purification kit to remove excess primers and nucleotides. The purified amplicons were sent to the Institute of Clinical Molecular Biology (IKMB) in Kiel, Germany, for Sanger sequencing [51,53]. Sequencing was performed in both forward and reverse directions to ensure accuracy. Upon receiving the raw sequencing data, chromatograms were analyzed and edited using BioEdit software to remove low-quality sequences. The cleaned sequences were subjected to BLASTn searches against the NCBI database to identify fungal strains based on similarity to known ITS sequences. Taxonomic classification was determined by comparing the query sequences with the closest matches, and ambiguous results were resolved using phylogenetic analysis in MEGA software. This ensured the accurate identification of fungal species or genera. Bioactivity Assay To evaluate the antimicrobial activity of fungal metabolites, an agar well diffusion assay was conducted. Following seven days of incubation, fungal metabolites were collected by adding 10 mL of ethyl acetate to the fungal cultures. The cultures were homogenized by an Ultra-Turrax for 30 seconds and transferred to a separation funnel. Equal volumes of ethyl acetate were added, and the mixture was mixed for 30 seconds. After phase separation, the lower phase was disposed of, and the process was repeated. The upper phase was drained into a round bottom flask and concentrated using a rotary evaporator at 40°C, with shaking at 150-200 rpm. The resulting extract was re-dissolved in methanol in two steps. First Step: Dissolve the concentrated extract in a small volume of methanol (e.g., 5 mL). This helps to fully dissolve the extract and prepare it for further testing. Ensure the extract is well-mixed. Second Step: After the extract is dissolved, additional methanol was added to achieve a final volume appropriate for the antimicrobial assays, such as 10 mL or based on the required concentration. This ensures the extract was at the correct concentration for the diffusion test and other assays. The concentrated extracts were tested against Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive) as model organisms. Nutrient agar plates were prepared, and 100 µL of bacterial suspensions (adjusted to 0.5 McFarland standard) were evenly spread on the surface [48,50]. Wells of 6 mm diameter were punched into the agar using a sterile cork borer, and 60 µL of fungal extracts were added to each well. Chloramphenicol (0.25 g/L) was used as a positive control, while WSP30 media served as a negative control [51,52]. The plates were incubated at 37°C for 24 hours, and zones of inhibition were measured using a ruler. The percentage zone of inhibition (%ZOI) was calculated relative to the positive control, providing a quantitative measure of antimicrobial activity. These results were used to identify fungal strains with the most promising bioactive potential. Results Morphological and molecular identification of fungal isolates Marine fungal strains were collected from the Flensburg Strain Collection of Marine Fungi (mFSC), which targets samples with distinct morphology, such as pigmentation that is often associated with secondary metabolite production. Out of the 60 strains collected, only 20 were successfully cultivated. This emphasizes the challenges of cultivating marine fungi under laboratory conditions. Many strains either failed to grow or had very slow growth rates under laboratory conditions. There were morphological differences between the two media cultivated on WSP30 and GPY. Both media were emphasizing the important impact on fungal appearance. WSP30 encourages the growth of features that are comparable to actual marine habitats by simulating marine conditions, including glucose, peptone, malt extract, and yeast extract, which support the growth of a wide range of fungi. The high NaCl concentration (30 g/L) simulates marine salinity and promotes rapid growth, hyphae and pigmentation. On the other hand, GPY media limited nutrients with a low concentrations of glucose, peptone ,and yeast extract that caused an effect on fungal growth, colonies may appear smaller, with thinner hyphae, and less pigmentation compared to those on WSP30. In addition, The environment with conditions of limited nutrients induces a stress response mechanism known as sporulation. According to Table 1, the results for MF48 showed that colonies grown on GPY media exhibited less pink pigmentation compared to those on WSP30 media. Similarly, MF50 demonstrated that colonies on GPY were smaller and rounder. The internal transcribed spacer (ITS) region is a commonly used marker for fungal identification [58, 59]. From the twenty cultivated strains, fifteen were selected for DNA extraction and PCR amplification shown in Table 1. ITS1 and ITS4 primers were applied for amplification, which mostly had a 100% success rate and were identified at the genus level by subsequent ITS BLAST analysis. A known limitation of this marker is that ITS primers were insufficient to resolve species-level. Table 1. Search Results for Nucleotide Sequence Homology ITS rRNA Region from Fungal Type and Reference Materials using MEGA BLAST and Morphology Characterization. Morphology Name of next Strain % Proposed Microscopy relative in bp NCBI Accession Number Identity Taxonomy WSP30 GPY BLAST Greenish White, MF2 pure colonies, Penicillium Penicillium smooth, - 100.00% 105 NR_163669.1 2A conidiogenous fuscoglaucum sp. fluffy cells phialidic Whitish colonies Septate hyphae, Paraphaeosphaer Paraphaeosp MF5 - 97.58% 432 NR_155698.1 with bright bright spores ia xanthorrhoeae haeria sp. green edges Dark Bright, circular Penicillium Penicillium MF9 green, - spores, greenish 99.74% 389 NR_121255.1 atrovenetum sp. powdery colonies White, Conidia on uniformly small spread, Fusarium MF12 - projections, 100.00% 345 NR_164597.1 Fusarium sp. creamy hainanense fertile hyphae layer, fragmenting fluffy  Dark Pinkish green, colonies; MF22 pure Penicillium Penicillium powdery, - phialides borne 100.00% 441 NR_138315.2 2B patris-mei sp. round directly on colonies hyphae Pinkish- white, Slightly Banana-shaped puffy pink, MF40 conidia, septate Fusarium boothii 100.00% 396 NR_121203.1 Fusarium sp. string-lik slightly hyphae e fluffy structure Green/gra y/white, Dark green, Phialides borne scattered powdery Penicillium Penicillium MF46 directly on 100.00% 441 NR_138317.1 colonies, scattered oledzkii sp. hyphae firm colonies structure White-gr Fluffy, een bright Conidiophores colonies, Aspergillus Aspergillus MF48 green with typical 100.00% 421 NR_135447.1 fur-like, tennesseensis sp. scattered apical swelling round, colonies bumped Green, scattered, Green, Dark grey, bumped small conidia on small colonies round, Penicillium Penicillium MF50 projections, 100.00% 441 NR_121248.1 with powdery, gladioli sp. hyphae with white cloudy conidia and asci fur-like colonies structures White, Blackish, Smooth-walled uniformly scattered septa, Penicillium Penicillium MF51 spread, 99.77% 435 NR_121255.1 colonies, flask-shaped atrovenetum sp. fluffy, cloudy phialides fur-like White center, gray Brownish, Phialides with a Penicillium Penicillium MF53 sides, 100.00% 450 NR_163679.1 spread short neck radiatolobatum sp. bumped surface, fur-like  White, firm, Hyaline hyphae, White, round, phialides smooth, Neoacremonium Neoacremoni MF55 scattered, directly on 100.00% 345 NR_189466.1 powdery, distortum um sp. surrounde hyphae, slimy scattered d by head conidia white Creamy Creamy white, white with separated darker Septate hyphae, Microascus Microascus MF56 , center, conidiophores 100.00% 428 NR_155415.1 atrogriseus sp. attached, slightly bearing annelids bumped bumped, surface separated Black/dark brown, Chlamydospore smooth, s, arthroconidia cylindrical, Xerochrysium MF58 - present, - - - - separated, sp. branched root-like conidiophores growth in agar Cloudy Corollospora Corollospora MF59 - green, spot - 94.95% 468 NR_173756.1 mediterranea sp. colonies The identified strains showed significant diversity Penicillium sp. was the most represented, with strains including MF2 Pure 2A, MF9, MF22 Pure 2B, MF46, MF50, MF51, and MF53. Other genera included Paraphaeosphaeria sp. (MF5), Fusarium sp. (MF12, MF40), Aspergillus sp. (MF48), Neoacremonium sp. (MF55), Microascus sp. (MF56), and Corollospora sp. (MF59). Strain MF59 was recognized as a probable new species due to its percent identity, which marginally surpassed 94.5%. Additionally, based on morphological characterization, MF58 is identified as Xerochrysium sp.. Antimicrobial Potential of Fungal Extracts Fast-growing marine fungi were isolated for further bioactive assay. Crude extracts of marine fungus were tested for antibacterial potential against Gram-positive bacteria (Bacillus subtilis) and Gram-negative bacteria (Escherichia coli). The reference antibiotic in this research is chloramphenicol concentration at 0.25 g/L. The best result compared to both bacteria was that the percentage zone of inhibition (ZOI) of Penicillium sp. (MF9) was more significant against E. coli (Fig. 1) and Fusarium sp. (MF40) similarly surpassed against E. coli. Based on the effectiveness against E. coli (Fig. 1), the crude extract from Penicillium sp. (MF9) exhibited the highest percentage zone of inhibition (%ZOI, approximately 120%), demonstrating significant antibacterial activity. Similarly, Fusarium sp. (MF40) displayed notable activity against E. coli, with a %ZOI exceeding 100%. In contrast, the crude extract from Aspergillus sp. (MF48) exhibited the least activity among the tested samples, though it still demonstrated some inhibitory effects. Furthermore, the %ZOI against B. subtilis was consistently lower than that observed against E. coli. Most samples showed %ZOI values below the standard of 100%, with an average significantly below this threshold. Figure 1. Percentage Zone of Inhibition (%ZOI) of Fungal Extracts Against Escherichia coli (Gram-Negative) and Bacillus subtilis (Gram-Positive) at 60 µL, Standardized Relative to Positive Control (Chloramphenicol at 100%). Discussion Cultivation Challenges and Adaptive Potential of Marine Fungi Despite marine fungals having the ability to grow in extreme marine environments, including high salinity (approximately 0.6 M NaCl), intense ultraviolet radiation, limited nutrient availability, and significant hydrostatic pressure , cultivating marine fungi in the laboratory remains challenging, cultivation success rates showed relatively low (33%, or 20 out of 60 strains), these fungi have the capacity for adaptation to nutrient-poor conditions in laboratory settings. Some strains demonstrate slow but consistent growth, indicating their ability to adapt under resource-limited conditions. Limitations of ITS region The ITS region is widely known as the fungal DNA barcode due to its high sequence diversity, multiple copies per cell, and plenty of database support. It covers transcribed (18S-5.8S-28S) and non-transcribed (ITS) regions, making it useful for comparing organisms. However, the ITS region has limitations, for example, short ITS sequences roughly between 400 to 600 bp and ITS1 and ITS4 enable only genus-level identification and require additional markers for species-level identification, such as RPB1, RPB2, the β-tubulin gene, and whole-genome sequencing for future more profound identification. Antimicrobial Potential of Fungal Extract According to the name of the following relative in BLAST, MF9 is Penicillium atrouenetum. This strand was reported to secrete a toxic antibiotic called 3-nitro propionate (3-NPA). 3-NPA is a nitro aliphatic compound found in plants and several fungi to defend against herbivores. 3-NPA was involved in synthesizing Propionate 3-nitronate (P3N), a potent inhibitor of the crucial enzyme succinate dehydrogenase in the Krebs cycle and electron transport chain. Inhibition of succinate dehydrogenase led to cellular metabolism disruption, directly connecting the Krebs cycle with the aerobic respiratory chain. MF40 from the Fusarium genus was known for its wide range of fusarium-derived antibacterial secondary metabolites (SM) according to its antibacterial properties. Butenolide, a Fusarium mycotoxin from an unknown origin strain Fusarium sp., showed selective inhibition against E. coli. Another strain, F. solani JK10, was reported to have antibacterial activity against E. coli through extensive chemical investigation of nine 2-pyrrolidone derivatives. A Butenolide, striatisporolide A. (SA) from Athyrium multidentatum (Doll.) Ching suggested it had fewer antibacterial targets and weak activity compared with eight butenolide derivatives from the previous study , which may have happened due to the variation of structure associated with the antibacterial compound. SA damaged cell membranes and cell walls, and altered protein levels against E. coli. The study of antibiotics in marine fungi could be conducted through metabolite profiling techniques for further investigation. Previous research reported the use of High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD) coupled with analytical Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (LC-QTOF-MS) to analyze fungal ethyl acetate crude extracts. Among the tested species, Fusarium venenatum exhibited seven distinct metabolite molecular weights. However, no specific metabolites were identified in that study, suggesting that the findings remain preliminary about this species. Conclusion Marine fungi have a remarkable ability to adapt to extreme environments such as high salinity, ultraviolet radiation, and nutrient scarcity. However, cultivating them in laboratory settings remains challenging, with only 20 out of 60 strains (33%) successfully cultivated. WSP30 and GPY media highlighted the significant influence of environmental conditions on fungal morphology. WSP30’s nutrient-rich and saline environment supports rapid growth, hyphae, and pigmentation. In contrast, the nutrient-limited GPY medium produces smaller colonies with thinner hyphae and less pigmentation while promoting sporulation as a stress response. For example, the results for MF48 showed that colonies grown on GPY media exhibited less pink pigmentation compared to those on WSP30 media. Similarly, MF50 demonstrated that colonies on GPY were smaller and rounder, further underscoring the role of media composition in shaping fungal characteristics. The identified strains showed Penicillium sp. is the most represented genus, including strains such as MF2 Pure 2A, MF9, MF22 Pure 2B, MF46, MF50, MF51, and MF53. Others identified included Paraphaeosphaeria sp. (MF5), Fusarium sp. (MF12, MF40), Aspergillus sp. (MF48), Neoacremonium sp. (MF55), Microascus sp. (MF56), Xerochrysium sp. (MF58) and Corollospora sp.(MF59). Molecular identification using the ITS region provided a genus-level classification for 15 cultivated strains. While ITS1 and ITS4 primers were effective, the ITS region's limitations, such as its inability to resolve species-level relationships and short sequence length (400–600 bp), highlight the need for additional genetic markers (e.g., RPB1, RPB2, β-tubulin) or whole-genome sequencing for greater taxonomic accuracy. The antimicrobial potential findings indicate that the crude extract from Penicillium sp. (MF9) inhibited the highest antibacterial activity against E. coli while Fusarium sp. (MF40) also demonstrated significant activity. In contrast, the crude extract from Aspergillus sp. (MF48) exhibited the lowest activity among the tested samples against E. coli. Overall, antibacterial effectiveness was markedly higher against E. coli compared to B. subtilis, reflecting potential differences in susceptibility and mechanisms of action among the tested fungal extracts. 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