Fungus Epicoccum Nigrum Research Articles

Cytotoxic naphtho- and benzofurans from an endophytic fungus Epicoccum nigrum Ann-B-2 associated with Annona squamosa fruits

Chemical exploration of the total extract derived from Epicoccum nigrum Ann-B-2, an endophyte associated with Annona squamosa fruits, afforded two new metabolites, epicoccofuran A (1) and flavimycin C (2), along with four known compounds namely, epicocconigrone A (3), epicoccolide B (4), epicoccone (5) and 4,5,6-trihydroxy-7-methyl-1,3-dihydroisobenzofuran (6). Structures of the isolated compounds were elucidated using extensive 1D and 2D NMR along with HR-ESI–MS. Flavimycin C (2) was isolated as an epimeric mixture of its two diastereomers 2a and 2b. The new compounds 1 and 2 displayed moderate activity against B. subtilis, whereas compounds (2, 3, 5, and 6) showed significant antiproliferative effects against a panel of seven different cancer cell lines with IC50 values ranging from 1.3 to 12 µM.

Biosynthesis of Epipyrone A Reveals a Highly Specific Membrane-Bound Fungal C-Glycosyltransferase for Pyrone Galactosylation.

Epipyrone A is a unique C-galactosylated 4-hydroxy-2-pyrone derivative with an antifungal potential from the fungus Epicoccum nigrum. We elucidated its biosynthesis via heterologous expression and characterized an unprecedented membrane-bound pyrone C-glycosyltransferase biochemically. Molecular docking and mutagenesis experiments suggested a possible mechanism for the heterocyclic C-glycosylation and the importance of a transmembrane helix for its catalysis. These results expand the repertoire of C-glycosyltransferases and provide new insights into the formation of C-glycosides in fungi.

Destruction of Fraxinus angustifolia and Fraxinus ornus seeds under storage conditions caused by Epicoccum nigrum

AbstractDuring the period of seed storage in 2019, stored Fraxinus angustifolia and F. ornus seeds showed signs of infection and fungus Epicoccum nigrum was isolated from mould and necrotic lesions on the seeds. In order to confirm the pathogenicity of E. nigrum towards F. angustifolia and F. ornus seeds, an experiment that included inoculation of seeds with E. nigrum and the assessment of germinability was performed. The inoculation with E. nigrum strain caused mould to around 20% of F. angustifolia and 58% of F. ornus, necrotic lesions to 96% of F. angustifolia and 92% of F. ornus, and decrease in germinability to 95% of F. angustifolia and 97% of F. ornus seeds. This study presented, for the first time, the ability of E. nigrum to cause high infection rates and reduced germinability of the F. angustifolia and F. ornus stored seeds. The transmission routes and possibilities for preventive strategies were discussed.

Gamma and electron beam irradiation use in the control of a radiation-resistant fungus (Epicoccum nigrum) for preservation of contaminated organic artefacts of traditional heritage

When artefacts of traditional cultural heritage are waterlogged due to floods and other disasters, they can be quickly protected from contamination by harmful fungi using irradiation treatments. In this study, we showed that Epicoccum nigrum has the potential to contaminate traditional heritage artefacts made of Korean paper and silk under waterlogged conditions. E. nigrum showed 60% lower mycelial growth in medium containing freshwater than in medium containing natural seawater. Control of this contamination was attempted using gamma and electron beam irradiation disinfection technology. The mycelial growth of E. nigrum irradiated with a 5 kGy dose of gamma rays was completely controlled at 15 kGy dose. Next, we evaluated the effect of irradiation on contaminated sample artefacts. E. nigrum growing on waterlogged sample artefacts of cultural heritage was completely controlled at 15 kGy in freshwater-immersed artefacts of Korean paper and silk and seawater-immersed silk. However, in Korean paper immersed in seawater, 15 kGy gamma-irradiation treatment did not control growth of the fungus. Our results suggest that at least 15 kGy of gamma radiation should be used to for treatment, to preserve waterlogged traditional cultural heritage artefacts of Korea, after flooding or other disasters involving water.

Epicoccanes A-D, Four Oxidative Dimers of Pyrogallol Analogues from Epicoccum nigrum.

Epicoccanes A-D (1-4) are four novel metabolites of an endophytic fungus Epicoccum nigrum. Their distinct unprecedented structures are hypothesized as oxidative dimers of pyrogallol analogues. Compounds 1 and 2 possess a novel spirobicyclo[3.2.1]octane-6,1'-cyclopentane or -cyclohexane core skeleton. Compound 3 is of a unique cage-like pentacyclic system, which unusually contained three continuous spiro-carbons. Compound 4 is a highly rearranged dimer with five contiguous chiral centers. The absolute structures of 1 and 2 were deduced by electronic circular dichroism (ECD) calculations, and those of 3 and 4 were determined by X-ray crystallography. Compounds 1 and 4 showed potential antiliver fibrosis activity.

Gamma and Electron Beam Irradiation Use in the Control of a Radiation-Resistant Fungus (Epicoccum Nigrum) for Preservation of Contaminated Organic Artefacts of Traditional Heritage

Gamma and Electron Beam Irradiation Use in the Control of a Radiation-Resistant Fungus (Epicoccum Nigrum) for Preservation of Contaminated Organic Artefacts of Traditional Heritage

Furobenzotropolones A, B and 3-Hydroxyepicoccone B with Antioxidative Activity from Mangrove Endophytic Fungus Epicoccum nigrum MLY-3.

Three new metabolites, furobenzotropolones A, B (1–2) with unusual benzene and dihydrofuran moieties and 3-hydroxyepicoccone B (3), together with seven known compounds (4–10) were obtained from the endophytic fungus Epicoccum nigrum MLY-3 isolated from the fresh leaf of mangrove plant Bruguiear gymnorrhiza collected from Zhuhai. Their structures were assigned by the analysis of UV, IR, NMR, and mass spectroscopic data. Compound 1 was further confirmed by single-crystal X-ray diffraction experiment using Cu Kα radiation. In antioxidant activities in vitro, compounds 2, 3, 5, and 8 showed promising DPPH· scavenging activity with IC50 values ranging from 14.7 to 29.3 µM. Compounds 2, 3, 5, 7, and 8 exhibited promising potent activity in scavenging ABTS· with IC50 values in the range of 18–29.2 µM, which was stronger than that of the positive control ascorbic acid (IC50 = 33.6 ± 0.8 µM).

Azacoccones F-H, new flavipin-derived alkaloids from an endophytic fungus Epicoccum nigrum MK214079

Three new flavipin-derived alkaloids, azacoccones F-H (1–3), along with six known compounds (4–9) were isolated from the endophytic fungus Epicoccum nigrum MK214079 associated with leaves of Salix sp. The structures of the new compounds were established by analysis of their 1D/2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectroscopy (HRESIMS) data. The absolute configuration of azacoccones F-H (1–3) was determined by comparison of experimental electronic circular dichroism (ECD) data with reported ones and biogenetic considerations. Epicocconigrone A (4), epipyrone A (5), and epicoccolide B (6) exhibited moderate antibacterial activity against Staphylococcus aureus ATCC 29213 with minimal inhibitory concentration (MIC) values ranging from 25 to 50 μM. Furthermore, epipyrone A (5) and epicoccamide A (7) displayed mild antifungal activity against Ustilago maydis AB33 with MIC values of 1.6 and 1.8 mM, respectively. Epicorazine A (8) showed pronounced cytotoxicity against the L5178Y mouse lymphoma cell line with an IC50 value of 1.3 μM.

Production of the anticancer drug taxol by the endophytic fungus Epicoccum nigrum TXB502: enhanced production by gamma irradiation mutagenesis and immobilization technique.

Taxol, a phyto-extracted diterpenoid, is the most commercially needed drug in cancer chemotherapy. In spite of the microbial production of taxol being successful and prospective, the reported yields are still not sufficient for large-scale production. Thus, the discovery of new taxol-producing microbial strains and production enhancement methodologies such as process optimization, strain improvement, and immobilization technique are the main objectives. In this paper, a taxol-producing start strain Epicoccum nigrum TXB502 (initial yield 61.35 μg L-1) was isolated from Taxus baccata and identified by morphological and molecular tools. The optimum cultivation and nutritional conditions were assessed by testing one parameter at a time approach that resulted in 88.59% significant production increase. In addition, a stable mutant with improved productivity (40.07% yield increase in comparison with the parent strain) was successfully developed after gamma irradiation mutagenesis of the start strain. The taxol titer was further improved via testing different immobilization carriers for both spores and mycelia of this mutant. Over taxol production was achieved using alginate-immobilized mycelia with the feasibility of conducting six successive production cycles in a semi-continuous form. The final total concentration reached 8187.77 μg taxol 6 L-1 which represents approximately 22-fold increase, as compared to the initial titer of the start strain. These findings can pave the way for the prospective industrial manufacturing of taxol, as the achieved taxol production in this study is the highest reported by academic laboratories for microbial cultures. KEY POINTS: • Discovery of a new taxol-producing endophytic fungus E. nigrum TXB502 strain. • Taxol yield was successfully improved via bioprocess optimization and strain mutagenesis. • Alginate-immobilized mycelia were efficient for a semi-continuous production of taxol. • The final total concentration of taxol showed approximately 22-fold increase as compared to the initial titer.

Cytotoxic Thiodiketopiperazine Derivatives from the Deep Sea-Derived Fungus Epicoccum nigrum SD-388.

Four new thiodiketopiperazine alkaloids, namely, 5’-hydroxy-6’-ene-epicoccin G (1), 7-methoxy-7’-hydroxyepicoccin G (2), 8’-acetoxyepicoccin D (3), and 7’-demethoxyrostratin C (4), as well as a pair of new enantiomeric diketopiperazines, (±)-5-hydroxydiphenylalazine A (5), along with five known analogues (6–10), were isolated and identified from the culture extract of Epicoccum nigrum SD-388, a fungus obtained from deep-sea sediments (−4500 m). Their structures were established on the basis of detailed interpretation of the NMR spectroscopic and mass spectrometric data. X-ray crystallographic analysis confirmed the structures and established the absolute configurations of compounds 1–3, while the absolute configurations for compounds 4 and 5 were determined by ECD calculations. Compounds 4 and 10 showed potent activity against Huh7.5 liver tumor cells, which were comparable to that of the positive control, sorafenib, and the disulfide bridge at C-2/C-2’ is likely essential for the activity.

Amphiepicoccins A-J: Epipolythiodioxopiperazines from the Fish-Gill-Derived Fungus Epicoccum nigrum HDN17-88.

Ten new epipolythiodioxopiperazines (ETPs), namely, amphiepicoccins A-J (1-10), were isolated from the fish-gill-derived fungus Epicoccum nigrum HDN17-88. Their structures were deduced from extensive spectroscopic data and electronic circular dichroism (ECD) calculations. Amphiepicoccin A (1) which contains an aromatic indole motif is unprecedented among the epicoccin type of ETPs. Compounds 1, 3, and 6 displayed anti-HSV-2 activities, with IC50 values of 70, 64, and 29 μM, respectively (acyclovir as positive control with an IC50 value of 31 μM), while 5 and 6 also revealed inhibitory activity against Bacillus subtilis with minimum inhibitory concentration (MIC) values of 13 and 25 μM, respectively.

Fused multicyclic polyketides with a two-spiro-carbon skeleton from mangrove-derived endophytic fungus Epicoccum nigrum SCNU-F0002.

A pair of uncommon fused multicyclic polyketides with a two- spiro-carbon skeleton, (±)-isoepicolactone, (±)-1, and one new isobenzofuranone monomer (4), together with four other known biosynthetically related compounds were isolated from the fermentation of an endophytic fungus, Epicoccum nigrum SCNU-F0002, which was isolated from the fresh fruit of the mangrove plant Acanthus ilicifolius L. Comprehensive spectroscopic analysis, X-ray crystallography, together with calculated ECD, were employed to define the structures. The antibacterial and COX-2 inhibitory activities of the compounds (1–6) were evaluated. A possible biogenetic pathway of (±)-isoepicolactone was confirmed.

Biotransformation of Host Plant Flavonoids by the Fungal Endophyte Epicoccum nigrum

AbstractFermentation of the fungus Epicoccum nigrum isolated from leaves of Salix sp. on green lentil solid medium yielded the flavonol kaempferol (3) as well as two kaempferol O‐diglycosides (1 and 2) including the new compound 1. The fungal flavonoids bear strong structural similarities to kaempferol derivatives such as kaempferol O‐glycoside (4) being present in green lentils. Furthermore, feeding experiments were conducted by adding flavonoids (kaempferol and rutin) as precursors to solid rice media followed by HPLC and LC‐MS analyses. Fermentation of the fungus on flavonoid free solid rice medium afforded flavonoid free extracts indicating that the fungal flavonoids originate through hydrolytic cleavage of kaempferol glycosides such as 4 followed by glycosylation and acetylation. This study suggests that E. nigrum is capable of biotransformation reactions of plant derived flavonoids whereas de novo biosynthesis of flavonoids is less likely.

A novel source of the cardiac glycoside digoxin from the endophytic fungus Epicoccum nigrum: isolation, characterization, production enhancement by gamma irradiation mutagenesis and anticancer activity evaluation.

Different endophytic fungi were isolated and screened for their digoxin-producing ability. Strain improvement and different culture conditions were studied for more effective production of digoxin. Among the isolated fungi, an isolate produced digoxin in a concentration of 2·07mgl-1 . The digoxin-producing fungal isolate was identified as Epicoccum nigrum Link according to the morphological features and phylogenetic analyses. The potentiality of the fungal strain for production enhancement of digoxin was performed by gamma radiation mutagenesis. Gamma irradiation dose of 1000Gy intensified the digoxin yield by five-fold. Using this dose, a stable mutant strain with improved digoxin productivity was isolated and the stability for digoxin production was followed up across four successive generations. In the effort to increase digoxin magnitude, selection of the proper cultivation medium, addition of some elicitors to the most proper medium and several physical fermentation conditions were tested. Fermentation process carried out in malt extract autolysate medium (pH 6·5) supplemented by methyl jasmonate and inoculated with 2ml of 6-day-old culture and incubated at 25°C for 10days stimulated the highest production of digoxin to attain 50·14mgl-1 . Moreover, cytotoxicity of digoxin separated from the fungal culture was tested against five different cancer cell lines. Based on the MTT assay, digoxin inhibited the proliferation of the five different cancer cell lines and the recorded 50% inhibitory concentration ranged from 10·76 to 35·14μgml-1 . This is the first report on the production and enhancement of digoxin using fungal fermentation as a new and alternate source with high productivity. These findings offer new and alternate sources with excellent biotechnological potential for digoxin production by fungal fermentation. Moreover, digoxin proved to be a promising anticancer agent whose anticancer potential should be assessed in prospective cancer therapy.

Isobenzofuranone monomer and dimer derivatives from the mangrove endophytic fungus Epicoccum nigrum SCNU-F0002 possess α-glucosidase inhibitory and antioxidant activity.

Four new isobenzofuranone monomers, (+)-epicoccone C ((+)-1), (-)-epicoccone C ((-)-1), epicoccone D (2), epicoccone E (3) and one new isobenzofuranone dimer, epicolactone A (4), together with four known related dimers were obtained from the fermentation of an endophytic fungus, Epicoccum nigrum SCNU-F0002, which was isolated from the fresh fruit of the mangrove plant Acanthus ilicifolius L. Their structures were elucidated on the basis of spectroscopic methods, single-crystal X-ray diffraction analysis, and electronic circular dichroism calculations. These isolated compounds (1-8) were evaluated for their antioxidant activity and α-glucosidase enzyme inhibitory activity. All of the compounds except 5 exhibited more potent α-glucosidase inhibitory effect than acarbose. Most of the compounds showed superior antioxidant activity with IC50 values ranging from 10.2 to 15.3 μM than positive control, gallic acid and vitamin C.

Two-Photon Absorption and Cell Imaging of Fluorene-Functionalized Epicocconone Analogues.

Epicocconone 1 is a natural chromophore isolated from the fungus Epicoccum nigrum that has shown applications in proteomics and fluorescent microscopy thanks to its unique pro-fluorescence properties. The modification of the skeleton of the natural product by replacing the triene side chain by a fluorenyl scaffold can noticeably increase the fluorophore's absorption coefficient. The synthesis of the analogues of the natural product has been made possible by the use of a palladium-catalyzed carbonylation reaction, allowing the construction of the β-keto-dioxinone key intermediate. Two-photon absorption cross-section measurements of the fluorenyl epicocconone analogues show a structure dependency with values ranging from 60 to 280 GM and live cell imaging show intense staining of intracellular vesicle-like structures around the nucleus.

The Purification, Characterization, and Biological Activity of New Polyketides from Mangrove-Derived Endophytic Fungus Epicoccum nigrum SCNU-F0002.

Six new polyketides, including one coumarin (1), two isocoumarins (2 and 3), dihydroradicinin (4), and two benzofuranone derivatives (7 and 8), together with seven known analogues (5–6 and 9–13) were isolated from the culture of the mangrove endophytic fungus Epicoccum nigrum SCNU-F0002. The structures were elucidated on the interpretation of spectroscopic data. The absolute configuration of Compounds 2 and 3 were determined by comparison of their ECD spectra with the data of their analogue dihydroisocoumarins described in the literature. The absolute configuration of 4 was determined by single-crystal X-ray diffraction. All the compounds were screened for their antioxidant, antibacterial, anti-phytopathogenic fungi and cytotoxic activities. Using a DPPH radical-scavenging assay, Compounds 10–13 showed potent antioxidant activity with IC50 values of 13.6, 12.1, 18.1, and 11.7 μg/mL, respectively. In addition, Compounds 6 and 7 showed antibacterial effects against Bacillus subtilis (ATCC 6538), Escherichia coli (ATCC 8739), and Staphylococcus aureus (ATCC 6538), with MIC values in the range of 25–50 μg/mL.

Morphological response to salinity, temperature, and pH changes by marine fungus Epicoccum nigrum.

Epicoccum nigrum (strain LQRA39-P) was isolated from sediments collected in Chilean Patagonian fjords using microscopy and molecular techniques. We analyzed adaptive responses of cell wall morphology to salinity, temperature, and pH in order to explain the ability of E. nigrum to co-inhabit both marine and freshwater environments. For this purpose, E. nigrum was cultured in a series of media with variations in salinity (freshwater and seawater), pH (acidic, neutral, and basic), and temperature (5 to 25°C). Changes were observed through transmission electron microscopy. A direct correlation between increased salinity and cell wall thickening (> 0.2μm) was observed, along with a significant relationship between pH and the presence of extracellular polymeric substances (EPS) on the outside of the cell wall. The observed morphological changes could confirm that an ubiquitous fungus such as E. nigrum requires adaptive responses to co-inhabit freshwater, marine, and terrestrial substrates.

Biosynthesis of Silver Nanoparticles Using the Compound Curvularin isolated from the Endophytic Fungus Epicoccum Nigrum: Characterization and Antifungal activity

Biosynthesis of Silver Nanoparticles Using the Compound Curvularin isolated from the Endophytic Fungus Epicoccum Nigrum: Characterization and Antifungal activity

Characterization of fluorescent synthetic epicocconone-based dye through advanced light microscopies for live cell imaging applications

Identification of new fluorescent biomarkers is a key issue to decipher mechanisms in living cells. Isolated from the fungus Epicoccum nigrum, natural epicocconone (NE) establishes a covalent and reversible link with primary amines and has been identified as a new fluorescent dye for cell imaging. In the present study, the properties of a synthetic and multifunctional analogue of epicocconone (SE) have been compared to those of NE through 1-photon (1P) and 2-photon (2P) advanced light microscopies. 1P Λλ confocal microscopy analyses revealed that, in cell culture medium, SE and NE displayed two peaks of excitation (ex) at 480 and 530 nm and a large band of emission (em) with a maximum at 610 nm. In living cells, SE presented sharper bands compared to NE with maxima at 545 nm (ex) and 605 nm (em). In 2P microscopy, SE and NE presented maxima around 790 nm (ex) and 595 nm (em) when diluted in cell culture medium. In living cells, SE did not present any large disturbing blue-shift for emission that was observed for NE. In addition, 2P bands for SE were sharper than the NE ones. SE was 2–3 times more fluorescent than NE as determined through 1P and 2P approaches. SE did not induce any cytotoxicity and was adapted for time-lapse acquisition. In PC12 cells, SE broadly labeled nuclear and plasma membranes, vesicles-like structures and organelles including the Golgi apparatus and mitochondria. Taken together, these data indicate that SE has appropriate properties for in vitro and in vivo cell biology studies and presents many advantages compared to NE for 1P and 2P microscopies.