First Report of Epicoccum nigrum Causing Brown Leaf Spot in Tea in Guizhou Province, China

Brown leaf spots were observed on tea [Camellia sinensis (L.) Kuntze] in Sinan County (27.74 °N, 108.35 °E) and Kaiyang County (27.96 °N, 107.34 °E), Guizhou Province, China, from 2018 to 2020. For the leaf spots with the typical symptoms, the disease incidence was estimated to range between 56% and 61%, respectively. The disease severity was estimated to range from 39 to 43 across 12 tea plantations, respectively. The disease initially occurred at the margins of leaf tips, and the lesions expanded gradually, being dark brown and irregularly shaped and became necrotic. To identify the causal organism, two leaves from each of 15 tea twigs, one or two per plantation, were detached from 8- or 10-year-old tea plants on each of 12 plantations. Samples taken from the lesion margins were sterilized with 75% ethanol followed by 0.5% NaOCl, placed on potato dextrose agar (PDA), and then incubated at 25oC in darkness for 5 days (Wang et al. 2020). For each sample, hyphal tips from the margin of a growing colony were successively transferred to fresh PDA, and pure cultures were obtained. Three representative strains were grown on PDA, malt extract agar (MEA), and oatmeal agar (OA) plates. The colonies had smooth margins and abundant mycelia on all three media, with the colony colors being from gray to light purple on PDA, white on MEA, and purplish-red on OA at 5 days post-inoculation. At 20 days post-inoculation on MEA, stromata began to gradually form, which were droplet-like, 100 to 2,000 μm in diameter, and semi-immersed on the medium’s surface. Black sporodochia were produced on the surfaces of stromata. Conidiophores were aggregated in sporodochia, densely compacted, and dark brown. Conidia were globose or pyriform, dark, multicellular, and measured 22.95 ± 3.59 × 19.82 ± 3.13 μm (n = 50) in diameter. The morphological characteristics of the mycelia and reproductive structures of the strains were identical to those of Epicoccum nigrum. The internal transcribed spacer (ITS) region of rDNA, and the partial 28S large subunit rDNA (LSU), RNA polymerase II second largest subunit (RPB2), and beta-tubulin (TUB) genes of these strains were amplified using the primers V9G/ITS4 (De Hoog and Gerrits van den Ende 1998; White et al. 1990), LR0R/LR5 (Rehner and Samuels 1994), RPB2-5F2/fRPB2-7cR (Sung et al. 2007), and TUB2Fd/TUB4Rd (Woudenberg et al. 2009), respectively, and deposited in GenBank (accession no. MW646378, MW291537, MW602293, and MW602295 for ITS, LSU, RBP2, and TUB, respectively). A maximum parsimony phylogenetic analysis indicated that the representative strains clustered with E. nigrum CBS 173.73 (Chen et al. 2017). Pathogenicity tests were performed on 5-year-old potted tea and on 10-year-old C. sinensis cv. Fuding-dabaicha in the field. Mycelial plugs (6-mm diam.) and a conidial suspension (106 conidial/mL) were applied on punctured leaves using a sterile needle and non-punctured leaves. Inoculation with only a PDA plug or sterile water served as controls. Brown spots appeared on the wounded sites of tea leaves at 2 days post-inoculation. No symptoms were observed on the non-wounded leaves or wounded leaves inoculated with PDA plugs lacking mycelia. The re-isolated pathogen from diseased plants was identical to the purified strain ACCC39731 used for inoculation, with re-isolation frequency being 85.0%. To our knowledge, this is the first report of E. nigrum causing leaf spot on tea plants in China, and our findings will be useful for its management and further research.

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).

Antimicrobial and Antibiofilm Activities of the Fungal Metabolites Isolated from the Marine Endophytes Epicoccum nigrum M13 and Alternaria alternata 13A

Epicotripeptin (1), a new cyclic tripeptide along with four known cyclic dipeptides (2–5) and one acetamide derivative (6) were isolated from seagrass-associated endophytic fungus Epicoccum nigrum M13 recovered from the Red Sea. Additionally, two new compounds, cyclodidepsipeptide phragamide A (7) and trioxobutanamide derivative phragamide B (8), together with eight known compounds (9–16), were isolated from plant-derived endophyte Alternaria alternata 13A collected from a saline lake of Wadi El Natrun depression in the Sahara Desert. The structures of the isolated compounds were determined based on the 1D and 2D NMR spectroscopic data, HRESIMS data, and a comparison with the reported literature. The absolute configurations of 1 and 7 were established by advanced Marfey’s and Mosher’s ester analyses. The antimicrobial screening indicated that seven of the tested compounds exhibited considerable (MIC range of 2.5–5 µg/mL) to moderate (10–20 µg/mL) antibacterial effect against the tested Gram-positive strains and moderate to weak (10–30 µg/mL) antibacterial effect against Gram-negative strains. Most of the compounds exhibited weak or no activity against the tested Gram-negative strains. On the other hand, four of the tested compounds showed considerable antibiofilm effects against biofilm forming Gram-positive and Gram-negative strains.

Epipyrone A, a Broad-Spectrum Antifungal Compound Produced by Epicoccum nigrum ICMP 19927

We have isolated a filamentous fungus that actively secretes a pigmented exudate when growing on agar plates. The fungus was identified as being a strain of Epicoccum nigrum. The fungal exudate presented strong antifungal activity against both yeasts and filamentous fungi, and inhibited the germination of fungal spores. The chemical characterization of the exudate showed that the pigmented molecule presenting antifungal activity is the disalt of epipyrone A—a water-soluble polyene metabolite with a molecular mass of 612.29 and maximal UV–Vis absorbance at 428 nm. This antifungal compound showed excellent stability to different temperatures and neutral to alkaline pH.