Identification and characterization of Epicoccum nigrum causing leaf spot on O'Neal blueberry in China

Soybean [Glycine max(L.) Merr.] holds significant global economic importance due to its considerable nutritional and commercial value. Since 2022, a leaf blight of unknown etiology has been observed in soybean-growing areas of Nehe City and Hongxinglong Farm in Heilongjiang Province, China. Although the disease has not yet reached epidemic levels, it warrants attention due to its potential to spread under favorable environmental conditions. Initial symptoms on infected leaves appear as small brown spots that gradually expand to form streaks of necrotic tissue delimited by the veins, with a brown necrotic center surrounded by a yellow margin. Through systematic sampling and isolation, twenty-six fungal isolates with similar morphologies were obtained. Morphological characterization, molecular identification, and phylogenetic analyses were used to identify the pathogen of this new soybean leaf blight as Epicoccum nigrum. This study represents the first report of E. nigrum as a pathogen of soybean in China. Pathogenicity tests showed that most isolates displayed moderate to high virulence on soybeans. In greenhouse spray inoculation assays,E. nigrumisolates infected several legume species but did not cause disease on maize or hyacinth bean under the conditions tested.In vitro sensitivity assays showed that E. nigrum isolates exhibited high sensitivity to pyraclostrobin and azole-tebuconazole, with an EC₅₀ value ≤ 0.50 μg mL-1. In pot experiments, both fungicides provided significant disease control, achieving 90.2% control efficacy at 450 μg mL⁻¹ (pyraclostrobin) and 91.7% at 600 μg mL⁻¹ (azole-tebuconazole). These results support the use of chemical fungicides as a key strategy for managing soybean leaf blight caused byE. nigrum. Overall, this study clarifies the causal agent of soybean leaf blight, thereby providing a scientific basis forits management in soybean production. Additionally, the identification of effective fungicides provides practical guidance for targeted disease management, which is essential for safeguarding soybeanyields and mitigating economic losses associated with this emerging disease. Furthermore, this study also establishes a foundation for future investigations into the pathogenic mechanisms and epidemiological dynamics ofE. nigrum.

Abstract This work aimed to bioprospect the antiproliferative activity of 13 ethyl acetate extracts of pigmented strains of phytopathogenic fungi of the genera Epiccocum and Fusarium . The antiproliferative activity of extracts was tested against six solid tumor cell lines: A-549 (lung), HBL-100 (breast), HeLa (cervix), SW1573 (lung), T-47D (breast), and WiDr (colon). Ten extracts from strains of Epicoccum sorghinum (Sacc.) Aveskamp, Gruyter & Verkley, Epicoccum nigrum Link, Didymellaceae, Fusarium graminearum Schwab, and Fusarium sp., Nectriaceae, showed antiproliferative activity against at least one cell line, with GI 50 values ≤ 50 μg/ml. Three of them exhibited a strong antiproliferative activity (GI 50 < 2.5 μg/ml) against two cell lines. Furthermore, the extract of E . sorghinum showed a total growth inhibition (TGI) value < 5 μg/ml against the SW1573 and WiDr cell lines. The mycochemical study of this strain of E. sorghinum allowed the isolation and confirmation of the presence of the pigments altersolanol A and macrosporin, fungal metabolites previously reported with cytotoxic activity against cancer cells. While this work presents preliminary results related to the antiproliferative potential of pigment-producing phytopathogenic fungi, it fully opens the possibility of continuing to investigate these fungi as a source of substances with antiproliferative potential against cancer cell lines. Graphical Abstract

Postharvest fungal decay is a primary cause of losses in blueberries, motivating the development of sustainable alternatives to conventional fungicides. This study aimed to develop and evaluate antifungal active films based on polylactic acid (PLA) enriched with citronella essential oil to control phytopathogenic fungi associated with blueberry spoilage. PLA films containing 7.5, 10, and 12.5% (w/w) citronella essential oil were produced by solvent casting and characterized for water vapor transmission rate and nanomechanical properties. The antifungal effect was tested in vitro against Epicoccum nigrum, Alternaria alternata, and Cladosporium herbarum. Active films exhibited concentration-dependent antifungal activity, with C. herbarum being the most sensitive fungus. The incorporation of citronella essential oil did not significantly alter the water vapor barrier properties of PLA, while mechanical analysis revealed a reduction in elastic modulus only at the highest concentration. The antifungal mechanism was elucidated using scanning electron microscopy, fatty acid profiling, absorbance at 260 nm, and conductivity measurements. The results indicate that the released citronella essential oil induced membrane disruption and morphological damage in fungal hyphae, with species-specific responses. Overall, PLA-citronella essential oil films represent a promising biodegradable packaging solution to control postharvest blueberry losses.

Endophytic fungi can influence plant development through diverse molecular mechanisms; however, their volatile organic compound VOC-mediated effects on agriculturally relevant crops remain insufficiently characterized. In this study, we examined the effects of VOCs produced by six grass-root-associated endophytic fungi-Cadophora fastigiata, Cordyceps fumosorosea, Chaetomium funicola, Epicoccum nigrum, Microdochium bolleyi, and Plectosphaerella cucumerina-on early growth of spring barley (Hordeum vulgare) and red clover (Trifolium pratense). In plate-in-plate VOC exposure assays, we assessed root system traits, root hair formation, and biomass accumulation. Responses to fungal VOCs were fungal species-specific but similar across barley and red clover. VOCs emitted by C. fastigiata and P. cucumerina were consistently associated with increased root growth, root hair proliferation, and seedling biomass, whereas VOCs from M. bolleyi and C. funicola resulted in neutral or growth-suppressing effects. A complementary seed inoculation experiment was conducted with barley, which showed fungal species-dependent contrasting effects consistent with the observations of VOCs treatment. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that C. fastigiata, the isolate associated with the strongest growth-promoting responses, emitted a diverse VOC profile dominated by sesquiterpenes, with 22 compounds identified. Together, these results demonstrate that VOCs emitted by grass-root-associated endophytic fungi exert reproducible, species-specific effects on early plant development occurring in phylogenetically distant species. The findings highlight the value of VOC-based assays for comparative functional screening of fungal isolates, providing a foundation for future studies that aim to link individual VOCs to plant growth responses.

The aim of this paper is the development of a chemical-biological method for reducing the toxicity of used-up electrolytic bath sludge from the galvanizing industry, which performs zinc electrodeposition on steel parts. Cyanide-degrading microorganisms were isolated from soil samples in the sludge discharge area and cultivated under controlled conditions. The most representative colonies were seeded in a continuous bioreactor. Then, the effect of various initial concentrations of glucose and cyanide in the input flow was evaluated. The reactor was fed with a culture medium containing the leachate obtained by alkaline washing of sludge as a nitrogen source, glucose as a carbon source, and mineral nutrients. The greatest cyanide removal efficiency (95%) corresponded to the smallest initial cyanide concentrations (between 11 and 15 mg/L) and the highest initial glucose concentration (5 g/L) in the bioreactor. In this study, we achieved the isolation of three fungal species that degraded a mixture of Fe, Zn, and Cu cyanide complexes, one of which is ferric (III) hexacyanoferrate (II) Fe4[Fe(CN)6]3. These species were identified as Cladosporium cladosporoides, Epicoccum purpurascens, and Penicillium verrucosum. This is the first evidence that these fungal genera can degrade cyanide complexes, representing a very important contribution to the development of technologies for the treatment of industrial waste.

Canine mammary tumors (CMTs) are among the most frequent neoplasms in female dogs, with current therapeutic options being limited and non-standardized, prompting the search for alternative treatments such as fungal secondary metabolites. In this study, the fungal pigment Epipyrone A (Epy A) was first isolated from Epicoccum nigrum and then tested in vitro on two CMT cell lines, P114 and CF33. The compound significantly reduced cell viability in both lines in a concentration- and time-dependent manner (p < 0.05), with the strongest effect observed at 175 µg/mL after 48 h (p < 0.0001), while showing no cytotoxicity in MDCK non-tumor cells. Epy A also inhibited cell migration and increased total antioxidant capacity in P114 cells, accompanied by a significant reduction in ROS levels. Western blot analysis revealed modulation of the PI3K/Akt/mTOR pathway, crucial in CMT biology. Specifically, P114 cells showed downregulation of mTOR and p-Akt, indicating inhibition of proliferative signaling, whereas CF33 cells exhibited increased Akt and p-Akt alongside reduced mTOR, consistent with a compensatory feedback mechanism, probably linked to the changing in oxidative balance after treatment. Overall, these results identified Epy A as a promising natural molecule with potential applications in innovative therapeutic approaches for veterinary and comparative oncology.

Tomato (Solanum lycopersicum L.) is one of the most widely cultivated vegetable crops globally. Still, its productivity is significantly constrained by tomato leaf curl virus (ToLCV), a devastating begomovirus transmitted by whiteflies. This study examined the diversity and plant growth-promoting potential of culturable endophytes associated with tomato cultivars differing in resistance to tomato leaf curl virus (ToLCV). A total of 59 fungal and bacterial endophytes were isolated. Resistant cultivars (Nandi, Sankranthi, and Vybhav) harboured more diverse and compositionally distinct communities than the susceptible cultivar Arka Vikas, as indicated by Shannon, Simpson, and Chao-1 indices and multivariate analyses. Several isolates, particularly from the genera Xylaria, Fusarium, Arcopilus, Epicoccum, Bacillus, Pseudomonas, Stutzerimonas, and Paenibacillus, displayed strong nutrient-solubilizing traits in vitro, highlighting their potential as plant growth-promoting candidates. Eleven promising isolates were further evaluated on the susceptible cultivar Arka Vikas. At 30 days after sowing, Epicoccum nigrum and Bacillus subtilis significantly increased seedling height, biomass, and leaf number relative to the control. Overall, the study reveals that resistant cultivars are associated with greater culturable endophyte diversity and identifies several isolates with strong potential for promoting plant growth. Future research should assess the antiviral potential of these endophytes under ToLCV challenge and employ metagenomic studies to elucidate their functional roles in enhancing plant health.

The fungal endophyte strain, Epicoccum nigrum, was studied to assess the antagonistic activity against fungal phytopathogens and their bioactive secondary metabolites. The strain was isolated from plant tissues (Avena sativa) and identified through morphological and molecular analysis. Antagonistic potential was assessed via dual culture bioassays against Fusarium verticillioides and Alternaria alternata. Results showed significant inhibition of pathogen growth: F. verticillioides radial growth was reduced by 33.9% after 48 h, 65.5% after five days, and 66.8% after fifteen days, whereas weak inhibition was detected against A. alternata. Epipyrone A and epicoccamide A were the main secondary metabolites produced, purified by reversed-phase HPLC and their structures were elucidated using mass spectrometry and spectroscopic data obtained by NMR techniques. Epicoccum nigrum may function as a biocontrol agent through the production of bioactive compounds, but further studies are needed to explore the mechanisms and applications in terms of sustainable plant disease management.

Sorghum bicolor (L.) is a vital coarse grain in China with high nutritional value and significant economic importance. In September 2023, a leaf spot disease was observed affecting S. bicolor in Heihe City (50°15’N, 127°27’E), Heilongjiang Province, China, with a disease incidence of 54%. The primary symptoms consisted of elliptical lesions (1-3 mm) on leaves, displaying dark brown necrotic centers surrounded by reddish-brown halos. Samples taken from the lesion margins were sterilized with 75% ethanol and 0.5% NaOCl, followed by three rinses in sterile distilled water. The sterilized tissues were placed on potato dextrose agar (PDA) and incubated at 25°C in darkness for 5 days (Wang et al., 2020). For each sample, pure cultures were obtained by sequentially transferring mycelial tips from the margins of actively growing colonies to PDA. Colony growth on PDA was slow with irregular margins and sparse aerial mycelium. Colonies initially appeared white before developing tan-yellow pigmentation, with reverse sides showing central black areas and orange-red peripheries. Conidia were solitary, globose to subglobose, tan-brown to dark brown, and measured 18.97 ± 2.72 × 20.09 ± 2.39 μm in diameter. Mature spores developed both transverse and longitudinal septa, while immature spores retained pale stalk cells. The morphological characteristics of the strain were identical to those of Epicoccum nigrum (Sheikhloo et al., 2011). The internal transcribed spacer (ITS) region of rDNA, and the partial 28S large subunit rDNA (LSU), RNA polymerase II second largest subunit (RPB2) genes were amplified using the primers ITS1/ITS4 (Rehner and Samuels 1994), LROR/LR7 (Carbone et al., 1999), and RPB2-5F2/fRPB2-7cR (Sung et al., 2007), respectively. The sequences were deposited in GenBank under accession numbers PP907644 (ITS), PQ151145 (LSU), and PQ496857 (RPB2). A multi-locus phylogenetic tree was reconstructed via neighbor-joining analysis, with maximum parsimony analysis revealing 99 to 100% sequence identity between the obtained sequences and reference entries. Healthy sorghum BTx623 leaves at the 4-5 leaf stage were selected for pathogenicity testing. Mycelial plugs (6 mm diameter) were inoculated onto pricked leaf surfaces using sterile needles. Controls included inoculation with PDA plugs alone or sterile water. After 7 days of incubation under conditions (25 ± 2°C, 12 h light/dark), brown necrotic lesions developed at inoculation sites on wounded leaves. No symptoms appeared on unwounded leaves or wounded leaves inoculated with PDA plugs lacking mycelia. Re-isolated strains from infected tissues displayed identical morphological characteristics to the original inoculum. To our knowledge, this is the first report of the occurrence of E. nigrum on S. bicolor in China causing leaf spot disease.

Nowadays, microwave ovens have become a staple in every home and at public gatherings where food is needed. The popularity stems from its superior cooking performance compared to other methods. Microwaves can preserve vitamin C and other nutrients that degrade when exposed to heat better than other kitchen appliances. The effectiveness of microwave cooking has led many users to overlook the importance of using microwaves safely when preparing food. This study investigated the presence of harmful microorganisms in food splatters left inside microwave ovens. Samples were collected from six microwaves at six distinct sites, with a total of 12 swabs cultured from each microwave and tested for microbial presence. A total of 142 DNA samples were extracted, and both 16s rDNA and ITS rDNA were analyzed to identify bacteria and fungi. The sequence analysis revealed the presence of uncultured Lactococcus sp., uncultured Legionella sp., and Neottia nidus- avis bacteria, and a significant number of fungi, including Trichosporum lignicola, Candida spp., Saccharomyces cerevisiae, Aspergillus pseudoglaucus, Paecilomyces tenuis, Cercophora acanthigera, Epicoccum nigrum, and Leiotrametes Latina. The findings indicate that food splatters left in microwave ovens for several (three to four) days can harbor harmful microorganisms, such as bacteria and fungi. Findings also acknowledge the possibility of food cross-contamination. We recommend regular, thorough microwave cleaning after noticing food splashes to help prevent the spread of these microorganisms. Findings from this study will provide valuable insights into ongoing research on food safety and public health, specifically regarding microwave oven use.

Mercury-driven fungal endophytic community and the role of endophytes in rice grain mercury accumulation

ABSTRACT Seven biologically synthesized silver nanoparticles (AgNPs) were produced using extremophilic fungal species from the phylum Ascomycota , including Aspergillus tubingensis , Aspergillus spp., Bionectria ochroleuca , Cladosporium pini‐ponderosae , Fusarium proliferatum , Epicoccum nigrum , and Exserohilum rostratum , all of which were isolated from the Brazilian biodiversity. The nanoparticles were designated based on the initials of the respective fungal species (AgNP‐AT, AgNP‐Asp, AgNP‐BO, AgNP‐CPP, AgNP‐FP, AgNP‐EN, and AgNP‐ER) and showed predominantly spherical morphology with hydrodynamic diameters consistent with protein capping (DLS), dimensions between 22 and 86 nm (TEM), negative Zeta potential, and FTIR bands indicative of a fungal‐derived biomolecular corona. Cytotoxicity was assessed by the MTT assay on MDA‐MB‐231 triple‐negative breast cancer cells (IC50) and normal human fibroblasts (FN1; CC50) at 24, 48, and 72 h, and the selectivity index (SI = CC50/IC50) was calculated. All AgNPs reduced the MDA‐MB‐231 viability, and AgNP‐AT, AgNP‐BO, and AgNP‐Asp exhibited the highest selectivity toward tumor cells. The AgNP‐AT was the most effective and selected to determine its effects on cell‐cycle distribution, apoptosis induction, and mutagenicity. Furthermore, AgNP‐AT at 0.03 mM did not induce structural chromosomal aberrations in V79‐4 cells. These findings indicate that fungal‐derived biogenic AgNP‐AT is a potent and selective candidate against triple‐negative breast cancer cells, without detectable genotoxicity at its effective concentration.

Fungal community dynamics play a fundamental role in plant health, influencing resilience, disease progression, and ecosystem stability. Monilinia fructicola, a quarantine pathogen affecting Prunus persica, is hypothesized to drive fungal dysbiosis by altering microbial diversity, ecological interactions, and functional roles. In this study, the impact on fungal community composition, trophic dynamics, and network structure was investigated using internal transcribed spacer metabarcoding based on high-throughput sequencing of flowers and branches from infected and noninfected P. persica trees. The results showed a pathogen-induced shift, most pronounced in flowers, in which M. fructicola increased richness (Chao1), diversity (Shannon), and dominance (Simpson). In wood samples, increased dominance suggested pathogen-associated ecological filtering. Network analysis demonstrated that M. fructicola disrupts fungal connectivity, leading to fragmentation of microbial interactions, reduced network complexity, and increased modularity, indicative of weakened ecological cohesion. Shifts in fungal primary lifestyles further suggested that M. fructicola accelerates tissue decomposition and induces an imbalance in functional guilds, potentially facilitating secondary infections. Taxonomic restructuring was marked by the decline of dominant genera and the emergence of stress-adapted or pathogen-associated taxa. The isolation of Epicoccum nigrum, a known biocontrol agent, highlights its potential as a nature-based solution for disease management. Overall, these findings identify M. fructicola as a driver of fungal dysbiosis in P. persica, with cascading effects on microbial function, network resilience, and plant health. Understanding these pathogen-induced shifts is essential to inform sustainable microbiome-based approaches to disease management.

Bromus tectorum (cheatgrass) is one of the most diffused invasive plants in North America. The fungus Epicoccum nigrum has been isolated from the rhizosphere of cheatgrass. Pathogenicity tests revealed the capacity of this strain to kill germinating seeds of the host plant under conditions of water stress. The organic extract obtained from liquid culture of the pathogen showed high toxicity on seeds of Portulaca oleracea. The main secondary metabolite (13S,9Z,11E)-13-hydroxyoctadeca-9,11-dienoic acid (i.e. 13-HODE, 1) produced by this strain has been isolated and identified, together with its methyl ester. In hypocotyl/radicle elongation bioassays on P. oleracea, 13-HODE showed a moderate phytotoxicity at 6.6 mM, reducing hypocotyl length by almost half and radicle length by a factor of ten, compared to the control. The bioactivity of this fungal compound has been compared with methyl and acetyl derivatives to determine the relevance of the main functional groups of 13-HODE in the observed toxicity.

To evaluate the inhibitory effect and application potential of <italic>Epicoccum nigrum</italic> Z01 fermentation broth against <italic>Saprolegnia</italic>, this study isolated a filamentous fungus from the eyes of grass carp (<italic>Ctenopharyngodon idella</italic>) and obtained an antagonistic fungus against <italic>Saprolegnia</italic> from culture plates. After morphological and molecular biological identification, the strains were confirmed as <italic>Saprolegnia ferax</italic> and <italic>Epicoccum nigrum</italic> Z01, respectively. The inhibitory effect of the fermentation broth on <italic>S. ferax</italic> was assessed using plate confrontation, agar dilution, and broth dilution assays. Safety was evaluated through histopathological observation, hematological parameter analysis, and survival rate statistics after fish immersion. Furthermore, the preventive efficacy of the fermentation broth was assessed in grass carp infected with <italic>Saprolegnia</italic> via injection, immersion, and oral administration, while its therapeutic effect was investigated through post-infection immersion. Results demonstrated that <italic>E. nigrum</italic> Z01 exerted a significant and stable inhibitory effect on <italic>Saprolegnia</italic> growth. In the agar dilution assay, no <italic>Saprolegnia</italic> growth occurred when the fermentation broth-to-PDA ratio exceeded 3:7, and no spore germination was observed in PDB supplemented with more than 0.1 mL of fermentation broth. Immersed fish exhibited normal blood cell morphology and higher survival rates than the control group. The prevalence of <italic>S. ferax</italic> infections in grass carp treated by immersion, injection, and oral administration was significantly reduced by 60%, 50%, and 40%, respectively. Importantly, <italic>E. nigrum</italic> Z01 markedly suppressed <italic>Saprolegnia</italic> growth without causing damage to the liver, spleen, trunk kidney, and gut of grass carp. The fermentation broth also reduced infection rates while promoting choroid thickening and increasing lamina propria cell counts in intestinal villi. These findings provide crucial insights for the biological control of <italic>Saprolegnia</italic>.

Exploring the pharmaceutical potential of the Antarctic fungus Epicoccum nigrum isolated from Kallymenia antarctica seaweed

A significant quantity of agro-industrial waste is generated globally across various agricultural sectors and food industries. Composed primarily of cellulose, hemicellulose, and lignin—known as lignocellulosic materials—this waste holds significant potential and can be repurposed as a nutrient-rich substrate for mushroom cultivation. Therefore, mushroom cultivation can be regarded as a promising biotechnological approach for the reduction and valorization of agro-industrial waste. This investigation is the first to explore the utilization of agro-industrial waste- and by-products for the cultivation of Epicoccum nigrum for the production of extracts with valuable biological activities. The logistic curve and autocatalytic growth models were applied to study the kinetics of the growth process on wheat bran, sunflower cake, wheat straw, pine sawdust, and steam-distilled lavender straw substrates. Through mathematical modeling, the optimal composition of a nutrient medium containing the selected substrates was determined and successfully validated in experimental conditions. Biologically active water extracts were obtained after solid-state cultivation with α-amylase and cellulase activity up to 10.6 ± 0.6 U/mL and 0.52 ± 0.03 U/g, respectively. The extracts exhibited antimicrobial activity against fungal strains from six different species, and the most susceptible was the phytopathogen Sclerotinia sclerotiorum, with a minimum inhibitory concentration of 0.156–0.313 mg/mL.

This study aimed to assess the probiotic potential of Leuconostoc citreum DMLC16 previously selected as a fermentation starter candidate based on safety and enzymatic activity evaluations by further examining its health-promoting properties and antimicrobial efficacy. Strain DMLC16 exhibited higher acid tolerance, bile salt resistance, and intestinal adhesion ability than its type strain L. citreum KACC 11860T. Moreover, it demonstrated antimicrobial activities against a range of foodborne pathogens and spoilage organisms, including Bacillus cereus, Enterococcus faecalis, Listeria monocytogenes, Staphylococcus aureus, Alcaligenes xylosoxidans, Flavobacterium sp., and Salmonella enterica. Strain DMLC16 also showed antifungal activities against Clonostachys rosea, Epicoccum nigrum, and Penicillium citrinum. Whole-genome analysis confirmed the safety of DMLC16, revealing the absence of toxin-encoding and plasmids containing acquired antibiotic resistance genes. It also identified genes associated with enzymatic function, salt tolerance, and survival under extreme conditions such as gastric acidity. Although no specific bacteriocin genes were detected, genes implicated in the production of antimicrobial substances were present. The complete genome sequence of DMLC16 provides valuable insight into genetic determinants underlying its probiotic traits and supports its potential application as a functional starter culture or probiotic in the food industry.

The biocontrol effect and mechanism of the marine-derived fungus Epicoccum nigrum against Alternaria alternata, the causal agent of tomato black spot disease.