Cultivation of Fungal Endophytes with Tissue Culture Grapevine Seedlings Reprograms Metabolism by Triggering Defence Responses.

In this study, the transcriptome profiles of tissue-cultured grapevine (Vitis vinifera L. × Vitis labrusca L.: Rose Honey) seedlings inoculated with fungal endophytes Epicoccum layuense R2-21 (Epi R2-21) and Alternaria alternata XHYN2 (Alt XHYN2), were analyzed at three different time points (6 h, 6 d, and 15 d). A total of 4783 differentially expressed genes (DEGs) was found, of which 1853 (6 h), 3878 (6 d), and 4732 (15 d) were differentially expressed relative to those of the control in endophyte Epi R2-21 treatments, while a total of 5898 DEGs, of which 2726 (6 h), 4610 (6 d), and 3938 (15 d) were differentially expressed in endophyte Alt XHYN2 treatments. DEGs enriched in secondary metabolic pathways, plant-pathogen interaction, and hormone signalling were further analysed. The upregulated DEGs in the Epi R2-21 and Alt XHYN2 treatments, both enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG), were mainly involved in flavonoid biosynthesis, phenylpropanoid biosynthesis, stilbenoid, diarylheptanoid and gingerol biosynthesis, phenylalanine metabolism, and circadian rhythms-plant and plant-pathogen interactions, similar to the trend observed in our previous study conducted on the cultivar 'Cabernet Sauvignon' (Vitis vinifera L.). Taken together with the results obtained from the cultivar 'Cabernet Sauvignon', it was found that tissue-cultured seedlings of the cultivar 'Rose Honey' induced a stronger defence response to fungal endophyte infection than that of the cultivar 'Cabernet Sauvignon', and inoculation with the endophyte Alt XHYN2 triggered a stronger response than inoculation with the endophyte Epi R2-21. In addition, the protein-protein interaction (PPI) network revealed that the genes VIT_16s0100g00910, encoding CHS, and VIT_11s0065g00350, encoding CYP73A, were involved in secondary metabolism and thus mediated in the resistance mechanism of grapevine on both the cultivars. The results showed that inoculation with the endophytes Epi R2-21 and Alt XHYN2 had a great ability to induce defence responses and reprogram the gene expression profiles in different grapevine cultivars, which deepens our knowledge of the interaction between fungal endophytes and grapevine and gives hints for grape quality management in viticulture using candidate fungal endophytes.

Genome sequences of three plastic-debris-inhabiting fungi isolated from New Zealand waste and recycling management plants.

Cladosporium and Epicoccum are cosmopolitan fungi of the class Dothideomycetes with few cultured and genomic representatives. Here, we report draft reference genome sequences of Epicoccum sp. F181 (GenBank accession number JAJSLS01), Cladosporium sp. F165 (JAJSLR01), and F190 (JAJSLT01) isolated from recycling and waste management facilities in New Zealand.

Stimulating Novel and Bioactive Metabolite Production by Cocultivation of Two Fungi─Aspergillus oryzae and Epicoccum dendrobii.

Fungi produce various bioactive secondary metabolites (SMs) as protective and weaponized tools to enhance survival in shared ecological niches. By mimicking a competitive ecosystem, cocultivation has been proven to be particularly successful in stimulating SM discovery. Here, we reported the identification of four novel metabolites, epiclactones A and B, epioxochromane and aoergostane, from the coculture of two biotechnologically important strains, Aspergillus oryzae and Epicoccum dendrobii. Transcriptome and metabolome analyses revealed widespread silent gene activation during fungal-fungal interaction. The majority of differentially expressed gene clusters were summarized for both strains. Based on these highly activated biosynthetic pathways, we suggested that a bidirectional chemical defense occurred under cocultivation. E. dendrobii enhanced the production of the spore inhibitor, fumigermin. Moreover, A. oryzae highly accumulated the antifungal agent kojic acid with a yield of up to 1.10 g/L. This study provides an excellent example for the discovery of hidden natural products by cocultivation.

Bioprospecting of exopolysaccharides from the endophytic fungi Epicoccum sorghinum AMFS4, for its structure, composition, bioactivities and application in seed priming.

The endophytic fungi, Epicoccum sorghinum AMFS4 was investigated for its metabolic components and composition of bioactive exopolysaccharides (EPS). Metabolic analysis of the ethyl acetate extract majorly detected sugars derivatives such as, 4-Cholesten-3-one semicarbazone (20.9%), d-Fructose (18.96%), and α-d-Galactopyranosiduronicacid (1.71%). The growth curve and EPS yield were determined as 12.22 ± 1.02 g/L and 7.41 ± 0.32 g/L (dry weight) respectively on day 8. The deproteined EPS has been characterised with pyranose ring linked by α-glycosidic bonds, composing fructose, galactose and glucose monosaccharides validated by HPLC. Total sugar content was found to be 93.18 ± 0.81% with detection of proteins and uronate. The viscous EPS appeared filamentous under SEM observation and behaves as emulsifier with notable antioxidant properties. Priming of EPS on tomato seeds showed early induction of secondary rooting than in the control seedlings. Thus, E. sorghinum AMFS4 synthesises bioactive EPS with simple carbohydrate structure, good water absorption and significant metabolic influence on seed germination.

Didymellaceae species associated with tea plant (Camelliasinensis) in China.

Tea plant is one of the most important commercial crops worldwide. The Didymellaceae fungi can cause leaf blight disease of tea plant. In this study, 240 isolates were isolated from tea plant leaves of 10 provinces in China. Combined with multi-locus (ITS, LSU, RPB2 and TUB2) phylogenetic analysis and morphological characteristics, these isolates were identified as 25 species of six genera in Didymellaceae, including 19 known species Didymellacoffeae-arabicae, D.pomorum, D.segeticola, D.sinensis, Epicoccumcatenisporum, E.dendrobii, E.draconis, E.italicum, E.latusicollum, E.mackenziei, E.oryzae, E.poaceicola, E.rosae, E.sorghinum, E.tobaicum, Neoascochytamortariensis, Paraboeremialitseae, Remotididymellaanemophila and Stagonosporopsiscaricae, of which 15 species were new record species and six novel species, named D.yunnanensis, E.anhuiense, E.jingdongense, E.puerense, N.yunnanensis and N.zhejiangensis. Amongst all isolates, D.segeticola was the most dominant species. Pathogenicity tests on tea plant leaves showed that E.anhuiense had the strongest virulence, while E.puerense had the weakest virulence. Besides, D.pomorum, D.yunnanensis, E.dendrobii, E.italicum, E.jingdongense, E.mackenziei, E.oryzae, E.rosae, E.tobaicum, N.mortariensis, N.yunnanensis, N.zhejiangensis and R.anemophila were non-pathogenic to the tea plant.

Epicoccum sorghinum Causing Leaf Spot on Polygonatum cyrtonema in China.

Polygonatum cyrtonema Hua (family Asparagaceae) is a traditional Chinese medicinal plant that is widely cultivated in various parts of China, including Hunan Province. In summer 2022, a leaf spot disease was observed in 10% of the P. cyrtonema plants (Huang jing) in 18 hectares of this crop in the Hongjiang District (27°18'4″N, 110°11'1″E) of Hunan Province. The initial symptoms of the disease were brown spots on young leaves, and adjacent tissues gradually changed from green to yellow. The entire leaf then became yellow, withered, and eventually exhibited a thn and black appearance. In total, 12 diseased plants from four sampling sites (three plants per site) were collected for laboratory analysis to address the concerns of P. cyrtonema growers. Symptomatic leaf samples were selected, and the leaf fragments containing infected parts of the plants were disinfected with 75% ethanol for 1 min, then immersed in 2.5% hypochlorite for 45 s. After disinfection, symptomatic leaf samples were rinsed three times with sterile water, placed on potato saccharose agar containing 50 µg/ml kanamycin and incubated at 25°C for 2 days. Subsequently, 12 fungal isolates were isolated from various leaf samples through hyphal tip transferring. Ten of the 12 fungal isolates had similar morphological features, and one of them (isolate hjh) was used as the representative isolate for the study. With a growth rate of 6.3 mm per day, its white colonies transformed into red concentric rings in five days; they gradually became black after 10 days of growth. The chlamydospores were round (4.0-9.9 × 3.1-9.3 µm, n = 30), whereas the conidia were ovate (8.0-12.1 × 3.2-6.5 µm, n = 30). The morphological features of the isolate hjh were similar to the features of Epicoccum spp. (Aveskamp et al. 2010). The internal transcribed spacer (ITS) region (including the partial ITS1 sequence and the 5.8S and ITS2 complete sequences), ß-tubulin (tub) gene, and large subunit (LSU) rRNA gene, were amplified from the isolate hjh using the primer pairs ITS5/ITS4, Bt2a/Bt2b, and LROR/LR5, respectively (Taguiam et al. 2021). BLASTn analysis showed that the ITS (OR253745), tub (OR253764), and LSU (OR253746) sequences generated from the isolate hjh were 98-99% similar to the sequences of E. sorghinum strains CBS 179.80 and CBS 627.68. Subsequently, the ITS, tub, and LSU sequences were combined using Sequence Matrix software; phylogenetic analysis via Bayesian and maximum likelihood methods (Vaidya et al. 2011; Li et al. 2021) classified the isolate hjh into the E. sorghinum clade. To fulfill Koch's postulates, pathogenicity tests were conducted on healthy (lesion-free and disease-free) 2-year-old P. cyrtonema plants. Three healthy plants were inoculated by spraying whole plant until run-off with a spore suspension of the isolate hjh (1 × 106 conidia/ml); Three other healthy plants were sprayed with sterile water as controls. The inoculated plants were incubated in a growth chamber at 25 ± 2°C with 85% humidity for 28 days(Chen et al. 2021). Leaves from the inoculated plants gradually became brown within 15 days. Finally, the plants died 28 days after inoculation. The control plants showed no symptoms throughout the experimental period. Isolates (isolate hjh1, hjh2 and hjh3) that were reisolated from the inoculated plants exhibited morphologically similar characteristics and molecularly identical to the original isolate hjh. To our knowledge, this is the first report of E. sorghinum causing leaf spot disease on P. cyrtonema. The results of this study may facilitate the production of P. cyrtonema in China.

First Report of Black Leaf Spot Caused by Epicoccum latusicollum on Chinese Yam in China.

Chinese yam (Dioscorea polystachya Turczaninow cv. Tiegun), which belongs to the family Dioscoreaceae, is widely cultivated throughout China due to its high economic and medicinal value. In June 2023, black leaf spots on Chinese yam (cv. Purple 1) were observed in Nanchang city (28.45° N, 115.49° E) of Jiangxi province, southeastern China. The incidence of the disease ranged between 70 and 85% of plants, and up to 30% of the leaves per plant were affected in the field over a 2-week period of study. Infected foliage displayed brown necrotic lesions, elliptical or irregular, with yellow halo at the edge of the lesion (0.5 to 3 cm diam.). To identify the causal agent, 32 symptomatic leaves of eight symptomatic plants were collected. Small pieces from the margin of necrotic leaf tissue (about 3 x 3 mm) were surface sterilized in 75% ethanol for 30 s followed in 0.1% HgCl2 for 1 min, and washed three times with ddH2O. Then, the pieces were transferred onto potato dextrose agar (PDA) plates and incubated at 26°C for 3 days with a 12-h light-dark cycle. From the 32 isolates, 21 exhibited similar morphology after hyphal tipping resulting in an isolation frequency of 65.6%. Colonies on PDA were initially white aerial hyphae but became grayish with age, and a reddish orange pigment on the underside. After 16 days of incubation, pycnidia were observed, which were dark, spherical or flat spherical, and 64.1 to 172.5 µm (n = 25) in diameter. Conidia were ellipsoidal, aseptate, hyaline, and 4.1 to 5.6 × 1.8 to 2.7 µm (n = 80). In addition, a blackish green discoloration was produced on malt extract agar (MEA) using the NaOH spot test. The isolates were tentatively identified as Epicoccum spp. based on morphological characteristics (Chen et al. 2017). Isolate AYZ-1 was randomly selected for identification and pathogenicity testing. Genomic DNA of the isolate (AYZ-1) was extracted and amplified by polymerase chain reaction (PCR) using ITS1/ITS4 for the internal transcribed spacer (ITS) region (White et al. 1990), Btub2Fd/Btub4Rd for the ß-tubulin (TUB) region (Woudenberg et al. 2009), LROR/LR7 for the large ribosomal RNA gene (LSU) region (Rehner and Samuels 1994), and RPB2-5F2/fRPB2-7cR for RNA polymerase II second largest subunit (RPB2) region (Liu et al. 1999), respectively. The concatenated sequences (GenBank Accession No. OR574165, OR567827, OR574166, OR567828, respectively) shared 99.8 to 100% identity with Epicoccum latusicollum (OP788080, MN329871, OR428532, and OL422485, respectively). A neighbor-joining phylogenetic tree was generated based on the concatenated sequences in MEGA7, placed isolate (AYZ-1) within E. latusicollum. To fulfill Koch's postulates, healthy leaflets from three one-year-old Chinese yam (cv. Purple 1) were used as inoculation materials, using isolate AYZ-1. Two sites of each leaf were wounded with a sterile needle and covered with a piece of cotton drenched with 200 µL spore suspension (106 conidia/mL) on the left sides, while sterilized water served as the control on the right sides of leaves. All inoculated leaves were covered with clear polyethylene bags for 24 h. Plants were grown outdoors at a daily average temperature of 26°C with relative humidity over 45%. After 7 days of incubation, the leaves showed the same symptoms as the original diseased leaves. The E. latusicollum isolate was re-isolated from diseased leaves and confirmed by morphology and sequencing analysis, fulfilling Koch's postulates. E. latusicollum has been previously reported to cause black root on yam in China's south-western province of Sichuan (Han et al. 2019). Meanwhile, leaf spot have been reported on many plants by this genus, such as tobacco (Guo et al. 2020) and banana (Liu et al. 2023). According to our knowledge, this is the first report of E. latusicollum causing black leaf spot on Chinese yam in China. This finding will provide an important reference for understanding the biology of E. latusicollum and the distribution of the disease, but more research is needed to determine if management is warranted.

Impact of Foliar Fungicide Application on the Culturable Fungal Endophyte Community of Soybean Seed in the Midwest United States.

The purpose of our study was to determine whether the application of quinone outside inhibitor (QoI) and pyrazole-carboxamide fungicides as a tank mix would impact the endophyte community of soybean seed. Field trials during 2018 in Iowa, South Dakota, and Wisconsin, U.S.A., investigated the impact of a single combination fungicide spray at early pod set in soybeans. The composition of culturable endophytic fungi in mature soybean seed was assessed on three cultivars per state, with maturity groups (MGs) ranging from 1.1 to 4.7. An unusually wet 2018 season delayed harvest, which led to a high level of fungal growth in grain. The survey included 1,080 asymptomatic seeds that were disinfested and individually placed on 5-cm-diameter Petri plates of acidified water agar. The survey yielded 721 fungal isolates belonging to 24 putative species in seven genera; taxa were grouped into genera based on a combination of morphological and molecular evidence. The dominant genera encountered in the survey were Alternaria, Diaporthe, and Fusarium. The study showed that the fungicide treatment reduced the incidence of Fusarium in Wisconsin seed, increased the incidence of Diaporthe in seed from all states, and had no impact on the incidence of Alternaria. This is one of the first attempts to characterize the diversity of seed endophytes in soybean and the first to characterize the impacts of fungicide spraying on these endophyte communities across three states. Our study provides evidence that the impact of a fungicide spray on soybean seed endophyte communities may be influenced by site, weather, and cultivar maturity group.

The Interactions between Two Fungal Endophytes Epicoccum layuense R2-21 and Alternaria alternata XHYN2 and Grapevines (Vitis vinifera) with De Novo Established Symbionts under Aseptic Conditions.

In this study, we focused on grapevine-endophyte interactions and reprogrammed secondary metabolism in the host plant due to defense against the colonization of endophytes. Thus, the transcriptional responses of tissue cultured grapevine seedlings (Vitis vinifera L. cv.: Cabernet Sauvignon) to two fungal endophytes Epicoccum layuense R2-21 (Epi R2-21) and Alternaria alternata XHYN2 (Alt XHYN2) at three different time points (6 h, 6 d, 15 d) were analyzed. As expected, a total of 5748 and 5817 differentially expressed genes (DEGs) were separately initiated in Epi R2-21 and Alt XHYN2 symbiotic tissue cultured seedlings compared to no endophyte treatment. The up-regulated DEGs at all time points in Epi R2-21- or Alt XHYN2-treated seedlings were mainly enriched in the flavonoid biosynthesis, phenylpropanoid biosynthesis, phenylalanine metabolism, stilbenoid, diarylheptanoid and gingerol biosynthesis, and circadian rhythm-plant pathways. In addition, the up-regulated DEGs at all sampling times in Alt XHYN2-treated tissue cultured seedlings were enriched in the plant-pathogen interaction pathway, but appeared in Epi R2-21 symbiotic seedlings only after 15 d of treatment. The down-regulated DEGs were not enriched in any KEGG pathways after 6 h inoculation for Epi R2-21 and Alt XHYN2 treatments, but were enriched mainly in photosynthesis-antenna proteins and plant hormone signal transduction pathways at other sampling times. At three different time points, a total of 51 DEGs (all up-regulated, 1.33-10.41-fold) were involved in secondary metabolism, and 22 DEGs (all up-regulated, 1.01-8.40-fold) were involved in defense responses in endophytic fungi symbiotic tissue cultured seedlings. The protein-protein interaction (PPI) network demonstrated that genes encoding CHS (VIT_10s0042g00920, VIT_14s0068g00920, and VIT_16s0100g00910) and the VIT_11s0065g00350 gene encoding CYP73A mediated the defense responses, and might induce more defense-associated metabolites. These results illustrated the activation of stress-associated secondary metabolism in the host grapevine during the establishment of fungi-plant endophytism. This work provides avenues for reshaping the qualities and characteristics of wine grapes utilizing specific endophytes and better understanding plant-microbe interactions.

First report of Epicoccum nigrum causing brown leaf spot of sweet cherry (Prunus avium) in China.

Sweet cherry (Prunus avium L.) has become an important economic fruit in China, mainly produced in Shandong Province. In recent years, the planting area of Aba Prefecture in Sichuan Province has increased. In June 2022, sweet cherry brown leaf spot was found in a cherry plantation (100ha) in Wenchuan County (30°54'50.21″N, 103°24'49.10″E), with an incidence of 50 - 70%. The symptoms appeared as brown circular spots on the leaf, gradually expanding until multiple lesions coalesced to form large irregular brown spots; eventually entire leaves were killed. To isolate the causal pathogens, 10 diseased trees were randomly selected from an orchard, one diseased leaf was taken from each tree, and samples (4×4 mm2) were cut from the border between diseased and healthy tissues of 10 diseased leaves, surface sterilized with 75% ethanol for 30 sec, washed three times with sterilized water, dried on sterilized filter paper and placed on potato dextrose agar (PDA). After 5d at 25℃, five morphologically similar colonies were obtained, colony appears yellow fluffy and released a large amount of red-orangepigment. Microscopy revealed circular to ovoid, verrucose, and multicellular conidia measuring 20×25 µm diameter (n = 30) were produced on the mycelia. The morphological characteristics were consistent with the description of Epicoccum nigrum (Lima et al 2011). To further identify the strains, the internal transcribed spacer (ITS), ß-tubulin, and RNA polymerase second largest subunit (RPB2) gene regions were amplified with ITS1/ITS4 , Bt2a/Bt2b, and 5f2/7cr (White et al. 1990; Glass and Donaldson 1995; Sung et al. 2007), respectively. BLAST analysis revealed that the ITS, ß-tubulin, and RPB2 sequences were 99.2%, 100% and 99.6% homologous, with those of E. nigrum (KU204750.1, OL782123.1, and MW602294.1), respectively. The sequences of the five isolates were identical; and those of representative strain TY3 were deposited in GenBank (ITS, OP410968; ß-tubulin, OR502448; RPB2, OP484927). Maximum likelihood phylogenetic analyses were performed for the combined data set with ITS , ß-tubulin and RPB2 using MEGA6 under the Tamura-Nei model (Tamura et al. 1993). Isolate TY3 clustered with E. nigrum type strain CBS 505.85. The pathogenicity of TY3 was tested on 10 sweet cherry trees aged 3 years (there were about 50 leaves per plant). Five plants were sprayed with 50 mL of spore suspension (1×105 spores/mL), while the controls (Five plants) were sprayed with 50 mL of sterile water. All plants were in closed plastic bags to maintain high humidity, placed in a greenhouse, and incubated at 25℃with a 12-h photoperiod. Twelve days after inoculation, 35% of the inoculated leaves showed lesions; that were consistent with those observed in the field, and the control group was asymptomatic. To confirm Koch´s postulates, two isolates were taken from the margins of leaf lesions and both were confirmed to be E. nigrum based on morphological observations and molecular identification using ITS ß-tubulin, and RPB2 sequences. This is the first report of brown leaf spot caused by E. nigrum on P. avium in China. This discovery needs to be considered in developing and implementing disease management programs in sweet cherry production.

High Diversity of Epicoccum Associated with Leaf Spot on Italian Ryegrass in Southwestern China: Three New Species and Six New Records.

Italian ryegrass is widely cultivated for the productions of forage, hay and silage, due to its high nutritional value and good palatability. Leaf spots caused by fungi pose a serious threat to forage crops. In order to expand knowledge of fungi causing leaf spot in ryegrass (Lolium multiforum) in Sichuan, Yunnan, Chongqing and Guizhou of southwestern China, a comprehensive survey was undertaken from 2015 to 2022. Survey discovered that Epicoccum leaf spot (ELS) was a common and widespread disease, more serious at the late stage of growth (after late May); symptomatic leaf samples collected from the four different provinces were analyzed, and a total of 202 Epicoccum isolates were obtained; based on both multilocus phylogeny (ITS, LSU, TUB2, and RPB2) and morphology, 10 Epicoccum species were finally identified, including three novel species (E. endololii sp. nov., E. lolii sp. nov. and E. loliicola sp. nov.), six new host records (E. draconis, E. endophyticum, E. oryzae, E. plurivorum, E. thailandicum and E. tobaicum), and an unknown species (Epicoccum sp.1). Pathogenicity tests showed that E. endophyticum, E. endololii and Epicoccum sp.1 were non-pathogenic to Italian ryegrass, which were confirmed as endophytes in this study; other six species could infect Italian ryegrass and cause leaf lesions to different degrees, of which E. draconis was more aggressive (P ≤ 0.05). Coupling with the isolation rates and geographical distributions of these species, E. plurivorum was the predominant pathogen in Yunnan while E. oryzae and E. tobaicum in other three provinces. This work provides an initial understanding of the taxonomies, virulence and distributions of Epicoccum species associated with ELS of southwestern China, and lays a solid foundation for the diagnosis in the field, and scientific control of ELS on Italian ryegrass.

Characteristics of Epicoccum latusicollum as revealed by genomic and metabolic phenomic analysis, the causal agent of tobacco Epicoccus leaf spot.

Epicoccum latusicollum is a fungus that causes a severe foliar disease on flue-cured tobacco in southwest China, resulting in significant losses in tobacco yield and quality. To better understand the organism, researchers investigated its optimal growth conditions and metabolic versatility using a combination of traditional methods and the Biolog Phenotype MicroArray technique. The study found that E. latusicollum exhibited impressive metabolic versatility, being able to metabolize a majority of carbon, nitrogen, sulfur, and phosphorus sources tested, as well as adapt to different environmental conditions, including broad pH ranges and various osmolytes. The optimal medium for mycelial growth was alkyl ester agar medium, while oatmeal agar medium was optimal for sporulation, and the optimum temperature for mycelial growth was 25°C. The lethal temperature was 40°C. The study also identified arbutin and amygdalin as optimal carbon sources and Ala-Asp and Ala-Glu as optimal nitrogen sources for E. latusicollum. Furthermore, the genome of E. latusicollum strain T41 was sequenced using Illumina HiSeq and Pacific Biosciences technologies, with 10,821 genes predicted using Nonredundant, Gene Ontology, Clusters of Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes, and SWISS-PROT databases. Analysis of the metabolic functions of phyllosphere microorganisms on diseased tobacco leaves affected by E. latusicollum using the Biolog Eco microplate revealed an inability to efficiently metabolize a total of 29 carbon sources, with only tween 40 showing some metabolizing ability. The study provides new insights into the structure and function of phyllosphere microbiota and highlights important challenges for future research, as well as a theoretical basis for the integrated control and breeding for disease resistance of tobacco Epicoccus leaf spot. This information can be useful in developing new strategies for disease control and management, as well as enhancing crop productivity and quality.

Biological production of epicoccamide-aglycone and its cytotoxicity.

Epicoccamide (EPC) is an O-d-mannosylated acyltetramic acid of Epicoccum origin and is a bolaamphiphilic fungal polyketide. EPC displays weak toxicity against Staphylococcus aureus and HeLa cell lines. The EPC biosynthetic gene cluster was previously identified in Epicoccum nigrum and knockout of the glycosyltransferase gene (epcB) abolished EPC production. EPC-aglycone was expected in the epcB knockout but was not found. This study demonstrates that extractive culture using the hydrophobic resin Diaion HP-20 resulted in the production of EPC-aglycone, which was isolated using chromatographic separation techniques, and its structural identity was substantiated by chemical analyses. EPC-aglycone displayed strong antibacterial activity against Staphylococcus aureus, with the minimal inhibitory concentration of 1 µg/mL (64 µg/mL for EPC). EPC-aglycone displayed higher levels of growth inhibition against HeLa cell line (the half inhibitory concentration, 19 µM) and WI-38 (15 µM) cell line than EPC (76 µM and 38 µM vs. HeLa and WI-38, respectively). The dose-response curve fit of growth inhibition indicated that EPC-aglycone adopted a shallow curve (low slope factor), which was different from that of EPC, suggesting that their cellular targets are distinct from each other. This study substantiates that the d-mannose attachment is the final step in EPC biosynthesis, showcasing a glycosylation-mediated modulation of the biological activity of simple acyltetramic acid. This study also highlights the usefulness of extractive cultures in mining cryptic microbial natural products.

Epicoccum sp. as the causative agent of a reddish-brown spot defect on the surface of a hard cheese made of raw ewe milk.

Colour defects can affect the appearance of cheese, its flavour, the safety of its consumption, and the price it can demand. This work reports the identification of five fungal isolates from a dairy plant where the surface of most cheeses was affected by patent, reddish-to-brown stains. One of these isolates was obtained from cheese, two from brine, and two from a bulk tank containing ewe milk. Molecular identification by partial amplification, sequencing, and database comparison of the concatenated sequence of the genes coding for the largest subunit of RNA polymerase II (RPB2), ß-tubulin (ß-TUB), and the large subunit of the rRNA molecule (LSU), plus the internal transcribed sequence (ITS) regions, assigned the isolates to Epicoccum layuense, Epicoccum italicum, and Epicoccum mezzettii. Features of the growth of these different species on different agar-based media, and of the morphology of their conidia following sporulation, are also reported. The strain isolated from cheese, E. layuense IPLA 35011, was able to recreate the reddish-brown stains on slices of Gouda-like cheese, which linked the fungus with the colour defect. In addition, two other strains, E. italicum IPLA 35013 from brine and E. italicum IPLA 35014 from milk, also produced stains on cheese slices. Epicoccum species are widely recognized as plant pathogens but have seldom been reported in the dairy setting, and never as human or animal pathogens.

Cadavers found outdoor in whom fungal growth was observed on the body surface: Consideration of the role of mycology in forensic medicine.

In forensic case work, fungal growth is occasionally observed on decomposed cadavers, however, the isolation of these organisms is not always carried out routinely. The usefulness of investigating fungi isolated from corpses in forensic settings, has been reported, such as providing trace evidence, estimating the time since death, and ascertaining the time of deposition. A 32-year-old male missing for 4 months, was found in a car in a forest far from his home and had died from blood loss, having been stabbed with a kitchen knife in the left side of his chest. Whitish or white greenish colonies ranging 5 cm to 8 cm in size were observed over his whole body. Penicillium commune and Cladosporium cladosporioides were isolated from the colonies. A 49-year-old male missing for one month, and was found dead at 500 m elevation in the mountains. An autopsy was conducted, but the cause of death could not be determined. Dark greenish or whitish colonies were found on his face and neck, and Epicoccum nigrum, Mucor sp.. Cladosporium sp., and Pestalotiopsis sp. were isolated. Penicillium sp. and Cladosporium sp. are major indoor fungi, and Epicoccum sp. and Pestalotiopsis sp. are plant pathogens found in outdoor environments. There was no discrepancy between the police statement and findings of the corpse such as place discovered, types of isolated fungi, and estimated time since death. Identifying fungi on cadavers may aid in forensic casework, and further studies are needed to develop for the use of fungi as a forensic tool.

The Sensitivity of Fungi Colonising Buckwheat Grains to Cold Plasma Is Species Specific.

Fungi are the leading cause of plant diseases worldwide and are responsible for enormous agricultural and industrial losses on a global scale. Cold plasma (CP) is a potential tool for eliminating or inactivating fungal contaminants from biological material such as seeds and grains. This study used a low-pressure radiofrequency CP system with oxygen as the feed gas to test the decontamination efficacy of different genera and species commonly colonising buckwheat grains. Two widely accepted methods for evaluating fungal decontamination after CP treatment of seeds were compared: direct cultivation technique or contamination rate method (%) and indirect cultivation or colony-forming units (CFU) method. For most of the tested fungal taxa, an efficient decrease in contamination levels with increasing CP treatment time was observed. Fusarium graminearum was the most susceptible to CP treatment, while Fusarium fujikuroi seems to be the most resistant. The observed doses of oxygen atoms needed for 1-log reduction range from 1024-1025 m-2. Although there was some minor discrepancy between the results obtained from both tested methods (especially in the case of Fusarium spp.), the trends were similar. The results indicate that the main factors affecting decontamination efficiency are spore shape, size, and colouration.

Ganodermasides E-H, four new ergosterol derivatives from the endophytic fungus Epicoccum poae DJ-F associated with Euphorbia royleana.

Ganodermasides E-H (1-4), four new ergosterol derivatives and two known ones (5 and 6) were isolated from the fermentation of the endophytic fungus Epicoccum poae DJ-F in the stems of Euphorbia royleana Boiss. Their structures were elucidated by spectroscopic analysis, including extensive 1D NMR, 2D NMR, and HRESIMS techniques. All the isolated compounds were tested for their vitro antibacterial activity. Compounds 1-6 showed weak inhibitory effects on Staphylococcus epidermidis, Pseudomonas syringae, and Ralstonia solanacearum with MIC values ranging from 0.4 to 3.6 mM.

First Report of Leaf Spot on Soybean Caused by Epicoccum sorghinum in Heilongjiang Province, China.

Soybean (Glycine max (Linn.) Merr.) is one of the important oil crops in China. In September 2022, a new soybean leaf spot disease was found in Zhaoyuan County, Suihua City, Heilongjiang Province, China. Symptoms of the initial formation of irregular brown lesions on the leaves, dark brown inside, the periphery is yellow, vein chlorotic yellow, severe leaf spots connected into pieces, late fall off, not the same as previously reported soybean leaf spot (Fig. 1A). The leaf samples of infected plants were collected, and the leaf tissue (5 × 5 mm) was cut from the edge of the lesion, and then surface sterilized with 3% sodium hypochlorite for 5 min, rinsed with sterile distilled water for 3 times, and inoculated on potato dextrose agar (PDA) at 28°C. Isolates growing around the tissues from samples were subcultured on PDA, and 3 isolates were obtained using the single-spore isolation method. The fungal hyphae were white or grayish white in early stage, and the hyphae with light green concentric ring appeared on the front of the colony after 3 days, appeared orange, pink or white convex, irregular shape, reddish brown on the front of the colony for 10 days and black spherical pycnidium can be produced in the hyphae layer for 15 days (Fig.1D, E). Conidia were oval, hyaline, unicellular, aseptate, and 2.3 to 3.7 × 4.1 to 6.8 µm (n=30, Fig. 1F). Chlamydospores were subglobose, light brown, unicellular or multicellular, and 7.2 to 14.7 × 12.2 to 43.9 µm (n=30, Fig. 1H, I). Pycnidia mostly spheroid, brown, and 47.1 to 114.4 × 72.6 to 167.4 µm (n=30, Fig. 1G). A cetyl trimethyl ammonium bromide method was used to extract DNA from 7-day-old. Internal transcribed spacer (ITS), RNA polymerase II (RPB2) and ß-tubulin (TUB) gene were amplified using ITS1/ITS4 (White et al. 1990), RPB2-5F/RPB2-7cR (Liu et al. 1999) and BT2a/Bt2b (O'Donnell et al. 1997) primers respectively. The sequences obtained by polymerase chain reaction (PCR) were sequenced and the results showed that the DNA sequences of the 3 isolates were identical. Therefore, the sequence of isolate DNES22-01, DNES22-02 and DNES22-03 was submitted to GenBank. According to BLAST search, the ITS (OP884646), RPB2 (OP910000) and TUB (OP909999) sequences showed 99.81% similarity to Epicoccum sorghinum strain LC12103 (MN215621.1), 99.07% to strain P-XW-9A (MW446946.1), and 98.85% with the strain UMS (OM048108.1), respectively. Phylogenetic analysis by maximum likelihood method (MEGA7.0) generated based on the ITS, RPB2 and TUB sequences indicated that the isolates formed a supported clade to the related E. sorghinum type sequences. Isolates was found to be most closely related to E. sorghinum and far from other species. Based on morphological and phylogenetic characteristics, isolates DNES22-01, DNES22-02 and DNES22-03 was identified as E. sorghinum (Bao et al. 2019; Chen et al. 2021; Zhang et al. 2022). At the 4-leaf-stage, 10 soybean plants were inoculated by spraying with a conidial suspension (1 × 106 spores·ml-1). Sterile water served as a control. The test was repeated 3 times. All samples were incubated in a growth chamber at 27°C. Symptoms typical developed on the leaves after 7 days, but control samples remained healthy (Fig.1B, C). The fungus was reisolated from symptomatic tissues and identified as E. sorghinum by morphology characteristics and molecular characterization. To our knowledge, this is the first report of E. sorghinum causing leaf spot on soybean in Heilongjiang, China. The results can provide the basis for future studies on the occurrence, prevention, and management of this disease.

First report of Epicoccum latusicollum causing leaf spot on Lophatherum gracile in Jiangsu Province of China.

Lophatherum gracile Brongn. is an important Chinese herbal medicine. Since 2016, a leaf spot disease has appeared on L. gracile seedlings in the traditional Chinese medicine resource garden of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province (32.06°N, 118.83°E). About approximately 80% of the seedlings suffered from the disease. The disease spot usually starts from the leaf margin, round or irregular, with yellow halo at the edge of the lesion. To isolate the pathogen, four diseased leaves were collected from four different seedlings and there are 6 sections from each diseased leaf. The leaf sections were surface sterilized in 75% alcohol for 30 s and 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA). Pure cultures were obtained by monosporic isolation. Eleven isolates were obtained (isolate rate of 55%) and identified as Epicoccum sp.. Thus, a representative isolate, DZY3-3 was used for the further study. After 7 days of culture, the colony produced white aerial hyphae, and reddish orange pigment on the underside. The chlamydospores were produced, either multicellular or unicellular. The colony produced pycnidia and conidia after nearly three weeks of cultivation on oatmeal ager OA. Conidia were unicellular, hyaline, oval, and were 4.9 to 6.4 x 2.0 to 3.3 µm (n=35). In addition, a brown discoloration was produced on malt extract agar (MEA) after using the 1 mol/L NaOH solution for 1 h. These characteristics were consistent with the description of Epicoccum sp. (Chen et al. 2017). To comfirm this identification, the internal transcribed spacer (ITS), large subunit ribosomal RNA (LSU), beta-tubulin (TUB) and RNA polymerase II second largest subunit (RPB2) regions were amplified using the detailed primer pairs described by White et al., Rehner and Samuels, Woudenberg et al. and Liu et al., respectively. They had 99.8-100% homology to the ITS (GenBank no. MN215613, 504/505 bp), LSU (MN533800, 809/809 bp), TUB (MN329871, 333/333 bp), and RPB2 (MG787263, 596/596 bp) sequences of E. latusicollum in the GenBank database. A neighbor-joining phylogenetic tree was generated based on the concatenated sequences of all the above regions in MEGA7. The DZY3-3 clustered in the E. latusicollum clade with 100% bootstrap support. Koch's postulates were performed by spray inoculation (1×106 spores/mL) on the left sides of leaves of three healthy L. gracile seedlings and detached leaves, using isolate DZY3-3, while sterilized water served as the control was sprayed on the right sides of leaves. All plants and detached leaves were covered with clear polyethylene bags to maintain about 80% relative humidity at 25℃. Whether in vivo or in vitro pathogenicity test showed similar symptoms to those occurred in the field after 5 days post inoculation. No symptoms occurred on controls. The experiment was repeated three times. Subsequently, the same fungus was reisolated and identified from leaves of three inoculated seedlings. The E. latusicollum has a very wide host range. For example, it has been reported to cause stalk rot on Maize (Xu et al. 2022) and cause leaf spot on Tobacco in China (Guo et al. 2020). To our knowledge, it is the first report of E. latusicollum causing leaf spot on L. gracile in the world. This study will provide an important reference for the biology of E. latusicollum and the distribution of the disease.

First Report of Leaf Tip Blight Caused by Epicoccum sorghinum on Phoebe bournei in Fujian, China.

Phoebe bournei, belonging to the family Lauraceae, is indigenous to China, where it is a protected species. In March 2022, ca. 90% of 20,000 P. bournei saplings suffered from leaf tip blight in a sapling nursery with an area of 200 m2 in Fuzhou, China. Initially, brown discoloration appeared on the tips of young leaves. The symptomatic tissue continued to expand as the leaf grew. To isolate the pathogen, 10 symptomatic leaves were randomly sampled from the nursery, and surface-sterilized in 75% alcohol for 30 s, followed by a 5% NaClO solution for 3 min, and then rinsed 3 times with sterile water. Twenty small pieces (0.3 x 0.3 cm) were excised from the margin of diseased and healthy tissue and transferred to five PDA plates amended with 50 µg/ml ampicillin. The plates were incubated at 25°C for 5 days. Finally, 17 isolates were obtained, and nine isolates with the highest isolation frequency shared the same morphological characteristics. On PDA, these colonies had aerial hyphae, white in the beginning, and became pale brown with the pigment production. Chlamydospores were observed after incubation for 7 days at 25°C, pale brown and nearly spherical, unicellular, or multicellular. Conidia were unicellular or bicellular, hyaline, and ellipsoidal, 5.15 to 9.89× 3.46 to 5.87 µm, n=50. The 9 fungi were identified as Epicoccum sp (Khoo et al. 2022a, b, c). Furthermore, strain MB3-1 was chosen randomly as the representative of the 9 isolates, and ITS, LSU, TUB sequences were amplified using the primers ITS1/ITS4, LR0R/LR5, Bt2a/Bt2b respectively (Raza et al. 2019). The sequences were submitted to NCBI and analyzed using BLAST. Results of BLAST showed that ITS (OP550308), LSU (OP550304), TUB (OP779213) sequences had 99.59% (490bp out of 492bp), 99.89% (870bp out of 871bp), 100% (321bp out of 321bp) identity to Epicoccum sorghinum sequences MH071389, MW800361, MW165323, respectively. ITS, LSU, TUB sequences were concatenated for phylogenetic analysis using the maximum likelihood method with 1000 bootstrap replicates in MEGA 7.0 software. The phylogenetic tree showed that MB3-1 was clustered together with E. sorghinum. Pathogenicity tests were performed on young leaves of healthy P. bournei saplings in vivo by inoculating with fungal conidia suspension. The conidia were eluted from the colony of MB3-1 and adjusted to 1×106 spores/ml. An amount of 20 µl conidia suspension (0.1% tween-80) was evenly sprayed on three leaves of one P. bournei sapling, 20 µl sterile water was sprayed on three other leaves of the same sapling as control, and three saplings were treated. All the treated saplings were kept at 25°C. MB3-1 caused leaf tip blight symptoms similar to those observed in nature at 6 days post inoculation (dpi). The pathogen was reisolated from inoculated leaves and identified as E. sorghinum. The experiment was repeated twice with the same results. Recently, E. sorghinum has been reported in Brazil (Gasparetto et al. 2017), Malaysia (Khoo et al. 2022a, b, c), and the United States (Imran et al. 2022). To our knowledge, this is the first report of E. sorghinum causing leaf tip blight on P. bournei. Wood from P. bournei is used to produce high-quality furniture due to its vertical grain and durability (Chen et al. 2020). And the demand for wood requires numerous saplings for afforestation. But this disease has the risk of causing insufficient saplings, which would affect the development of the P. bournei timber industry.