First Report of <i>Epicoccum nigrum</i> Causing Leaf Spot on <i>Sorghum bicolor</i> in Heilongjiang Province, China

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.