First Report of Soybean Seed Disease Caused by <i>Epicoccum italicum</i> in North Dakota

In October 2024, moldy and healthy seeds were collected during harvest from a soybean (Glycine max (L.) Merr.) field in Barnes County, North Dakota. The sampled area (~1.6 ha) was sheltered by a tree grove. Moldy seeds were brown, with some appearing shriveled and covered in whitish mycelium. A total of 400 seeds were sampled using the hand-halving method described for Glycine spp. (ISTA International Rules for Seed Testing) and plated on potato dextrose agar (PDA). Seeds were surface sterilized by soaking in 0.5% NaOCl solution (40 s), followed by 70% ethanol (1 min), then rinsed twice in autoclaved water (30 s each). Plates were incubated at 23±2°C for 7 days. Six morphologically distinct fungal colonies emerged from the diseased seeds with an isolation frequency of 1.5%, alongside Diaporthe spp., which had a 10.5% recovery rate. Hyphal tips from six colonies were subcultured in PDA plates. Colonies were white with cottony, aerial hyphae and exhibited irregular margins. In five days, the colony surface turned reddish-orange, while the reverse side showed characteristic orange pigmentation. By 10 days, blackish-brown, globular, multicellular conidia [11.9–23.6 × 12.2–25.4 µm (n=100)] were observed. One representative isolate (24-EPI-ND) was randomly selected, and DNA was extracted from the mycelium. The internal transcribed spacer (ITS), partial large subunit (LSU), RNA polymerase II second largest subunit (RPB2), and β-tubulin (TUB) genes were amplified (Rodríguez et al. 2023) and deposited in NCBI GenBank (ITS: PV763250; LSU: PV789017; RPB2:PV826789; TUB: PV826790). The ITS sequence shared 99.8% identity, while LSU, RPB2, and TUB sequences showed 100% identity with type material of E. italicum LC:8150 (NR 158264, KY742253, KY742172, and KY742341). Multi-gene phylogenetic analysis confirmed the identity as Epicoccum italicum Chen et al. Pathogenicity was evaluated on soybean cultivar ‘Hawkeye’, modifying the seed inoculation method of Duvnjak et al. (2007) and repeated once. One D. longicolla isolate (16-OP-SB-DIA-052) was included to compare disease symptoms. Mature seeds (n=100) were soaked for 2 h in 100 mL of conidial suspension (106 conidia/ml) of D. longicolla and E.italicum. Control seeds were soaked in autoclaved water. Seeds were placed on moist filter paper in four 90 mm Petri dishes (25 seeds/dish) and incubated at 23±2°C under a 12 h light/dark cycle. In four days, seeds inoculated with E. italicum showed brownish-black discoloration and white mycelial growth on the seed surface without internal discoloration. These symptoms resembled those observed in the field. In contrast, seeds infected with D. longicolla exhibited internal and external brown discoloration and appeared chalky. No symptoms were observed on control seeds. Average germination rates for seeds inoculated with E. italicum and D. longicolla were 70% and 44% respectively, across both experiments, while control seeds had 100% germination. Fungi were reisolated and confirmed as E. italicum by morphology and ITS sequence analyses, fulfilling Koch’s postulates. Species of Epicoccum have been reported as seed-borne field fungi in soybeans (Kulik and Sinclair 2015) and E. italicum as an endophyte in soybean pods (Batzer et al. 2023). This study presents the first report of E. italicum associated with soybean seed disease in North Dakota, suggesting the potential of E. italicum as a pathogen and highlighting the need to determine its prevalence in soybean fields.