First Report of Epicoccum Tobaicum on Urdbean ( Vigna Mungo ) in Iowa

The expanding U.S. crop production of urdbean (Vigna mungo L. Hepper.), a protein-rich East Asian legume (Gowda et al. 2013), presents a unique opportunity to document Vigna pathogens. In August 2022, small chlorotic foliar lesions with brown to black centers and yellow halos were observed on urdbean accession lines PI518264 and PI377394 in a field plot of 0.006 hectares in Boone County, IA. Disease incidence ranged from 30% to 75% of affected plots. Symptomatic leaves with >10% lesion severity were collected for analysis. Leaf surfaces were disinfected with a 5% NaOCl wash, rinsed with autoclaved distilled water, and sectioned into 1 x 1 mm fragments containing infected tissue. Fragments were placed on Potato Dextrose Agar (PDA) and incubated at room temperature under ambient light. After 7-14 days, orange to red velvety colonies emerged. Mycelia were transferred to Water Agar for 2 days, then hyphal-tipped and transferred to PDA. Plates were incubated at room temperature for 7-14 days, and five representative isolates were selected for morphological and molecular identification. The isolates exhibited velvety mycelium ranging from orange to brown, with an orange pigment diffusing into the PDA. After 14-21 days, black sporodochia formed, producing globular conidia measuring 15-20 µm. Based on morphology, the isolates E_A2, and E_EHB were identified as Epicoccum sp. DNA was extracted using 50 µl of Prepman Ultra Sample Reagent (Applied Biosystems, Foster City, California, USA). The rDNA internal transcribed spacer (ITS), large subunit (LSU), RNA polymerase II second largest subunit (RPB2), actin (ACT), β-tubulin 2 (TUB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) regions were amplified by PCR using the primer pairs ITS-1F/ITS-4, ITS5/TW13 FRPB2-5F/FRPB2-7R, ACT-512F/ACT-783R, Bt2A/Bt2B, and GDF/GDR, respectively. Phylogenetic trees of concatenated sequences, the best fit-partition scheme, and substitution models were selected by PartionFinder2 (Lanfear et al. 2016) and Bayesian Inference done by MrBayes 3.2 (Ronquist et al. 2012) both integrated in Phylosuite v2 (Zhao et al. 2025) with all its dependencies. Sequences were deposited in GenBank (accession nos. PZ357005:PZ357006, PZ437652:PZ437653, PZ452804:PZ452805, PZ787846:PZ787847, PZ452811:PZ452812, and PZ787844:PZ787845). To confirm the pathogenicity of the Epicoccum strains, 10 urdbean plants from accessions PI518264 and PI377394 were grown in 10 x 10 cm pots with professional greenhouse potting mix (Sungrow, Agawam, MA) under standard greenhouse conditions. At 25 days (V2 growth stage), plants were inoculated with a spore suspension (10 6 conidia/mL) sprayed twice, two days apart. Control plants received autoclaved distilled water. Inoculated plants were covered with plastic domes for 96 h, misted every 4-8 h, and kept in darkness for 24 h post-inoculation. After dome removal, plants were placed into a growth chamber (25ºC, 16 h light cycle, 85% RH) for 7-30 days. One week post-inoculation, brown to red spots appeared on inoculated leaves, while controls remained symptom-free. Epicoccum tobaicum was reisolated from symptomatic tissue with a 78% frequency, confirming its role as the pathogen. Studies have reported E. tobaicum (syn. E. layuense) as a brown leaf spot-causing on hosts such as Camellia sinensis (Chen et al. 2020), and Avena sativa (Jeong et al. 2023). Epicoccum tobaicum belongs to E. nigrum complex species and was formally described as a new species within the genus Epicoccum in 2017 (Chen et al. 2017). This is the first report of E. tobaicum causing disease on urdbean in the U.S., posing a potential threat to crop productivity.