Soybean (Glycine max) is the predominant legume cultivated in South Korea. This legume is susceptible to pod and stem blight disease mainly caused by D. longicolla, D. sojae, D. phaseolorum, and D. caulivora (Rahel et al. , 2025; Sun et al. , 2012). In September 2024, 64 soybean samples showing reddish-brown stem lesions, black pycnidia on pods, and poor seed quality were collected from different regions in South Korea. Stem fragments (5 × 5 mm), pods, and seeds from infected tissues were surface-sterilized with 1% sodium hypochlorite for 1 minute, followed by three rinses with sterile distilled water. After air-drying, tissues were cultured on water agar and incubated at 25°C for 7 days. Single-spore isolates were transferred to oatmeal agar medium and incubated at 25°C for 21 days to enhance sporulation. Different morphological appearances were observed among isolates. Isolate NS₇2354 collected from Nonsan exhibited white, cottony aerial mycelia that turned gray and produced black pycnidia within two weeks. Oval, aseptate, hyaline α-conidia measuring 3. 31 to 4. 43 × 1. 57 to 2. 11 µm were observed, while β-conidia were absent. For molecular analysis, genomic DNA from NS₇2354 was extracted, and PCR was performed using primers targeting the internal transcribed spacer (ITS1/ITS2, ITS1/ITS4), translation elongation factor 1-α (TEF-F/TEF-R), beta-tubulin (Bt2a/Bt2b), and calmodulin (Cal-F/Cal-R) regions (Udayanga et al. , 2014; Zhang et al. , 1997). The sequence analysis identified the isolate as Diaporthe pseudooculi, with BLASTn results showing 99. 1–100% sequence similarity to the reference sequences. All sequences were submitted to GenBank under accession numbers PX226075, PX854921, PX872756, PX872757, and PX842374. A phylogenetic analysis using the maximum likelihood method was performed on partial sequences from ITS1/ITS2 (281 bp), ITS1/ITS4 (293 bp), β-tubulin (400 bp), TEF1-α (347 bp), and Cal (378 bp). NS₇2354 was clustered within the same clade as the reference D. pseudooculi. Pathogenicity test involved stem inoculation of soybean seedlings (Li, 2018; Rahel et al. , 2025) and pod inoculation at the R5 growth stage (Nascimento et al. , 2025) across 24 cultivars. For stem inoculation, seedlings had their apex cut 25 mm above the first trifoliolate node, and a 0. 5 mm diameter mycelial plug was placed on the surface. Control plants received sterile agar plugs, and inoculated sites were sealed for 2 days. Symptoms appeared 4 days post-inoculation, with monitoring continuing until 14 days. The control group exhibited no symptoms. For pod inoculation, pods were surface sterilized, rinsed, air-dried and placed on Petri dishes. Each pod was inoculated with 1 x 10⁶ conidia/ml suspension, while control pods were treated with sterile water. The plates were incubated at 25°C with ≥ 95% humidity for 21 days, with disease assessed at 4, 7, 9, 14, and 21 DAI using a 0-5 scale. By day 21, inoculated pods were fully colonized, whereas controls remained asymptomatic. The pathogen was re-isolated from diseased tissues and confirmed by morphological and molecular methods to fulfill Koch’s postulate. D. pseudooculi has been reported as a human pathogen and recently linked to hazelnut defects (Arciuolo et al. , 2020). To our knowledge, this is the first report of pod and stem blight of soybean caused by D. pseudooculi, which expands the known host range and geographic distribution of the pathogen and highlights its potential significance as an emerging pathogen of soybean.
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