First Report of Angular Leaf Spot Caused by <i>Pseudocercospora kakiicola</i> on <i>Diospyros kaki</i> in China

Persimmons (Diospyros kaki Thunb.‌) are widely cultivated in China due to their economic and medicinal values. In October 2023, angular leaf spot of persimmon was observed in Luoning County (34.47°N, 111.57°E), Henan Province, China, with an incidence rate of approximately 50% observed in a 1.5-ha persimmon orchard. Angular and brown lesions were observed on upper leaves of persimmon, and black on the lower surface. Six symptomatic leaves were collected from three trees. Small tissue pieces (2 × 3 mm) excised from lesion margins were surface sterilized in 2% sodium hypochlorite for 2 min, rinsed three times with sterile distilled water, and plated onto potato dextrose agar (PDA). Plates were incubated at 25°C in the dark. After 7 days, five isolates with similar morphology were obtained and were further transferred to fresh PDA plates by hyphal tip isolation. After incubating at 25°C in the dark for 28 days, colonies reached 30-35 mm in diameter, were radially wrinkled, lanate, pale brown to yellow green, with undulate to irregular lobate margins; The reverse side was dark gray to dark green. Hyphae were branched, smooth, septate, colorless to pale brown. Conidia were solitary, pale olivaceous, obclavate or acicular, straight to curved, with 4 to 10 septa. Conidial size ranged from 21.17 to 71.41 μm long and 2.40 to 4.32 μm wide (n = 35). Morphological characteristics were consistent with those described for Pseudocercospora kakiicola (Braun et al. 2020). DNA was extracted from the representative isolate SZJB23-48 using the CTAB method. The internal transcribed spacer region (ITS), translation elongation factor 1-α (TEF), actin (ACT), and RNA polymerase II second largest subunit (RPB2) loci were amplified with the primer pairs ITS1/ITS4, EF1-728 F/EF1-986R, ACT-512 F/ACT-783R, and fRPB2-5F/fRPB2-7cR, respectively (Nakashima et al. 2016). The sequences were deposited in GenBank under the accession numbers: PX056953 (ITS), PX403056 (TEF), PX403065 (ACT), and PX961854 (RPB2). BLASTn against the GenBank showed 99.58% ITS, 100% TEF, 99.40% ACT, and 100% RPB2 identity with P. kakiicola MUCC 900 (ITS: GU269729; TEF: GU384442; ACT: GU320431; RPB2: LC738891). Multilocus phylogenetic analysis based on the concatenated sequences indicated that the representative isolate was clustered with P. kakiicola MUCC 900 into one branch with a bootstrap value of 100. Therefore, these isolates were identified as P. kakiicola based on morphology and phylogeny. Pathogenicity tests of the isolate SZJB23-48 were carried out on three five-year-old persimmon plants (cv. Niuxinshi). Six healthy leaves of each plant were inoculated in a greenhouse by spraying a conidial suspension (1 × 10 5 spores/ml). Sterilized water was used as a control. Inoculated leaves were wrapped in plastic bags to maintain high humidity for two days. The tests were replicated three times. Four weeks after inoculation, all inoculated leaves developed symptoms similar to the primary diseased leaves, whereas control leaves remained asymptomatic. The same pathogen was reisolated from the inoculated leaves and confirmed through morphological and molecular analysis, thereby fulfilling Koch’s postulates. This fungus has ever been reported as a pathogen of D. kaki and D. lotus in Japan (Braun et al. 2020). To our knowledge, this is the first report of angular leaf spot caused by P. kakiicola on D. kaki in China, which lays the foundation for developing effective management strategies against this disease.