First Report of Brown Spot of Dekopon Fruit Caused by Cladosporium tenuissimum in China

Dekopon citrus (Citrus reticulata Shiranui) is a three-way hybrid (Citrus unshiu Marcov. × C. sinensis Osbeck × C. reticulata Blanco) developed in Japan in 1972. This citrus is popular in China due to its sweet and tender taste (Lim 2012). In November of 2021, a brown spot disease on Dekopon fruits with about 20% disease incidence was observed in an orchard of the Institute of Citrus Research in Ganzhou, Jiangxi Province, China. Initially, the symptoms appeared as slightly sunken deep red to purple spots on the fruit surface, with the disease progression, lesions became brown to brown-black large necrotic regions covered with a fluffy layer of gray spores. Infected fruits were surface sterilized with 70% ethanol for 30 sec and rinsed three times with sterile distilled water. Diseased tissues from the edge of lesions were cut into small segments, placed onto potato dextrose agar and incubated at 25℃ for 7 days. Ten single-spore isolates were obtained in total. Fungal colonies were olive green to dark green, velvet-like in texture and sporulated abundantly, surrounded by grayish-white hyphae. Conidiophores were subcylindrical, straight, septate, solitary or in clusters of two to three, and ranged in size from 65 to 550 × 3.8 to 6.3 µm (x ̅= 261.7 ± 60.5 × 5.2 ± 0.4 µm, n=50). Ramoconidia were cylindrical，aseptate, and 10 to 22 × 2.8 to 4.5 µm (x ̅= 15.5 ± 1.4 × 4.0 ± 0.9 µm, n=50). Conidia were lemon-shaped to oval-shaped, smooth-walled, and 1.8 to 5.0 × 1.4 to 2.5 µm (x ̅= 3.9 ± 0.4 × 2.2 ± 0.2 µm, n=50). The morphological characteristics of the pathogen were consistent with those of Cladosporium tenuissimum Cooke (Li et al. 2021). For further identification, DNA was extracted from two representative isolates. The internal transcribed spacer (ITS) region, translation elongation factor (EF1-α), and actin (ACT) were amplified by using primers ITS1/ITS4, EF1-728F/EF1-986R, and ACT-512F/ACT-783R (Bensch et al. 2012), respectively. ITS (OM232067, OM232068), EF1-α (OM256525, OM256526) and ACT (OM256529, OM256530) sequences were deposited in GenBank. Multi-gene (combined data set of ITS, EF1-α and ACT) phylogenetic analysis was conducted using the Maximum Likelihood method (Nguyen et al. 2015). Based on the morphological characteristics and the molecular data, two fungal isolates were identified as C. tenuissimum. To evaluate pathogenicity, fifteen fruits were surface sterilized with 1% NaClO solution for 30 sec, rinsed twice with sterile distilled water and dried