First Report of Phyllosticta capitalensis Causing Leaf Spot on Chinese Bayberry ( Myrica rubra ) in China

Chinese bayberry (Myrica rubra Siebold & Zucc.) is an economically important fruit crop in southern China. In May 2025, a leaf spot disease was observed on M. rubra trees (Anhaibian Ruansi, native variety) in a commercial orchard (24°16′36″ N, 117°58′23″ E) in Longhai District, Zhangzhou City, Fujian Province, China, with approximately 30% incidence. Symptoms began at the leaf margins and spread across the entire leaf, resulting in reddish-brown, blighted foliage. Microscopic examination of tissue sections from field-infected leaves revealed the presence of pycnidia and perithecia on the lesions. Within these structures, pycnidia contained subglobose to ovoid conidia, and perithecia contained clavate asci with fusoid ascospores. Symptomatic leaf tissues were cut into small pieces (3 to 4 mm²), surface-sterilized with 70% ethanol for 30 s followed by 5% NaClO for 1 min, rinsed three times with sterile distilled water, and then plated on potato dextrose agar (PDA). Nine morphologically similar fungal isolates (MPC001-MPC009), which developed consistent grayish-black colonies on PDA, were obtained. Detailed observation of a representative isolate (MPC001) revealed the production of hyaline, aseptate, subglobose to ovoid conidia sized (11.5 ± 1.0) × (7.8 ± 0.5) µm (n = 60), matching the morphological description of Phyllosticta spp. (Wikee et al. 2013). For molecular identification, the internal transcribed spacer (ITS) region, actin (ACT) gene, and translation elongation factor 1-alpha (TEF1-α) gene were amplified and sequenced from all nine isolates using primers ITS1/ITS4 (White et al. 1990), ACT-512F/783R (Carbone and Kohn 1999), and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. These loci were selected because they are commonly employed for species delimitation in the genus Phyllosticta, and have been demonstrated to provide sufficient phylogenetic resolution to distinguish closely related taxa within the Phyllosticta capitalensis Henn. species complex (Guarnaccia et al. 2017; Wikee et al. 2013). The obtained 27 sequences have been deposited in GenBank under the following accession numbers: ITS (PV992391-PV992395, PX069328-PX069331); ACT (PX024942-PX024946, PX099139-PX099142); TEF1-α (PX025638-PX025642, PX092827-PX092830). BLASTn analysis revealed >99% nucleotide identity between our sequences and the ex-epitype strain of Phyllosticta capitalensis (CBS 128856) across all three loci. Phylogenetic analysis based on concatenated ITS-ACT-TEF1-α sequences, which included the ex-epitype strain of P. capitalensis (CBS 128856) and the closely related species P. paracapitalensis (Guarnaccia et al. 2017), placed all nine isolates within a well-supported clade (93% bootstrap) with P. capitalensis, clearly separating them from P. paracapitalensis. These results are consistent with recent studies emphasizing the importance of multilocus phylogeny for resolving species boundaries within the P. capitalensis species complex (Norphanphoun et al. 2020; Tran et al. 2019). Pathogenicity was confirmed by inoculating leaves of asymptomatic 2-year-old potted M. rubra plants. For wound inoculation, three plants were inoculated and one plant served as a control; for non-wound inoculation, another three plants were inoculated and one plant served as a control. On each plant, five mature leaves were selected. Prior to inoculation, leaves designated for wound treatment were pricked with a sterile needle, while non-wound leaves were left intact. A 10 μL droplet of conidial suspension (10⁶ conidia/mL) of isolate MPC001 was placed on each inoculated leaf (Zhao et al. 2025). Control leaves received a droplet of sterile distilled water. All plants were kept in a sealed, humid environment (85% RH) for 24 h and then transferred to standard growth conditions at 28°C. After 14 days, typical leaf spots developed on all wounded inoculated leaves and on most non-wounded inoculated leaves, whereas all control leaves remained asymptomatic. The fungus was re-isolated from lesions, fulfilling Koch’s postulates. No fungal colonies were obtained from control leaves. To our knowledge, this is the first report of P. capitalensis causing leaf spot on M. rubra. Given the economic importance of Chinese bayberry in southern China and relatively high disease incidence observed in the surveyed orchard, this finding has significant implications for disease management and biosecurity strategies in bayberry production areas.