Chimonanthus praecox (L.) Link is a deciduous aromatic shrub known for its unique ornamental value and fragrance (Guo et al. 2024). In August 2025, symptoms of leaf spot disease were observed on the leaves of C. praecox at Nanjing Xiaozhuang University (31°53'34.22″N, 118°54′6.59″E) in Jiangsu Province, China, with a disease incidence of 15% to 25%. The disease begins as small yellow spots that gradually expand, with the edges darkening to deep brown and the center turning grayish-white. Some lesions develop concentric ring-like patterns; in severe cases, they cause the leaves to yellow and fall off. To isolate the pathogens, five leaves with typical symptoms were collected. Tissue blocks (5×5 mm) were excised from the margins of the lesions, surface sterilized with 75% ethanol for 30 s and 2% NaClO for 5 min, and then rinsed with sterile water. Then it was transferred onto potato dextrose agar (PDA) plates and incubated at 25°C for 5 days for primary isolation. The mycelium was subsequently subcultured for purification, and after a further 7 days of incubation at 25°C, all purified colonies produced olive green aerial mycelia on PDA. A representative strain (NXU-LM1) was selected for morphological and molecular identification. The conidia were obclavate or elliptical, with 3 to 5 transverse septa and 1 to 2 longitudinal septa, measuring 24 to 34 × 10 to 12 µm, with an average length of 29.0 ± 3.2 µm and an average width of 11.0 ± 0.7 µm (n=30). The morphological characteristics of these colonies and conidial structures were consistent with those of the genus Alternaria (Dang et al. 2023). For molecular identification, genomic DNA was extracted from the NXU-LM1 isolate. Four genomic DNA regions were targeted for amplification and sequencing: the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2), and translation elongation factor 1-α (TEF1-α) regions. The sequences were deposited in the GenBank database (ITS, PX495952; GAPDH, PX911610; RPB2, PX911611; TEF1-α, PX911612). In a BLAST search, the sequences were 99% to 100% identical to the corresponding sequences of Alternaria alternata (accession numbers MG250610.1, KP124211.1, KP124825.1, and KP125133.1). Based on the concatenated sequences of the ITS, GAPDH, RPB2, and TEF1-α genes, phylogenetic analysis was performed in MEGA 7.0 using the maximum likelihood method with 1000 bootstrap replicates. The results revealed that strain NXU-LM1 clustered together with known reference strains of A. alternata (CBS 118814, CBS 102598) in the phylogenetic tree. Combining morphological observations and multigene phylogenetic analysis, NXU-LM1 was identified as A. alternata. Verifying the pathogenicity of strain NXU-LM1 according to Koch's postulates: Healthy leaves from 2–3-year-old C. praecox plants were surface-disinfected with 75% ethanol. 5 mm mycelial blocks from 7-day-old PDA cultures were attached to the leaves, with sterile PDA blocks as controls. The inoculated leaves were incubated in a moist chamber at 25°C with 90% relative humidity and a 12 h light/dark photoperiod. Five days later, typical symptoms appeared on inoculated leaves, while no symptoms on control leaves. The pathogen was re-isolated from lesions, with identical morphological and molecular characteristics to NXU-LM1. No pathogen was isolated from control leaves, confirming that this A. alternata strain is the causal agent of C. praecox leaf spot. To our knowledge, this is the first report in China of A. alternata infecting C. praecox and causing leaf spot disease. This study identifies the pathogen of the disease and provides important reference value for the monitoring and control of C. praecox leaf spot.
Zhang et al. (Thu,) studied this question.