First report of leaf spot caused by <i>Diaporthe vaccinii</i> on <i>Michelia figo</i> in China

Michelia figo (Lour.) Spreng. (banana magnolia), which is widely cultivated in gardens and landscapes in southern China, is an evergreen shrub or small tree in the Magnoliaceae family. From July to August 2025, severe leaf spot disease was observed on the leaves of multiple M. figo plants at the Fangshan Campus of Nanjing Xiaozhuang University in Jiangsu Province (31.89°N, 118.90°E), China. Disease severity was estimated at 20-30% based on symptoms observed in the field. The initial symptoms appeared as small, circular, tan to dark brown spots, which later expanded into irregular lesions with yellowish halos. Severe infections caused defoliation. For isolation of the causal agent, 30 symptomatic leaves were collected from 20 plants as representative samples. From each sample, three to five tissue pieces (approximately 1 × 0.5 cm) were excised from the lesion margins via a sterilized blade. The tissues were surface sterilized with 75% ethanol for 40 s, followed by 2% NaClO for 5 min, rinsed with sterile water, and transferred onto potato dextrose agar (PDA) medium. The samples were incubated at 25°C for 5 days (Xue et al. 2025). Pure cultures from six isolates with similar colony characteristics were obtained via hyphal tip transfer. A representative isolate (strain NXU-HX5) was selected for morphological analysis, molecular identification, and pathogenicity tests. On PDA, colonies were initially grayish-green with fluffy aerial mycelia. After approximately 13 days of incubation, the colonies expanded to the plate edges and were covered with dense, white to gray, cottony aerial mycelia. Microscopic observation revealed that the vegetative hyphae were hyaline, septate, and branched, with branching predominantly lateral. Hyphae were straight to slightly curved, uniform in width. Conidia were hyaline, filiform to cylindrical, and predominantly curved or arcuate, 23.57 × 2.14 to 15.32 × 1.84 μm (average 20.23 × 2.02 μm, n = 50). Partial sequences of the internal transcribed spacer region (ITS) and translation elongation factor 1-alpha (TEF1), calmodulin (CAL), and beta-tubulin genes were amplified with primers ITS1/ITS4, EFl-728F/EFl-986R, CAL228F/CAL737R and βt2a/βt2b, respectively, (Gomes et al. 2013). All sequences were deposited in GenBank (ITS, PX494256; TEF1, PX911607; CAL, PX911608; and beta-tubulin, PX911609). BLAST results revealed that the ITS, TEF1, CAL and beta-tubulin sequences were highly similar to those of Diaporthe vaccinii (&gt;99.9%). Multigene phylogenetic analysis was performed via the maximum likelihood method in MEGA. According to results of the phylogenetic and morphological analyses, the strain was identified as D. vaccinii. For pathogenicity tests, following the protocol of Xue et al. (2020), 10 leaves of M. figo plants were inoculated with a spore suspension (1.5 × 104 CFU/mL) while 10 control leaves were treated with sterile distilled water. The inoculated plants were maintained under natural field conditions for 7 days (average daily temperature 25°C), with the inoculation sites covered by moist cotton and plastic film for the first 48 h to maintain &gt;90% relative humidity. All inoculated leaves developed symptoms consistent with those observed in the field, whereas no symptoms appeared on the control leaves. The experiment was conducted three times. The pathogen was reisolated from the inoculated leaves and confirmed to be D. vaccinii through morphological and molecular analyses. This first report of the effects of D. vaccinii on M. figo in China provides insights into the host adaptation and spread risks of this pathogen.