First Report of <i>Neopestalotiopsis clavispora</i> Causing Leaf Spot on <i>Cornus racemosa</i> in China

Cornus racemosa Lam. is valued for its ornamental appeal in naturalistic and restoration landscapes. In June 2024, leaf spots were observed on C. racemosa at Qujing Normal University (25.53°N, 103.75°E), with an incidence of 40–60%. The disease led to premature leaf senescence and defoliation, reducing the plant's horticultural value. Symptoms were irregular to circular leaf spots, initially purplish with yellow-green margins, later turning dark brown. To identify the causal agent, 15 symptomatic leaves were collected. Tissue samples (5 × 5 mm) were excised from lesion margins, surface sterilized in 75% ethanol for 30 s and 1% sodium hypochlorite for 60 s (Suo et al. 2024), rinsed three times with sterile water, and dried on sterilized filter paper. Sterilized tissues were placed on potato dextrose agar (PDA), and incubated at 25 °C for 3 days. Emerging colonies were subcultured and purified using the hyphal tip method (Senanayake et al. 2020). Three representative isolates (LM01, LM02, and LM03) were selected for analysis. Colonies on PDA grew rapidly, forming white, cottony mycelia with undulate margins and dense aerial hyphae. Sporulation occurred after 21 days at 25°C in darkness. Conidia were five celled, clavate to fusiform, smooth-walled, and measured 17.9-24.8 × 5.5-8.4 µm (n = 50). The three median cells were dark brown to olivaceous, with the central cell darker than the adjacent ones; both apical and basal cells were hyaline. A single basal filiform appendage (4.6–8.2 µm; n = 50) was observed. Morphological features were matched descriptions of Neopestalotiopsis clavispora (Maharachchikumbura et al. 2014). For molecular identification, genomic DNA was extracted, and the internal transcribed spacer (ITS), β-tubulin (TUB 2), and translation elongation factor 1-alpha (TEF1-α) genes were amplified using primers ITS1/ITS4, T1/Bt2b, and EF1-728F/EF1-986R (Maharachchikumbura et al. 2014). Sequences were deposited in GenBank (accession numbers PV937560- PV937562 (ITS), PV980051- PV980053 (TEF1-α), and PV980054- PV980056 (TUB 2). BLAST analysis showed 99.42% (ITS), 100% (TUB), and 99.02% (TEF1-α) identity with N. clavispora strain MFLUCC 12.0281. A phylogenetic tree (maximum likelihood) based on concatenated sequences clustered isolates with other N. clavispora strains with 95% bootstrap support, confirming their identity. To assess pathogenicity, ten healthy, mature leaves (one per twig) were punctured with a sterile needle and inoculated with 20 μL of a conidial suspension (1 × 10⁶ conidia/mL). As a control, ten similarly punctured leaves were treated with sterile water. Leaves were enclosed in plastic bags for 48 h to maintain high humidity. Symptoms similar to field observations developed on inoculated leaves within 7 days, while control leaves remained asymptomatic. The pathogen was reisolated from symptomatic leaves, and its identity was confirmed by both morphology and DNA sequence, fulfilling Koch’s postulates. The experiment was repeated three times with consistent results. N. clavispora was previously reported causing fruit brown spot in pomegranate, calyx and receptacle blight on strawberry, and leaf spot in Photinia bodinieri (Shi et al. 2022; Xu et al. 2023; Kong et al. 2024). However, this is the first report of P. clavispora causing leaf spot on C. racemosa in China. This study broadens the host range of N. clavispora and provides a basis for risk assessment and management of this emerging disease.