First Report of <i>Neofusicoccum parvum</i> Causing a Leaf Spot Disease on <i>Callerya cinerea</i> in China

Callerya cinerea (Benth.) Schot is an ornamental shrub species cultivated in southern China. In October 2024, severe leaf-spot symptoms were observed on C. cinerea plants at Qujing Normal University (25.527°N, 103.744°E), Qujing, Yunnan, China. The estimated incidence of the disease was approximately 70% of the trees on campus. Light brown lesions initially emerged at the leaf tips. Subsequently, these lesions expanded and turned grayish-white, presenting numerous black, dot-like pycnidia. To identify the causal agent, six symptomatic leaves were collected from three plants. Lesion margins (4 × 4 mm) were excised, surface-sterilized in 70% ethanol for 30 s followed by 1% NaClO for 1 min, rinsed three times in sterile distilled water, dried on sterilized paper, placed on potato dextrose agar (PDA), and incubated at 28°C for 3 days. Developing colonies were retransferred to new PDA and purified by the hyphal tip technique. Eight out of ten isolates were morphologically identical, and three isolates (M1, M2 and M3) were used for molecular identification and pathogenicity assay. Initially, fungal colonies exhibited a white aerial mycelium. Subsequently, the center of the colony turned gray, and ultimately the entire colony assumed a mouse gray color. Conidia formed on leaf spots were hyaline, unicellular, non-septate, and smooth-walled, measuring 5.4–8.6 × 2.5–4.4 μm with an average of 6.8 × 3.2 μm (n=50). No pycnidia were observed on PDA. Molecular identification was performed by partially sequencing the internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (tef1-α), and beta-tubulin (tub2) gene regions using primer pairs ITS1/ITS4, EF1-728F/EF1-986R, and Bt2a/Bt2b, respectively (White et al. 1990; Carbone and Kohn 1999; Glass and Donaldson 1995). Sequences from three representative isolates were almost identical to one another. The generated sequences of ITS (PX453001-PX453003), tub2 (PX514764- PX514766), and tef1-α (PX514767-PX514769) were deposited in GenBank. BLAST analyses for each gene revealed 99-100% identity between the query sequence and its closest match (PQ240103, PQ166409, HQ859954) from Neofusicoccum parvum isolates. Based on the concatenated sequences, Bayesian phylogenetic analysis placed the isolates in a strongly supported clade with reference N. parvum group. Based on morphological characteristics, BLAST results, and the phylogenetic tree, the pathogen was determined to be N. parvum. Pathogenicity was confirmed by inoculating nine healthy C. cinerea leaves (punctured with a sterile needle) of three C. cinerea plants with mycelial plugs of M1, M2 and M3. Control leaves received sterile PDA plugs. All inoculated leaves were enclosed in plastic bags for three days. After 7 days, black spots developed on these leaves, while controls remained asymptomatic. The inoculation experiment was conducted three times with consistent results. Based on morphological characteristics and ITS sequencing, the same pathogen was reisolated from lesions, fulfilling Koch’s postulates. N. parvum was reported to cause leaf spots on multiple hosts like Camellia japonica (Chang et al. 2024), Geodorum eulophioides (Du et al. 2021) and Acer truncatum (Dong et al. 2025). To our knowledge, this is the first report of N. parvum infecting C. cinerea in China. This finding highlights the need for monitoring and management to protect this ecologically valuable species.