First report of Ceratobasidium sp. AG-P causing black rot on leaves of Camellia sinensis in China

Camellia sinensis (L.) O. Kuntze is an important specialty crop in China. In June 2024, a severe black rot was observed on leaves of C. sinensis cultivar ‘Jiukeng’ in a 1.1-hectare tea plantation in Hangzhou city (30°21′1″N, 119°51′58″E), Zhejiang province. Of 180 surveyed plants, 42 plants showed typical symptoms, with a disease incidence over 20%. Early symptoms were dark brown to blackish lesions; as the disease progressed, lesions expanded and coalesced into irregular spots with grayish-white centers and grayish-brown margins, eventually causing leaf black rot. Twenty diseased leaves were randomly collected from ten infected plants for pathogen isolation. 5-mm tissue fragments from lesion margins were surface-sterilized with 70% ethanol for 30 s, rinsed three times with sterile distilled water, inoculated onto potato dextrose agar (PDA), and incubated at 26°C for 48 h. Thirteen single-hypha strains were isolated. After 7 days, colonies were white to gray with sparse mycelium and distinct concentric rings. Hyphae were septate, branched at nearly right angles, and constricted at branch points. Hyphae ranged from 3.5 to 7.8 μm in width (n=60; mean = 4.8 μm). Binucleate cells were observed via 4′,6-diamidino-2-phenylindole (DAPI) staining, which is characteristic of Ceratobasidium sp. (Sneh et al., 1991). Four strains were selected for molecular identification. The internal transcribed spacer (ITS) and the second largest subunit of RNA polymerase II (rpb2) genes were amplified using primer pairs ITS1/ITS4 (White et al., 1990) and rpb2-980F/rpb2-7cR (Gónzalez et al., 2016), respectively. ITS sequences (GenBank: PX271204-PX271207) and rpb2 sequences (GenBank: PZ295856-PZ295859) showed 99% similarity to Ceratobasidium sp. (ITS: JX514384, MK848669; rpb2: PP665479, MT150071). Phylogenetic analysis showed these strains clustered in the Ceratobasidium sp. AG-P clade (bootstrap=100). Strains JS21 and JS3 were used for pathogenicity testing. The strains were cultured in potato dextrose media on a shaker (26°C, 200 rpm) for 7 days, homogenized with a sterile blender, and adjusted to 10⁶ fragments/mL (Guidry and Trelles, 1962). Four-month-old ‘Jiukeng’ seedlings were inoculated with 10 μL mycelial suspension per leaf; sterile water served as the negative control. Seedlings were incubated in a greenhouse (26°C, 85% RH) for 3 days. The experiment was triplicated with five seedlings per replicate. Three days post-inoculation, necrotic lesions similar to field symptoms developed on inoculated leaves, while control leaves remained healthy. Re-isolated pathogens matched the original strains morphologically and molecularly, confirming Koch’s postulates. Ceratobasidium sp. was previously reported to cause tea black rot in Japan (Ezuka & Andou, 1994), and Rhizoctonia solani caused tea black blight in India (Mulder et al., 1962). To our knowledge, this is the first report of Ceratobasidium sp. AG-P causing black rot on C. sinensis in China, providing a basis for disease early warning and control strategy development.