First Report of Anthracnose on Caused by in Hunan, China.

Camellia oleifera, known as the tea-oil tree, is a multifunctional woody oil crop renowned as the "Oriental olive". During 2023-2024, in Jishou City (28°17'N, 109°29'E), Hunan Province, China, 100% of tea-oil tree plants and over 40% of the leaves on each plant exhibited typical anthracnose symptoms. Initially, infected areas displayed yellowish regions. As the disease progressed, brown necrotic lesions with dark brown margin and tiny, dark dots emerged. Diseased leaves were collected, rinsed under running water, and cut into small pieces (5×5 mm) from the edges of the lesions. These pieces were surface-disinfected with 70% ethanol for 30 seconds, followed by 2% NaOCl for 2 minutes, and then rinsed three times with sterile water. The disinfected samples were placed on potato dextrose agar (PDA supplemented with 100 mg/L streptomycin sulphate) and incubated at 28℃ in the dark. After 3 days, mycelia plugs from the infected tissues were transferred to fresh PDA for subculturing. Twenty isolates displaying two distinct morphological types were obtained. One representative strain from each morphology type (designated as LinC-A and LinC-B) was randomly selected for further study. On PDA, LinC-A developed cottony, raised colonies that were initially white on the obverse and yellowish on the reverse. Over time, the colonies turned reddish to deep red at the center, with red pigmentation on the reverse side. LinC-B colonies were initially white to yellowish on the obverse and deep yellow on the reverse, later turning yellowish to deep pink on both sides, producing pink pigmentation. Conidia of both strains were single-celled, hyaline, fusiform with acute ends, and smooth-walled. The average conidial size of LinC-A was 12.9 ± 1.6 × 5.8 ± 1.6 μm (n=100), while that of LinC-B was 15.6 ± 2.2 × 6.4 ± 0.7 μm (n=100). Additionally, brown setae were observed surrounding dark brown acervulus. For molecular identification, the ITS region and four other genes (GAPDH, CHS-1, ACT, and TUB2), were amplified and sequenced. The sequences were deposited in GenBank (ITS: PQ825943, PQ825944; GAPDH: PQ827013, PQ827014; CHS-1: PQ827015, PQ827016; ACT: PQ827017, PQ827018; TUB2: PQ827019, PQ827020). Phylogenetic analysis based on the combined ITS, GAPDH, CHS-1, ACT, and TUB2 sequences confirmed that both LinC-A and LinC-B clustered with C. fioriniae strains, consistent with the homology search results. Therefore, based on morphological and molecular evidence, LinC-A and LinC-B were identified as C. fioriniae. To fulfill Koch's postulates, pathogenicity tests were conducted using conidial suspensions of 1×106 conidia/mL on tea-oil tree plants. A 50 μL aliquot of the suspension conidia was applied on each leaf, while control leaves were inoculated with an equivalent volume of sterile distilled water. Inoculated plants were maintained in humid chambers at 28℃ under a 12-hour light-dark cycle. Six leaves were used for each of the three replicates. After five days, all inoculated leaves developed anthracnose symptoms, whereas control leaves remained asymptomatic. C. fioriniae was re-isolated and re-identified from the inoculated leaves, confirming its pathogenicity. Previous studies have reported C. fructicolca, C. gloeosporioides, C. camelliae, C. aeschynomenes, C. karstii, C. siamense, C. kahawae, and C. horii as pathogens of tea-oil tree in China (Li et al., 2016, 2017, 2019; Wei et al., 2023). This is the first report of C. fioriniae causing anthracnose on tea-oil tree plants.