First Report of <i>Colletotrichum citricola</i> Causing Anthracnose on <i>Rhododendron mucronatum</i> in China

Rhododendron mucronatum is highly valued for its ornamental qualities and is widely cultivated in many cities within its distribution range, including Guangdong, Guangxi, Sichuan and Yunnan Province of China. Additionally, it is a traditional Chinese medicinal plant with significant therapeutic potential. In May 2025, a severe anthracnose disease was found in Songming county (‌25°33′ N, 102°56′ E) in Kunming City, Yunnan Province, China, with a disease incidence of 62% in field. The leaves were the primary infection tissues, exhibiting typical anthracnose symptoms. Initially, small necrotic spots appeared, which rapidly expanded into irregular grayish-brown lesions with a reddish-brown outer ring. A total of 18 diseased samples were randomly collected from three nursery gardens. Single-spore isolation method was used to isolate the pathogen (Choi et al. 1999), and three single-spore isolates (DJTJ03/06/11) were obtained. The strains exhibited a circular, white, cottony appearance with yellowish-brown discoloration after 5-day of culture on PDA. Conidia were hyaline, aseptate, cylindrical, oval, and ranged in size from 10.37 to 17.58 × 4.51 to 9.24 μm (n = 50). Morphological characteristics were consistent with published descriptions of the Colletotrichum boninense species complex (Liu et al. 2022). For molecular identification, the ribosomal internal transcribed spacer (ITS) (White et al. 1990), beta-tubulin 2 (TUB2) (Glass and Donaldson 1995), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Templeton et al. 1992), chitin synthase 1 (CHS-1), beta-actin (ACT), and histone3 (HIS3) (Carbone and Kohn 1999) genomic regions were amplified and sequenced. The sequences were deposited in GenBank (ITS: PV833928, PV833929; TUB2: PV857554, PV857555; GAPDH: PV857550, PV857551; CHS-1: PV857548, PV857549; ACT: PV857546, PV857547; HIS3: PV857552, PV857553). Multilocus phylogenetic trees were constructed using Maximum Likelihood (ML) method, which showed that the two isolates clustered with C. citricola with 98% bootstrap support. All isolates were clearly distinguished from other species. Pathogenicity tests were conducted using three healthy 1-year old seedlings and the strain DJTJ06. Conidia were collected and resuspended in sterilized distilled water (1×106 conidia/mL). Five leaves per seedling were selected for the test, 30 μL each of conidial suspension was inoculated on two sites of the half leaf separated by the midrib and 30 μL each of sterile water was inoculated on the other half of the leaf as control. Plants were incubated at 25℃ and 90% humidity with a 12 h photoperiod. Anthracnose symptoms were observed on all inoculated sites of the leaves after 5 days. No symptoms were observed on control leaves. The re-isolated strains had the same morphological characteristics and sequences as the original isolates, and no isolates were obtained from the control. The entire experiment was completed three times. Thus the Koch’s postulates were fulfilled. The Colletotrichum boninense species complex contains several closely related species, including C. citricola, that cause severe production losses to agricultural and forestry plants. C. citricola has been reported on Cavendish Bananas (Huang et al. 2022), Italian Ryegrass (Xu et al. 2024), among others. To our knowledge, this is the first report of anthracnose caused by C. citricola on R. mucronatum in China. This disease causes massive defoliation, severely reducing the ornamental value of plants. Therefore, preventive measures must be implemented to mitigate its impact.