First Report of <i>Colletotrichum fructicola</i> Causing Leaf Spot on <i>Rhododendron lapponicum</i> in China

Rhododendron lapponicum belongs to the genus Rhododendron. It has a wide range of colours and flowers, which is of great economic and ornamental value. In March 2024, symptoms of leaf spot were observed on R. lapponicum in Liaoning Province, China. The lesions on infected leaves usually have a characteristic circular or irregular shape, often with a brown halo surrounding them. As the disease progresses, the lesions enlarge and may coalesce, causing extensive leaf browning. Eventually the leaves dry out and wilt and the plant dies. To isolate the pathogen, tissue pieces were cut from the junction of the diseased and healthy tissues on azalea leaves with a sterile blade, soaked in 70% alcohol for 1 min, and immersed in sterile water three times for 30 s each. The pieces were transferred to synthetic nutrient-poor agar medium (SNA). Cultures were incubated for 10 d at 20 °C under near UV light with a 12 h photoperiod (Liu et al. 2014). One isolate (SNPF202401) was subcultured on fresh SNA plates for further study. The colonies on SNA grew white aerial mycelium. The conidia were unicellular, colorless, hyaline, cylindrical, obtusely rounded at both ends, and measured 14.1 to 19.5 × 4.4 to 5.4 µm. The morphological features were consistent with Colletotrichum fructicola (Prihastuti et al 2009, Rojas et al 2010). The pathogen was confirmed as C. fructicola by amplification and sequencing of the internal transcribed spacer region (ITS), a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a partial sequence of the actin (ACT), and beta-tubulin (TUB2) genes using primers ITS1/ITS4, GDF1/GDR1, ACT-512F/ACT-783R, and T1/Bt-2b, respectively (Weir et al. 2012). Sequences were deposited in GenBank (ITS: PQ608623, GAPDH: PQ642047, ACT: PQ642048, TUB2:PQ642049). Using the ClustalW algorithm in MEGA11 for nucleotide sequence alignment, phylogenetic trees were constructed with ITS, GAPDH, ACT, and TUB2 with the maximum likelihood method, confirming the pathogen species as C. fructicola. The pathogen was thus identified as C. fructicola based on morphological characterization and molecular analyses. To test pathogenicity, two healthy R. lapponicum plants were scratched with a sterile needle (0.45 mm in diameter) on five leaves per plant. Plants were inoculated by spraying a conidial mixture suspension (105 conidia ml−1) onto leaves; control leaves were sprayed with sterile water. Plants were kept at 25°C and 95% relative humidity in a growth chamber. The test was repeated five times. After 14 days, treated leaves developed a characteristic circular or irregular shape, often with a brown halo surrounding them like those in the field; controls had no symptoms. The pathogen reisolated from infected leaves was the same as the original isolate based on morphological characteristics and molecular identification. To our knowledge, this is the first report of leaf spot on R. lapponicum caused by C. fructicola in the China . This is valuable information for prevention and management of leaf spot disease associated with R. lapponicum.