First Report of Anthracnose Caused by Colletotrichum gloeosporioides on Cinnamomum camphora in Korea.

Abstract

Cinnamomum camphora, known as the camphor tree, is an evergreen tree widely cultivated in Asia as an ornamental plant (Singh and Jawaid, 2012). In June 2023, several leaves on a total of 10 trees planted on a street in Suncheon, Jeonnam Province, Korea showed black spots. Disease incidence was observed in at least 15% of the 10 trees. The symptoms included circular spots with a light ash-colored center and dark brown borders. The size of lesions varied depending on the progress of the disease. The disease progressed by 30% on the tree leaves. To isolate the pathogen, we cut out the lesions on the leaf surface sterilized with 70% ethanol for one minute, washed three times with sterilized distilled water, dried, and placed on water agar. Then, it was incubated at 25°C for three days. Emerging hyphae from the samples were subcultured on potato dextrose agar (PDA), resulting in three independent isolates (SYP-F1226-1 to SYP-F1226-3) after single spore isolation from 3 independent trees. The isolates exhibited grayish fluffy mycelium in the center of the colony, while the edges were white on PDA. Conidia had rounded cylindrical shape and were 4.9 to 8.4 µm  1.4 to 3.1 µm (avg. 5.9  2.1 μm, n = 100) in size. Appressoria were round, dark gray, produced at the tip of the germ tube after a septum formed the conidium. The morphological characteristics matched those of Colletotrichum species complexes. (Damm et al., 2012; Weir et al., 2012). For molecular identification, ITS (OR647338 to 40), GAPDH (OR657042 to 44), CHS-1 (OR657045 to 47), ACT (OR657048 to 50), and CAL (OR657051 to 53) sequences from isolates SYP-F1226-1~3 showed a 99.65%, 98.56%, 99.00%, 99.28%, and 99.52% identity with that of type strain C. gloeosporioides ICMP 17821 (JX010152, JX010056, JX009818, JX009531, and JX010445, respectively). Using the MEGA X program (Kumar et al. 2018), maximum likelihood analysis based on the concatenated sequences placed the isolates within a clade comprising C. gloeosporioides. Pathogenicity of SYP-F1226-1 was tested using three leaves from a 1-year-old branch of three independent healthy C. camphora plants. The leaf surfaces were sterilized by rubbing a cotton pad soaked in 70% ethanol and then wiping them with a sterilized cotton pad. The leaves per plant were inoculated with 5 mL of a conidial suspension (1 × 105 conidia/mL), both with and without wounding. Another three control leaves were inoculated with sterile distilled water, both with and without wounding. The inoculated leaves were wrapped in a plastic bag for 48 hours under conditions of 100% relative humidity. Spot symptoms were observed on both wounded and non-wounded leaves 21 days after inoculation. No symptoms were observed in the control on either of the wounded leaves. Pathogenicity tests were performed three times. The pathogen was re-isolated from the lesion after treatment, and its identity was confirmed using the five genes and morphological characteristics. This confirms the fulfillment of Koch's postulates. C. fioriniae (Liu et al, 2022) and C. siamens (Liu et al, 2022; Khoo et al, 2023) have been reported as the causal pathogen of anthracnose in C. camphora, but C. gloeosporioides has not been reported as a pathogen in C. camphora. To our knowledge, this is the first report of anthracnose caused by C. gloeosporioides on C. camphora in Korea. This study will provide symptomatic, mycological, and molecular biological information for the early detection of anthracnose disease in C. camphora plants.