First report of Colletotrichum gloeosporioides causing leaf spot on Cyclobalanopsis glauca in China.

Abstract

Cyclobalanopsis glauca (Thunb.) Oerst. is one of the most widely distributed species of evergreen broad-leaved tree in subtropical areas of China. It is also grown in Korea, Japan, and India. Because of its beautiful shape, C. glauca is commonly used for greening gardens and walkways. In July 2018, leaf spots on C. glauca were observed in Zhejiang province (Lishui, N: 28°26' 6.75";E: 119°54'11.22), China. About 70% of the trees were found to be diseased, with approximately 50% of leaves showing symptoms. The symptoms on C. glauca leaves initially appeared as small brown-yellow spots which gradually expanded, developing a light brown central and dark brown to black margin. The spots ranged from 4 to 15 mm in diameter. Ten symptomatic fragments measuring approximately 5×5 mm from each leaf were surface disinfested with 70% ethanol for 30 s, and then they were rinsed in sterile distilled water and placed on potato dextrose agar (PDA) medium at 25 °C in the dark for five days. Segments of colony perimeters were then transferred to new plates. The colonies initially produced white mycelia that later turned gray-white with pink and occasionally black dots scattered on the surface of the mycelium. Spores were aseptate, cylindrical, 8 to 15 μm in length, and 3 to 5 μm wide, most with rounded ends, a few with one apex round and the other fusiform, as described for Colletotrichum gloeosporioides (Penz.) Sacc. (Agostini et al. 1993). The internal transcribed spacer rDNA (ITS: MK758005) and two nuclear protein-coding genes (CHS: MK784770, ACT: MK784769) were amplified with ITS1/ITS4, CHS-79F/CHS-345, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). The sequence had 99.61% identity to GQ485605 for ITS, 99.56% to GQ856782 for ACT, and 100% to GQ856733 for CHS of C. gloeosporioidesCBS 953.97 in GenBank, respectively. To fulfill Koch's postulates, spores (1×108) of the isolate were sprayed onto leaves of twelve 2-year-old C. glauca plants (at least six leaves per plant). The fungus was inoculated on one side of each leaf, and distilled water was used as a mock inoculated control on the other side. The plants were cultivated in the greenhouse to maintain high humidity and a temperature near 25 °C. After 9 days, 100% of the leaf halves that had been inoculated had symptoms identical to those observed onaffected C. glaucaleaves in the field, while no symptoms were observed on the mock inoculated half of each leaf. The fungus was reisolated from the symptoms and identified as C. gloeosporioides using techniques previously described. To our knowledge, this is the first report ofC. gloeosporioidesinfecting C. glauca in China. This study will establish a foundation for the further study of C. gloeosporioides to address the disease effectively.