First Report of Colletotrichum siamense Causing Anthracnose on Dioscorea alata in China.

Dioscorea alata is an annual or perennial dicotyledonous plant which is a vegetatively propagated tuberous food crop (Mondo et al. 2021). In 2021, symptoms of leaf anthracnose occurred on D. alata plants at a plantation in Changsha, the Hunan Province of China (28°18' N; 113°08'E). Symptoms initially showed as small, brown water-soaked spots on the leaf surface or margins, and enlarged to irregular, dark brown or black, necrotic lesions, with a lighter center and darker edge. At latter, lesions extended to most of the leaf surface causing leaf scorch or wilting. Almost 40% of the plants surveyed were infected. Symptomatic leaf samples were collected, and small pieces were taken at their disease-healthy junction, sterilized with 70% ethanol for 10 s, 0.1% HgCl2 for 40 s, rinsed three times with sterile distilled water, and then placed on potato dextrose agar (PDA) for incubation at 26 °C for 5 days in the dark. Fungal colonies with similar morphology were observed and, in total, 10 isolates were obtained from 10 plants. On PDA, colonies were initially white with fluffy hyphae, and later became light to dark gray, showing faint concentric rings. Conidia were hyaline, aseptate, cylindrical, and rounded at both ends, measuring 11.36 to 17.67 × 3.45 to 5.9 μm (n = 50). Appressoria were dark brown, ovate, globose, measuring 6.37 to 7.55 × 10.11 to 12.3 µm. These morphological characteristics were typical of Colletotrichum gloeosporioides species complex (Weir et al. 2012). For molecular identification, the internal transcribed spacer (ITS) region of rDNA, and partial sequences of actin (ACT), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes of a representative isolate Cs-8-5-1 were amplified and sequenced using the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and GDF/GDR as described previously (Weir et al. 2012). These sequences were deposited in GenBank (accession nos. OM439575 for ITS, OM459820 for ACT, OM459821 for CHS-1, and OM459822 for GAPDH). BLASTn analysis showed 99.59 to 100 % identity to the corresponding sequences of C. siamense strains. A Maximum likelihood phylogenetic tree based on the concatenated ITS, ACT, CHS-1 and GAPDH sequences was generated by MEGA 6. It revealed that the Cs-8-5-1 was clustered with the C. siamense strain CBS 132456 with 98% bootstrap support. For pathogenicity test, conidia suspension (105 spores/ml) was prepared by harvesting conidia from 7-day-old cultures growing on PDA, and 10 uL was dropped onto leaves of potted D. alata plants (8 droplets per leaf). Leaves treated with sterile water were served as controls. All the inoculated plants were placed in humid chambers (with 90% humidity) at 26°C with a photoperiod of 12 h. The pathogenicity tests were performed twice, with each had three replicated plants. Seven days after inoculation, the inoculated leaves showed symptoms of brown necrosis resembling that observed in fields, however, the control leaves remained symptomless. The fungus was specifically re-isolated and identified by morphological and molecular methods, thus fulfilling Koch's postulates. To our knowledge, this is the first report of C. siamense causing anthracnose on D. alata in China. Since this disease might seriously affect the photosynthesis of the plants, which can influence the yield, prevention and management strategies should be adopted to control this new disease. Identification of this pathogen will provide a foundation for the diagnosis and control of this disease.