Lin, CH; Wu, WQ; Liao, XM; Liu, WB; Miao, WG; Zheng, FC
Water yam (Dioscorea alata [Dioscoreaceae]) is a vegetatively propagated tuberous food crop cultivated widely in the tropics and subtropics (Mignouna et al. 2003). It is the most widely distributed species of yam in China and eaten mainly as a vegetable. During July 2016, anthracnose-like lesions were observed on the leaves of cultivar Da56 at a plantation in Danzhou City, Hainan Province. Leaf symptoms began as small, brown, pinpoint spots <1 mm in diameter and enlarged to dark brown to almost black, irregular blotches >10 mm diameter with yellow haloes. Approximately 35% of the plants showed the same symptoms on their lower leaves. Symptomatic leaves were collected randomly from different parts of the field. Tissue was removed from the margin of three lesions per leaf. It was then surface sterilized in 75% ethanol, air-dried, plated on potato dextrose agar (PDA) plates, and incubated at 28°C and a 12-h photoperiod. Colonies with similar characteristics were produced after 4 days. Four isolates (DA1605-1608) from different leaves were subcultured on PDA using the single-spore method. After 4 days, the slow-growing colonies produced numerous, discrete colonies. The colonies contained dark-based acervuli with bright orange conidial spore masses and irregular sectors of pale gray pigment when viewed from below. Conidia were hyaline, aseptate, straight, and cylindrical with both ends rounded. They measured 12.7 to 21.7 × 5.4 to 8.9 µm (average 18.0 × 7.0 µm, n = 100). Appressoria were simple, elliptic to fusoid in shape. Perithecia were not seen. Morphological characteristics of the isolates matched descriptions of the slow-growing gray group of Colletotrichum alatae (Abang et al. 2002). To confirm the morphological identification, internal transcribed spacer region (ITS), chitin synthase (CHS-1), actin (ACT), and glyceraldehydes-3-phosphate dehydrogenase gene regions were amplified using universal primers ITS1/ITS4, CHS-79F/CHS-354R, ACT-512F/ACT-783R, and GDF/GDR and were sequenced (Weir et al. 2012). All four gene sequences from the four isolates shared 100% identity. These four gene sequences of isolate DA1605 were deposited in GenBank (accession nos. KY689723, KY689725, KY689724, and KY689726). A multilocus phylogenetic analysis performed with the reference sequences (Weir et al. 2012) revealed that the isolate clustered within C. alatae. Isolate DA1605 was deposited in the Agriculture Culture Collection of China as ACCC39281. For the pathogenicity test, a conidial suspension (1 × 106 spores/ml) of each isolate was prepared by harvesting conidia from 5-day-old cultures growing on PDA. The suspension was sprayed onto 10 detached, unwounded, healthy young leaves. The same number of control leaves was treated with sterile water. All leaves were kept in plastic boxes under moist conditions at 28°C and a 12-h photoperiod. Seven days after inoculation, black spots were observed on all inoculated leaves. No symptoms were observed on the control leaves, and no fungus was isolated. The fungus was reisolated from inoculated leaves, fulfilling Kochs postulates. The pathogen has been previously reported only on yam (D. alata) from Nigeria, Barbardos, India, and Guadeloupe (Abang et al. 2002, 2003; Weir et al. 2012). To our knowledge, this is the first record for C. alatae in China.
; actin; air drying; anthracnose; appressoria; chitin synthase; Colletotrichum; conidia; cultivars; culture media; Dioscorea alata; ethanol; food crops; fungi; genes; harvesting; internal transcribed spacers; leaves; pathogenicity; pathogens; perithecia; photoperiod; phylogeny; subtropics; tropics; vegetative propagation; yams; China; Guadeloupe; India; Nigeria/