First Report of Epicoccum sorghinum Causing Black Leaf Spot on Dioscorea polystachya in China.

Abstract

Chinese yam (Dioscorea polystachya Turcz.), known for its nutrient-rich underground tubers, is both a food source and a traditional Chinese medicinal plant. It offers significant nutritional and medicinal benefits. In 2023, the cultivation of Chinese yam, specifically the Wencheng Waxy Yam variety, exceeded 400 hectares in Wencheng County, Zhejiang Province, China, with an annual yield surpassing 9,000 tons (Chen et al. 2024). This crop has become a vital industry for local farmers in mountainous areas. In July 2023, a black leaf spot disease was detected in the Wencheng County Chinese Yam planting base on almost 21 hectares (Huangyang Village, Huangtan Town, 120.09°E, 27.79°N). The disease affected between 70% and 85% of the plant area, with symptomatic leaves constituting 45% of the total leaf count. Initially, black spots appeared on the leaves, which later expanded into dark brown spots with yellowing of the edges. In severe cases, the centers of the spots became necrotic. To investigate the nature of the pathogen, eight symptomatic leaves were randomly collected, cut into small pieces (about 0.3 cm × 0.3 cm), and subjected to a series of treatments: soaking in 75% ethanol for 1 minute, followed by 5 minutes in 2% sodium hypochlorite, and finally washed three times with sterile distilled water. The pieces were then dried on sterile filter paper and incubated on potato dextrose agar (PDA) at 28°C under a 12-hour light/dark cycle. After 5 days, mycelial edges were transferred to fresh PDA plates for future purification. Finally, five pure colonies with consistent morphology were obtained through repeated separations. When cultured on PDA for 7 days, colonies exhibited gray-white aerial hyphae on the front and brown on the back. Pycnidia were dark, mostly spheroid, and measured 44.5 to 81.2 × 66.9 to 107.3 µm (n = 20). Chlamydospores were nearly spherical, either unicellular or multicellular, and measured 7.16 to 10.35 × 15.82 to 28.92 μm (n = 30). While conidia were ellipsoidal, single-celled, aseptate, and ranged from 1.8 to 3.5 × 3.6 to 6.9 μm (n = 30). Genomic DNA from fresh colonies was extracted using the DNA Secure Plant Kit (Tiangen Biotech, Beijing, China). Polymerase chain reaction (PCR) amplification was carried out using standard primers and procedures for the rDNA internal transcribed spacer (ITS), β-tubulin (TUB), and actin (ACT). Sequences were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), and ACT512F/ACT783R (Carbone and Kohn 1999), respectively. These sequences were deposited in GenBank under accession numbers PQ197602 (ITS), PQ201634 (TUB), and PQ201635 (ACT). BLAST analysis revealed 100% identity with Epicoccum sorghinum (MT125854 (544/544 bp), OP555979 (344/344 bp), MK044832 (274/274 bp), respectively). A phylogenetic tree constructed using the Maximum Likelihood method in MEGA6 confirmed that the concatenated sequence (WY01) clustered with E. sorghinum. To satisfy Koch's postulates, healthy Chinese yam (Waxy Yam) leaves were inoculated with WY01 by spraying spore suspension (1 × 10⁶ conidia/mL). Meanwhile, sterile water was used as control. A total of three plants were inoculated. Three leaves from the same plant were used for inoculation, while the other three leaves were used as controls. All inoculated leaves were covered with humidified defatted cotton and clear polyethylene bags for 24 hours and then grown in the field at an average daily temperature of 28°C. Seven days post-inoculation, the leaves inoculated with WY01 developed disease spots similar to those observed initially, whereas the control leaves remained unaffected. The pathogen was reisolated from the inoculated leaves and identified as E. sorghinum, confirming Koch's postulates. While Epicoccum latusicollum has been reported to cause black root (Han et al. 2019) and black leaf spot (Yan et al. 2024) on yam in China, and E. sorghinum has been reported on Zea mays (Chen et al. 2021), white chrysanthemum (Chen et al. 2021), and Paeonia suffruticosa (Yang et al. 2023), this is the first documented case of E. sorghinum causing black leaf spot on Chinese yam (Waxy Yam) in China. The disease mainly breaks out during the plum rain season, leading to a significant decrease in the yield and quality of Waxy Yam if not managed properly. This report might provide the basis for further research to develop effective disease management strategies and mitigate future losses.