First Report of Leaf Blight Caused by Curvularia verruculosa on Zoysiagrass (Zoysia japonica) in Korea

Zoysia japonica Steud. is widely used for turfgrass, particularly on golf courses and teeing areas in East Asia (China, Japan, and Korea). In Korea, zoysiagrass use increased about threefold between 2003 and 2012 (Kang et al. 2016). In October 2016, zoysiagrass showing leaf spot symptoms, with disease incidence of less than 5% of the plants, was observed in a public park (37°20′39″N, 126°56′50″E) in Gyeonggi Province, Korea. Infection began as yellow to brown spots on leaf sheaths. The spots measured 0.1 to 0.3 cm in diameter and often enlarged up to 0.5 cm. Diseased leaf pieces were rinsed in distilled water and placed on potato dextrose agar (PDA) with streptomycin (50 µg/ml), and after 3 days, white mycelia were observed. Pure cultures were obtained from transferring the agar plugs onto corn meal agar (CMA), PDA, and V8 agar (V8A). After 1 week at 25°C, colonies were dark greenish to grayish black, velvety, and measured 2, 5, and 7 cm in diameter, respectively, in the three media. Conidiophores were brown to pale brown, simple but rarely branched, septate, thick-walled, variable in length, and measured 50 to 100 × 3 to 6 µm. Conidia were dark brown, ellipsoidal to fusiform, mostly 3-septate, straight or slightly curved, with a rough verrucose surface, and measured 21 to 36 µm × (9‒) 10 to 15 µm. The terminal cells were smaller, paler, and less verrucose than the central cells. All morphological characteristics fit the description of Curvularia verruculosa Tandon & Bilgrami (Ellis 1966). A representative culture was preserved in the National Institute of Biological Resources (NIBR, Incheon, Korea), with accession no. ZEVCFG0000000014. To support the morphological identification, molecular phylogenetic identification was also performed. Genomic DNA was extracted from conidia and mycelia harvested from the PDA plate. PCR was used to amplify the ITS rDNA region and glyceraldehyde-3-phosphate dehydrogenase (gpd) gene (Berbee et al. 1999). The sequences of the two regions were deposited in GenBank (accession nos. MF959538 for ITS, MF959539 for gpd). A comparison with the GenBank sequences revealed that the present Korean pathogen is identical to C. verruculosa available in GenBank (ITS: HF934909, JN116709, KT923466; gpd: HG779110, HF565480, KP645347). Therefore, both morphology and sequence comparison identified the assumed pathogen as C. verruculosa. Pathogenicity was demonstrated by spraying 5 ml of conidial suspension (1.5 × 10⁶ conidia/ml in sterile water) onto the leaves of healthy zoysiagrass in five pots. Five control pots were sprayed only with sterile water. All pots kept in a plant growth chamber (25°C, 70% relative humidity, and in darkness). Initial symptoms of foliar spots appeared 7 days after inoculation. Within 2 weeks, these spots turned yellow-brownish, followed by leaf dieback and necrosis, from which an identical fungus was successfully reisolated, fulfilling Koch’s postulates. Control plants remained symptomless. Curvularia affects a large number of grasses worldwide, out of which Z. japonica has been listed as a host plant of four Curvularia species in Korea, the United States, and Venezuela (Farr and Rossman 2017), but there was no report of infection by C. verruculosa. To our knowledge, this is the first report of leaf blight caused by C. verruculosa on zoysiagrass in Korea. Although its distribution is restricted at the area where it was first discovered, nationwide disease surveys for preventive phytosanitary and quarantine measures are ongoing.