First Report of Curvularia oryzae Causing Leaf Spot of Coconut Seedling in China

Abstract

Coconut (Cocos nucifera L.) is one of the most important perennial tropical crops of southern China. In June 2018, a new leaf spot disease was detected in nearly 100% of 1-year-old golden coconut seedlings at the nursery base in Qionghai, Hainan. At the beginning of the disease, the leaves exhibited water-stained chlorotic spots; as the symptom progressed, the edges of the lesions appeared orange-yellow to pale yellow-brown. Lesions were long oval, almost round, and sometimes irregular with dark brown center and distinct yellow halos. As the lesions continued to expand, several lesions coalesced forming a taupe irregular large spot. The center of the lesion was grayish white, and distinct dark brown necrotic lines were observed at the edges. Under high humidity conditions, thin gray-white mycelia were observed. Overall, 10 leaves with typical symptoms were randomly collected. Small tissue sections (5 × 5 mm) were cut from the lesion margin and surface sterilized in 75% ethanol for 30 s, following which the tissue surface was disinfected with 0.1% HgCl₂ for 2 min, washed thrice with sterile distilled water, plated on potato dextrose agar (PDA), and incubated at 28°C. Five days after incubation, fast-growing fungal colonies with gray-white mycelia were observed, which then changed to dark green with a neat colony edge on PDA medium. Conidiophores were terete, straight or slightly curved, unbranched, often nodular, dilated, and light-to-moderately brown with smooth outer walls. Conidia were 27.1 to 43.3 × 19.0 to 23.4 µm (average 38.1 × 20.1 µm, n = 100), terminal, ovoid, obclavate, and three-distoseptate. The second cell at the base was the largest, taupe to dark brown in color, and often swollen; cells at both ends were slightly smaller and light in color. Based on morphological characteristics, the fungal isolates were preliminarily identified as Curvularia oryzae (Sivanesan 1987). Single-spore isolates were cultured on the PDA medium, and three representative strains were selected for DNA extraction. The identity of the isolates was confirmed via sequence analysis of the internal transcribed spacer (ITS) gene amplified using primers ITS1/ITS4 (White et al. 1990), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene amplified using gpd1/gpd2 (Berbee et al. 1999), and translation elongation factor 1-alpha (EF1a) gene amplified using 983F/2218R (Schoch et al. 2009); sequence data were deposited in GenBank (MN180224, MN180225, and MN481482). BLAST analysis demonstrated that these sequences were 99 to 100% similar to the ITS (MG238513), GAPDH (HG779156), and EF1a (KM196590) sequences of C. oryzae. Moreover, phylogenetic tree analysis using ClustalX and MEGA 7.0 software with the maximum likelihood method placed the isolates in the clade of C. oryzae (CBS169.53) with 98% bootstrap support. Based on the morphology, sequence data, and phylogenetic analysis, these isolates were determined as C. oryzae. To validate the results a pathogenicity test was performed. Twenty leaves were divided into two groups; the first group was sprayed with spore suspension (1 × 10⁶/ml) of the isolate, whereas the other group was sprayed with sterile water as a control. All plants were incubated at 28 ± 2°C and 100% relative humidity. Ten days after inoculation, leaves sprayed with the spore suspension exhibited the typical symptoms with 100% of leaf spot similar to the original samples, whereas leaves sprayed with sterile water showed no symptoms. Further, C. oryzae was reisolated from the leaf spot. Pathogenicity tests were repeated thrice with the same results. Koch’s postulates were achieved by the reisolation of C. oryzae from the inoculated leaves. C. oryzae has previously been reported to cause leaf spot of oil palm seedlings in Thailand (Sunpapao et al. 2014). To the best of our knowledge, this is the first report of leaf spot of coconut seedlings caused by C. oryzae.