First Report of Curvularia pseudobrachyspora Causing Leaf Spots in Areca catechu in China

The areca palm (Areca catechu L.) is among the most important crops grown in Southeast Asia, with a planting area of over 100,000 ha in Hainan, China. In July 2017, we observed black-brown leaf spots on areca leaves in Wanning, Hainan Province, China. Although only small-scale occurrence of this disease has been observed to date, it causes serious symptoms in the areca palm. Initial symptoms appeared as 2- to 4-mm oblong, dark red lesions on lower leaves. Lesions reached 6.0 mm and gradually developed into spindly, dark brown spots. The internal area of these lesions was shallow, medium-dark brown, and had a dark brown edge and yellow halo. Over time, the necrotic spots enlarged, gradually turned black, and then coalesced to form larger necrotic areas. To isolate the pathogen, leaf surfaces were sterilized by wiping with 75% (v/v) ethanol. Leaf samples (3.0 × 5.0 mm) were cut from borders between symptomatic and healthy tissues. Diseased tissues were transferred onto potato dextrose agar (PDA) medium supplemented with 0.01% streptomycin sulfate and incubated at 28°C for 4 days. Colonies grown from these tissues were dark with even, villous edges. Conidia were brown, smooth, fusiform or hook-shaped, and measured 18 to 25 μm long × 8.0 to 12.0 μm wide. The conidia had three to four transversal septa, with the middle one to two cells larger and darker than the others, which were borne from the apex of geniculate conidiophores singly or in groups, showing very similar characteristics to Curvularia pseudobrachyspora (Manamgoda et al. 2012; Marin-Felix et al. 2017). Pathogenicity tests were performed on new leaves of healthy areca seedlings (0.6 to 0.8 m in height). Healthy leaves were rinsed with sterilized water and gently punctured with a sterile pin. Five mycelial disks taken from the edges of 4-day-old PDA cultures were placed on each wounded leaf, covered with wet sterile blotting paper, and wrapped in a plastic bag. As a control, PDA agar was placed on leaves of the same seedlings. Three areca seedlings were inoculated, and the symptoms were observed after 15 days. Although no symptoms appeared on the control leaves, typical symptoms (i.e., circular brown lesions surrounded by yellow halos) were observed on leaves inoculated with the isolate. The pathogen was reisolated from symptomatic leaves. The ribosomal internal transcribed spacer (ITS) region, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and translation elongation factor 1-alpha (EF1a) were amplified using the primers ITS5p and ITS4 (White et al. 1990), gpd1 and gpd2 (Berbee et al. 1999), and 983F and 2218R (Schoch et al. 2009), respectively. The combined ITS (MH516132), GAPDH (MH516133), and EF1a (MH516134) sequences were compared with published sequences of 49 Curvularia isolates and six Bipolaris isolates by phylogenetic analysis. The HNWN001 isolate from Areca in Hainan showed 100% similarity with an isolate (CPC 28808) of C. pseudobrachyspora from Eleusine indica but slight difference from other Curvularia isolates (Manamgoda et al. 2012). Surprisingly, HNWN001 showed distinct difference from a C. oryzae strain isolated from oil palm (CBS169.53). Based on morphology, pathogenesis, and multigene sequence analysis, the causal agent of leaf spots on A. catechu was identified as C. pseudobrachyspora. This is the first report of C. pseudobrachyspora causing leaf spots on areca in China and worldwide.