First Report of Curvularia lunata Causing Leaf Spot on Oryza sativa in Sabah, Malaysian Borneo

Rice (Oryza sativa L.) has been farmed in Malaysia since ancient times and is one of the most important commercial crops (Ma'arup et al. 2020). Throughout January to August 2022, chlorotic spots with brown halos ranging 2 to 10 mm wide were found on upper leaves of rice variety Mahsuri in the vegetative stage with a severity and incidence of approximately 60% and 100%, respectively in Kampung Tagas, Sabah, Malaysian Borneo (06°09'41.8"N, 116°13'45.1"E). As the disease developed, the spots coalesced into larger chlorotic spots. Three leaf pieces (5 x 5 mm) were excised from lesion margins, surface sterilized based on Khoo et al. (2022a), before plating on water agar (WA) at 25°C. Purification of fungi was conducted on WA using hyphal tip isolation. When three pure cultures were obtained, the fungi were cultured on potato dextrose agar (PDA) and WA for 7 days in 12 h light and 12 h dark at 25°C for the macro- and micro-morphological characterization, respectively. The colonies of the three isolates on PDA were initially gray, later becoming dark. Conidia (n=30) were fusiform, smooth-walled, dark-brown, and melanized with three transverse septa, measuring 7.3 to 11.4 × 16.2 to 27.2 µm. The isolates were named Tagas01, Tagas02, Tagas03. Genomic DNA was extracted from fresh mycelia of the pathogens based on the extraction method described by Khoo et al. (2022b). The primers ITS1/ITS4 (White et al. 1990), GPD1/GPD2 (Berbee et al. 1991), and EF1-983F/EF1-2218R (Schochet al. 2009) were used to amplify the internal transcribed spacer (ITS) region of rDNA, partial fragments of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and translation elongation factor (EF-1α) region, respectively based on PCR conditions as described previously (Khoo et al. 2022a). The sequences were deposited in GenBank under accession numbers OP268402, OP271304, OP271305 (677/677 bp) (ITS), OP270699, OP270703, OP270704 (609/613 bp) (GAPDH), OP270700-OP270702 (928/930 bp) (EF-1α). They were 99.35-100% similar to the Curvularia lunata ITS (HF934911), GAPDH (LT715821), and Curvularia dactyloctenicola EF-1α (MF490858) type sequences. Although C. dactyloctenicolais related to C. lunata, the conidia of the former are much wider making them easier to differentiate (Marin-Felix et al. 2017). Phylogenetic analysis using maximum likelihood based on the combined ITS, GAPDH and EF-1α sequences indicated that the isolate formed a supported clade to C. lunata. The pathogens were identified as C. lunata based on morphological and molecular characterization. Koch's postulates were performed. Three replicate healthy rice at the vegetative stage were sprayed with a spore suspension of 1 × 106 spore/ml in distilled sterilized water, prepared from 1-week-old fungal culture, grown in the dark on WA. Three replicate rice plants were sprayed with distilled sterilized water as control. Plants were covered with transparent polyethylene bags to keep moisture, and kept in a greenhouse at ~27°C. Bags were removed after 4 days of incubation. Monitoring and incubation were performed in greenhouse based on Iftikhar et al. (2022). The pathogenicity test was also performed using isolate Tagas02 and Tagas03. All inoculated leaves developed symptoms as described after 6 days post-inoculation, whereas no symptoms occurred on controls. The experiments were repeated twice. The reisolated fungi were identical to the pathogen morphologically and molecularly, thus fulfilling Koch's postulates. C. lunata has been reported in Peninsular Malaysia (Lee et al. 2012). This is the first report of C. lunata causing leaf spot on Oryza sativa in Sabah, Malaysian Borneo. This illness not only reduces yields and lowers milling quality, but it may also be mistaken for rice blast, necessitating needless fungicide spraying.