First Report of Leaf Spots on Oat Caused by Alternaria Alternata in South Korea

Oat, a gluten-free source of vitamins, minerals, fiber, and antioxidants required for human health (Rasane et al., 2015), is increasingly being cultivated in South Korea (Ju et al. 2011). In May 2020, ~30 % Avena sativa (cv. Samhan) leaves of a commercial oat field in Jeongeup, Korea (35.3859°N, 126.5607°E), displayed a leaf spot disease at the tillering growth stage. Small, brown spots appeared initially on the lower leaves, but progressed to dark-brown oval or irregular, necrotic spots with a light-yellowish border. To isolate the pathogen, tissue excised from growing edges of about 50 lesions was surface sterilized (immersion in 70 % ethanol then 1 % sodium hypochlorite), rinsed and transferred to potato dextrose agar. Five isolates were purified using the single-spore culture method on water agar plates (Choi et al. 1999). Conidia from the PDA cultures (n = 50) were elongated, ellipsoidal, and obclavate, with one to five septa (primarily transverse), 18 to 35 μm × 7 to 12 μm. The morphological characteristics of the fungus from each culture were consistent with those observed for Alternaria alternata (Chen et al. 2020; Simmons 2007). To confirm the identity of the fungal pathogen, the internal transcribed spacer (ITS) region was amplified with universal primers ITS1/ITS4 and sequenced (White et al. 1990). BLAST analysis of the resulting nucleotide sequence of one representative isolate, Oaa-2 (GenBank MT786424), showed 100 % identity with A. alternata-type strains, such as CBS916.96. Further, nine nuclear protein-coding genes from the Oaa-2 isolate were amplified using previously described primers (Lawrence et al. 2013; Wouderengerg et al. 2015). These sequences were deposited under the following GenBank accession numbers: MT795660 (rpb2), MT795656 (His), MT795657 (CML), MT795658 (ACT), MT787343 (CHSD), MT795655 (GAPDH), MT795661 (endoPG), and MT795662 (TEF1). The nine sequences of the Oaa-2 isolate showed 99%-100 % identity to those of A. alternata-type strain CBS916.96 in NCBI. Fig. S2 shows the phylogenetic trees for the ITS and GAPDH sequences indicating high homology. Pathogenicity of this isolate was confirmed by spraying leaves of surface-sterilized, glasshouse-grown, "Samhan" oats with suspensions (5 × 106 spores/ml) of Oaa-2 conidia. Experimental seedlings were grown for 2 weeks in a chamber at 20 ± 3 °C in 60 % relative humidity with a 10-h light/14-h dark cycle (200 µmol/m2/s). Conidial suspensions were prepared for inoculation by flooding with sterile water, followed by filtering the suspension through three layers of sterile cheesecloth to remove debris. Three pots each containing twenty oat seedlings were sprayed with the Oaa-2 isolate conidial suspension (5 × 106 conidia/ml) and incubated for 10 days at 20 ± 3 °C in 100 % humidity with a 10-h photoperiod. Three healthy seedlings were sprayed with sterile water as a control. After 10 days, dark brown spots developed in the inoculated plants; however, no spots were observed in the controls. Fungi with the same conidial characteristics as Oaa-2 were isolated from the infected plants, and the ITS sequence and the nine nuclear protein-encoding genes of these plant isolates was identical to those of Oaa-2. Pathogenicity tests also were conducted with isolate Oaa-2 with two oat cultivars, Sunyang and Joyang, with the same result. To our knowledge, A. alternata has not been previously reported to cause disease on oats in South Korea, although it does cause economic losses in China (Chen et al. 2020).