First Report of Corynespora cassiicola Causing Target Spot on Soybean in Taiwan

Soybean (Glycine max [L.] Merr.) is an important crop in Taiwan. In October 2020, an unknown leaf spot disease was counted (n = 100) to occur over 70% of soybean cultivar 'Hualien No.1' in the Shoufeng Township of Hualien County, eastern Taiwan. Initial symptoms on leaves as tiny lesions approximately 3 mm in diameter, which later enlarged and developed into round, irregular, and reddish-brown spots with concentric rings surrounded by a yellowish halo. The symptoms appeared on both young and old leaves, but rarely on the stem or pods. The lesions at the margin of healthy and infected tissues were surface-disinfested in 1% NaOCl for 30 seconds, washed twice in sterilized distilled water, dissected and plated on potato dextrose agar (PDA) to isolate the potential pathogen. Colonies on PDA exhibited light to dark brown color at 24°C with 12-hours light after 7-days incubation. The average growth rate was 3 mm per day. Conidia were light brown in color and obclavate to cylindrical in shape. The size of a conidium was measured with an average of 110.8 ± 28.2 μm in length and 15.2 ± 2.8 μm in width, typically with 3 to 18 septa (n = 50). To confirm the pathogenicity of this fungus, conidial suspension (104 conidia/mL) of two isolates, HL_GM-6 and HL_GM-7, were sprayed on the healthy leaves of 4-weeks-old soybean. Plants sprayed with sterile distilled water were used as a control. After inoculation, the plants were covered with plastic bags to maintain a high humidity for 24 hours before moving into a greenhouse with a condition of 20 to 25°C and relative humidity of 75 to 80%. After 7 days of inoculation, foliar symptoms began to appear and which were identical with the field observations. To complete the Koch's postulates, pathogen isolation was attempted and the identical fungus was retrieved from the foliar spots of the inoculated leaves. The foliar symptoms as well as the morphology of the conidiophores and conidia suggested the pathogen to be Corynespora cassiicola (Ellis et al. 1971). Molecular characterization was performed using the sequences of internal transcribed spacer (ITS) region of rDNA, actin (act1), tubulin, and translation elongation factor 1 alpha (tef1) genes after a PCR with ITS1/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn, 1999), BT2a/Bt2b (Udayanga et al. 2012), EF1-728F/EF1-986R (Udayanga et al. 2012), respectively. BLASTN sequence analyses of the ITS, act1, tubulin, and tef1 genomic regions of the isolate HL_GM-7 (GenBanK accessions MW548097 MW961420, MW961419 and MW961421) showed high similarity with the isolates of C. cassiicola including 99.58% with sequence KF810854 (Deon et al. 2014), 99.11% with FJ853005 (Dixon et al. 2009), 99.34% with MH763700 (Duan et al. 2019), and 99.33% with KY112719 (Zhang et al. 2018) respectively. Based on the morphology, pathogenicity, and sequence results, this study becomes the first report of C. cassiicola causing target spot on soybean in Taiwan. C. cassiicola is known to infect a broad host range (Dixon et al. 2009; Lopezet al. 2018), and it has been found to infect tomato, cucumber, papaya, and Salvia miltiorrhiza in Taiwan (Lu et al. 2019; Tsai et al. 2015). Therefore, the emergence of soybean target spot should be aware to avoid potential damage to soybean production in Taiwan.