First Report of Purple Seed Stain Caused by Cercospora cf. flagellaris on Soybean in Korea.

Abstract

In November 2021 and 2022, soybean seeds exhibiting purple staining were collected from Suwon, Gimje, and Yeoncheon in Korea. The symptoms first appeared on aging leaves and eventually covered the entire leaf. Initial infection manifested as small yellow spots that turned light brown or white, accompanied by yellow halos, with visible clusters of conidia at the center. The seeds showed shallow purple discoloration on the seed coat and deep purple staining within the seed. Infected leaves were surface sterilized with 10% chlorine solution and cultured on general isolation media. Colonies appeared white to gray after incubation in a growth chamber at 25°C with a 12-hour photoperiod. Single-spore isolates were transferred to PDA with 6% sucrose, showing a color change to brown or dark shades. Three isolates were selected for identification and pathogenicity tests. Conidiophores were brown, straight or slightly curved, and uniformly wide, forming clusters of 5-12 on the leaf's lower surface, measuring 300-700 μm (average = 560 μm, n = 25). Conidia were hyaline, needle-shaped, straight or slightly curved, and tapered at the base, with 3-15 indistinct septa, measuring 60-240 μm (average = 180 μm, n = 25). DNA was extracted from the three isolates, and sequences of the internal transcribed spacer (ITS) regions 1 and 2, actin (ACT), HIS3, and calmodulin (CAL) were obtained. These sequences were deposited in GenBank (PQ578698, PQ583605, PQ583606, PQ583607 and PQ583608). BLAST search results revealed 99-100% identity with Cercospora flagellaris for ITS and ACT sequences. To fulfill Koch's postulates, the isolates were grown in a growth chamber (25°C, 12-hour photoperiod) for 20 days. Spore suspensions (10^4 spores/ml) with 0.01% Tween 20 were sprayed on Nuriol and Jangol cultivars. Inoculated plants were maintained in a humid environment for 72 hours and then transferred to a greenhouse. Control plants were sprayed with sterilized water and kept under the same conditions. Five weeks post-inoculation, all inoculated plants exhibited purple seed stain symptoms, while control plants remained healthy. The same pathogen was re-isolated from the inoculated plants. Cercospora flagellaris is known to affect a wide range of plant species in various countries, including the USA and Brazil. This is the first report of C. flagellaris causing purple seed stain on soybean in Korea. Soybean is a critical food and oil crop in Korea. Therefore, further studies are needed to assess the epidemiology, cultivar responses, fungicide sensitivity, and management strategies to support the sustainability and future potential of soybean cultivation in Korea.