First Report of Verticillium dahliae causing Verticillium Wilt on Radish in South Korea.

Abstract

Radish (Raphanus sativus L.) is an important crop grown in Korea primarily for its use in kimchi, and is cultivated on an area of about 23,000ha. In September 2016, radish leaves were observed with yellowing and wilting symptoms in Gangneung (37.55406°N, 128.84871°E) and Jeongseon (37.42895°N, 128.85882°E), Gangwon province, South Korea. Disease incidence was estimated at approximately 10% in two fields, respectively. About 30% of radish plants (cv. Gwangdongyeoreum) with foliar symptoms exhibited vascular discoloration in the roots. Small pieces of discolored root tissue were surface sterilized in 1% NaOCl for 30s, and then rinsed in sterile water. The tissue pieces were placed on water agar and incubated at 25°C for 10 days. Eight isolates were obtained through single spore isolation, and a representative isolate NC16557 (from Gangneung) was selected for identification. After 14 days, colonies on potato dextrose agar (PDA) were 3.7 cm in diameter and becoming dark due to the formation of microsclerotia. Aerial hyphae were smooth-walled, 3 to 4 μm wide. Conidiophores were erect or slanted, verticillately branched or unbranched, and hyaline. Conidia were hyaline, smooth-walled, non-septate, cylindrical to oval, 4.8 to 6.6 × 2.6 to 3.4 μm (mean 5.7 × 2.9 μm, l/w =2.0, n =80). Microsclerotia were immersed in the agar, composed of rounded, brown-pigmented cells and elongate or irregular in shape, 42 to 60 μm diam. Based on morphology, NC16557 isolate was tentatively identified as Verticillium dahliae (Hawksworth and Talboys, 1970; Isaac, 1967). To confirm taxonomic placement, DNA extracted from mycelia of the same isolate was PCR amplified and sequenced targeting internal transcribed spacer (ITS) regions of rDNA, translation elongation factor 1 alpha (TEF), actin (ACT), tryptophan synthase (TS), and glyceraldehyde-3-phosphate dehydrogenase (GPD) genes (Inderbitzin et al., 2011). The sequences were deposited in GenBank with accession numbers MZ723402, and MZ735720-MZ735723, respectively. The sequencing results showed 100% (ITS=479/479, TEF=500/500, ACT=185/185, and TS=295/295) and 99.61% (GPD=257/258) similarity with the sequences of V. dahlia type strain PD322 (LR026889, HQ414624, HQ206921, HQ414909, and HQ414719) by BLAST. Based on the morphology and multigene sequence analysis, the isolate was identified as V. dahliae. Pathogenicity of two isolates (NC16557 and NC16547) was carried out in the greenhouse using ten 10-day-old seedlings (cv. Gwangdongyeoreum) by root-tip cutting and then soaking the roots in a fungal spore suspension of 106 conidia mL-1 for 1 hour. Ten seedlings were treated with sterile distilled water as a control. All plants were placed in the greenhouse at 15°C (night)/25°C (day) with natural light. After 6 weeks, all inoculated plants showed vascular discoloration in the roots while control plants remained asymptomatic. However, the above-ground symptoms of inoculated plants, such as yellowing and wilting, were indistinguishable from control plants. V. dahliae was consistently re-isolated from the symptomatic root tissues and the pathogen identity was confirmed by observing morphological characteristics. Verticillium wilt of radish has been reported from China(Yan et al., 2019). In Korea, this is the first report that V. dahliae causes Verticillium wilt of radish, although Dumin et al. (2020) already reported on Verticillium wilt of Chinese cabbage in Gangwon province, the main production area of Chinese cabbage and radish in summer. With these findings in Chinese cabbage and now radish, it will be critical to identify or develop Verticillium-resistant varieties and other management strategies for the stable production of these crops in Korea.