First Report of Chrysanthemum stem necrosis virus on Chrysanthemum morifolium in Korea.

Chrysanthemum (Chrysanthemum morifolium), one of the most important ornamental cut flower crops in the world, is cultivated on more than 540 ha in South Korea. In September 2013, necrosis on stems, chlorotic and necrotic ring spots on leaves, and leaf distortions were observed on chrysanthemum plants (cv. Jimba) in a greenhouse (~60% incidence) in Changwon, Korea. Symptoms on the diseased chrysanthemum plants were similar to those caused by Tomato spotted wilt virus (TSWV) on chrysanthemum (Chung et al. 2006). Thrips (Frankliniella occidentalis) were observed on symptomatic plants, so each symptomatic leaf of five different chrysanthemum plants was tested using TSWV-immunostrip kits (Agdia, Elkhart, U.S.A.). All samples were negative for TSWV. To identify a causal virus, each symptomatic leaf sample of five different chrysanthemum plants was analyzed using transmission electron microscopy of leaf dip preparations. Tospovirus-like spherical particles that were 80 to 100 nm in diameter were found in all symptomatic leaf samples. Samples of the five infected leaves were individually ground in 0.01 M phosphate buffer (pH 7.0) and mechanically inoculated onto six replicate plants of each species of Capsicum annuum, Chenopodium quinoa, Chrysanthemum morifolium (cv. Jimba), Datura stramonium, Nicotiana benthamiana, N. tabacum (cv. Samsun), Physalis floridana, and Solanum lycopersicum. All inoculated plants displayed local lesions in the inoculated leaves 4 to 5 days post inoculation (dpi). Subsequently, N. benthamiana and D. stramonium showed systemic symptoms typical of Tospovirus infection 10 dpi. All inoculated plants, except for these plant species, did not show systemic symptoms, as reported previously (Momonoi et al. 2011). Each systemic leaf of the five infected chrysanthemum, the six infected N. benthamiana, and the six infected D. stramonium plants was tested with Chrysanthemum stem necrosis virus (CSNV) antiserum (DSMZ, Germany) by DAS-ELISA. All samples reacted strongly with the antisera, suggesting that the plants were infected with CSNV. To further confirm these results, total RNA was extracted from a naturally infected chrysanthemum, and from each six infected indicator plants, D. stramonium, and N. benthamiana and tested using RT-PCR with primers CSNV-Up1 and CSNV-Lo1 specific to CSNV (Boben et al. 2007). Approximately 360-bp RT-PCR products were amplified from all the tested plant species. Subsequently, the amplification products of ~360 bp were purified, cloned, and sequenced. The virus was identified as CSNV (the Korean isolate was named as CSNV-Kr). A multiple alignment of the obtained CSNV sequence (accession no. LC126118) with a reference CSNV isolate (NC_027719) using MEGA6.0 showed 98.3% nucleotide identity. Taken together, these results clearly show that CSNV-Kr is the authentic cause of the disease in chrysanthemum (cv. Jimba) plants. CSNV may pose a major threat for production of chrysanthemum plants since chrysanthemum is produced as vegetative propagation and the farming of chrysanthemum plants is continuously performed in Korea. To our knowledge, this is the first report of CSNV in Korea. © The American Phytopathological Society.