First report of impatiens flower break virus infecting impatiens walleriana in south Korea

Abstract

Impatiens walleriana Hook. f. (Balsaminaceae), also known as busy Lizzie, balsam, or simply impatiens, is a flowering herbaceous perennial and very popular annual bedding plant. In June 2011, I. walleriana with virus-like symptoms-mottling and mosaic leaves on dwarfed plants-was observed in Asan-si Chungcheongnam-do, South Korea. To confirm the presence of virus, symptomatic leaves were tested for five viruses (Cucumber mosaic virus, Tobacco ringspot virus, Pepper mild mottle virus, Pepper mottle virus, and Tomato spotted wilt virus) in separate reverse transcription (RT)-PCR assays (Choi et al. 2015; Kim et al. 2011; Lee et al. 2015) using virus-specific primers. After extracting total RNA from symptomatic leaves using TRI Reagent (Molecular Research Center, OH), amplification was performed using a One-Step RT-PCR kit (Qiagen, Hilden, Germany); however, negative results were obtained even after repeated attempts. To identify the potential causative agent, total RNA from symptomatic leaves was sequenced on an Illumina HiSeq2500 next-generation sequencing platform (Theragen Bio Institute, Suwon, South Korea) as described in Lim et al. (2015), with the raw sequencing data processed by SeqGenesis (Daejeon, Korea). A de novo assembled contig of 9,409 bases was identified as the nearly complete sequence of Impatiens flower break virus (IFBV). The sole IFBV isolate (GenBank accession no. AY864851) from New Guinea impatiens in the United States (partial sequences) shared 99% identity, with 68% coverage. We next performed rapid amplification of 5′- and 3′- complementary DNA ends (RACE) using the 5′-/3′- RACE system (Invitrogen, Calsbad, CA) with primers IFBV-Asan_5R1 (5′-GTTCCACACTTCATGAGGGA-3′), IFBV-Asan_5R2 (5′-CCTTACAATTGTAGGTCGCT-3′), and IFBV-Asan_3R (5′-ATGAGGAACTCTGAAGGACT-3′). All PCR products were cloned into a T&A cloning vector (RBC Bioscience, Taipei, Taiwan), and the sequences of the clones were analyzed by Macrogen (Daejeon, South Korea). The complete genome sequence of this new isolate, which was 9,622 nucleotides long, was deposited in GenBank as IFBV isolate Asan (KU981084). IFBV belongs to the genus Potyvirus, which may cause significant losses in agricultural, horticultural, and ornamental crops (Gibbs et al. 2008). To confirm the host range of IFBV-Asan, sap from IFBV-Asan-infected I. walleriana was inoculated onto test plants, including Nicotiana tabacum, Capsicum annuum, Cucumis melo, Chenopodium quinoa, and I. balsamina. Two weeks after sap inoculation, only I. balsamina developed symptoms (mild mosaic and dwarfing). All inoculated plants were tested by RT-PCR with the following primers: IFBV CP 149_F (5′-GAACAATAGGGCGTGTCGTG-3′) and IFBV CP448_R (5′-TGCCATCCATCATCACCCAC-3′) and confirmed infection of IFBV only to tested I. balsamina. Because many potyviruses are transmitted by aphids and also via seeds, IFBV can potentially be transmitted to different agricultural or horticultural crops to cause problematic disease. To the best of our knowledge, this is the first report of the full genome sequence of IFBV derived from I. walleriana and the first report of IFBV in South Korea