First Report of Tomato yellow leaf curl virus Infecting Eustoma (Eustoma grandiflorum) in Korea.

Eustoma (Eustoma grandiflorum), also called lisianthus, belongs to the family Gentianaceae and is cultivated for flower production globally (1), including in Korea. At least 10 viruses can infect eustoma, including Cucumber mosaic virus (genus Cucumovirus), Tobacco mosaic virus (genus Tobamovirus), Tomato spotted wilt virus (genus Tospovirus), and Tomato yellow leaf curl virus (TYLCV, genus Begomovirus) (1,2). In December 2012, disease symptoms such as leaf curling and stunting were observed on eustoma plants grown in Gumi, Korea, where TYLCV outbreak was reported on tomato farms. In a eustoma greenhouse, about 5% of eustoma plants showed the leaf curling and stunting symptoms. Total DNA was isolated from 15 symptomatic eustoma plants with a Viral Gene-spin Viral DNA/RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea) and viral DNA was amplified by rolling circle amplification (TempliPhi Amplification Kit, GE Healthcare Life Sciences, Uppsala, Sweden) following the manufacturer's instructions. All amplicons were digested with the restriction enzyme SacI (TaKaRa Bio, Shiga, Japan) and 2.8-kb DNA fragments were verified on an agarose gel. Fifteen digested DNA fragments were purified from the gel, ligated into pGEM-T easy vector (Promega, Madison, WI), and sequenced (Macrogen, Seoul, Korea, GenBank Accession No. KF225312.1). A BLAST search exhibited a 99% identity to TYLCV previously reported in Korea (GenBank HM856911.1). This is the first report of TYLCV in eustoma plants in Korea. To identify the movement and replication of TYLCV in infected eustoma plants, PCR and Southern hybridization analysis were performed with samples from four organs (flower, leaf, stem, and root) of three individual TYLCV-infected plants. TYLCV TYL DNA from each organ sample was amplified using 2× Taq PCR MasterMix (Bioneer, Daejeon, Korea) with TYLCV-specific primers (TYLCV-F: 5'-ATATTACCGGATGGCCGCGCCT-3', CV-R: 5'-TCCACGGGGAACATCAGGGCTT-3'). Single-stranded as well as double-stranded TYLCV DNA were identified from all organs of symptomatic eustoma, indicating TYLCV can replicate and move systemically in eustoma plants. Whitefly (Bemisia tabaci)-mediated plant-to-plant viral transmission was performed with one TYLCV-infected eustoma plant and five healthy eustoma plants and revealed that 80% (4 of 5) of the eustoma plants were infected by whitefly-mediated transmission. These results indicate that TYLCV-infected eustoma plants could act as virus reservoirs to healthy eustoma plants as well as other potential TYLCV hosts, such as tomatoes. In Korea, TYLCV has been the most notorious plant virus since 2008 (3), but, until now, TYLCV infection in eustoma plants has not been reported in Korea. References: (1) C. C. Chen et al. Plant Dis. 84:506, 2000. (2) A. Kritzman et al. Plant Dis. 84:1185, 2000. (3) H. Lee et al. Mol. Cells 30:467, 2010.