First Report of the Begomovirus Tomato yellow vein streak virus Infecting Tomato in Uruguay

During 2013, a survey of greenhouse tomato plants with virus-like symptoms was conducted in Salto (Northwestern Uruguay), the main greenhouse tomato-producing area in Uruguay. High populations of the whitefly Bemisia tabaci (Hemiptera:Aleyrodidae) were present, with a number of tomato plants displaying mosaic and slight crumpling in young leaves. Leaf samples were collected from six symptomatic plants from three greenhouses (two plants were sampled per greenhouse) as well as from asymptomatic (putative healthy) plants used as negative controls. Total DNA was isolated from each sample and subjected to PCR using PAL1v1978/PAR1c496 primer set (Rojas et al. 1993), since begomovirus (genus Begomovirus, family Geminiviridae) infection was suspected. Amplification of the expected 1,100-bp fragment was observed in all samples from symptomatic plants whereas no such amplification was observed in samples from asymptomatic plants. The PCR fragments were directly sequenced, and 98 to 99% nucleotide sequence identity was found among the six sequences obtained. BLAST sequence analysis revealed ca. 94% identity to the sequence of the begomovirus Tomato yellow veins streak virus (ToYVSV) reported from Argentina (GenBank Accession No. KJ413253). DNA from one of the samples (SLT_43) was used for rolling-circle amplification (RCA) using ϕ29 DNA polymerase (TempliPhi kit, GE Healthcare, Little Chalfont, UK). Restriction pattern of the RCA product using HpaII suggested a bipartite nature for the genome of this begomovirus yielding four fragments that total approximately 5.1 kbp in size (1.9, 1.8, 0.8, and 0.6 kbp each). Putative DNA-A and DNA-B genome components were then cloned using RCA products digested with PstI and BamHI endonucleases that have single-cleavage sites in each component, respectively. The corresponding inserts for each component were cloned and completely sequenced (KR024026 and KR024027, respectively). The cloned components exhibited the typical genome organization of New World bipartite begomoviruses (Brown et al. 2012), with a 160-nt common region exhibiting 98% identity between A and B components and indicating that both represent a cognate pair. The DNA-A sequence was aligned with available begomovirus sequences from the GenBank database using MUSCLE, and pairwise identity scores were calculated using SDT (Sequence Demarcation Tool) (Muhire et al. 2014). The analysis showed that the DNA-A sequence displayed maximum (96%) nucleotide sequence identity with that of a ToYVSV tomato isolate from Argentina (KJ413253). Similar analysis for DNA-B also indicated maximum (89%) sequence identity with that of several isolates of ToYVSV available in databases. Therefore, based on the latest species demarcation criteria for begomoviruses (Brown et al. 2015), the characterized virus is an isolate of ToYVSV. To our knowledge, this is the first report of this virus infecting tomato in Uruguay. © 2016 The American Phytopathological Society.