A Lycopersicon esculentum (tomato) plant from a commercial property in New Zealand was submitted to the Investigation and Diagnostic Centre for diagnosis in 2003. Fruits had faint yellow ringspots but no obvious symptoms were observed on leaves. No virus particles were observed from tomato and symptomatic herbaceous plants crude sap preparations. Mechanically inoculated Nicotiana clevelandii and N glutinosa developed systemic chlorosis, whereas pinpoint necrotic local lesions were observed on Chenopodium amaranticolor. Chlorotic local lesions were also observed on C. quinoa followed by systemic necrosis. No symptoms were observed on Cucumis sativus, Gomphrena globosa, N. benthamiana, N. sylvestris, or N. tabacum cv. White Burley. Total RNA was extracted from N. glutinosa and C. quinoa leaf samples using the Qiagen (Qiagen Inc., Valencia, CA) Plant RNeasy Kit. Reverse transcription (RT) was carried out by using random hexamer primers and SuperScript II reverse transcriptase (Invitrogen, Frederick, MD) followed with PCR using broad-detection primers targeting the genera Carmovirus, Dianthovirus, Ilarvirus, Tospovirus, (Agdia Inc., Elkhart, IN) and Tombusvirus (2). A positive RT-PCR amplification was obtained only with Ilarvirus primers. The 450-bp product (GenBank Accession No. DQ457000) from the replicase gene had a 97.4% nt and 98.6% aa identity with Spinach latent virus (SpLV; Accession No. NC_003808). An RT-PCR protocol was developed for the specific detection of SpLV. Primers were designed from three SpLV RNA sequences (RNA1: NC_003808; RNA2: NC_003809; RNA3: NC_003810) using the Primer3 software (3). Primers SpLV-RNA1-F (5′-TGTGGATTGGTGGTTGGA-3′) and SpLV-RNA1-R (5′-CTTGCTTGAGGAGAGATGTTG-3′) anneal to the replicase gene from nt 1720 to 2441. Primers SpLV-RNA2-F (5′-GAACCACCGAAACCGAAA-3′) and SpLV-RNA2-R (5′-CCACCTCAACACCAGTCATAG-3′) bind to the polymerase gene from nt 603 to 1038. Primers SpLV-RNA3-F (5′-GCCTTCATCTTTGCCTTTG-3′) and SpLV-RNA3-R (5′-CATTTCATCTGCGGTGGT-3′) amplify the movement protein gene from nt 724 to 936. The predicted amplified product sizes were 722, 436, and 213 bp from RNA1, RNA2, and RNA3, respectively. RT was carried out as described above. PCR was performed in a 20-μl reaction containing 2 μl cDNA, 1× Taq reaction buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 μM of forward and reverse primers, and 1 U Taq polymerase (Promega, Madison, WI). The PCR amplification cycle was identical for the three primer pairs: denaturation (95°C for 3 min) followed by 37 cycles of 95°C (20 s), 60°C (30 s), and 72°C (30 s) with a final elongation step (72°C for 3 min). The amplified products were analyzed by gel electrophoresis, stained with SYBR Green, and their identities confirmed by sequencing. The tomato sample was grown from seed imported from the Netherlands where SpLV occurs (4). The virus is of potential importance for the tomato industry because of its symptomless infection and high frequency of seed transmission in many plant species (1,4). SpLV has never been detected in other submitted tomato samples. Consequently, SpLV is not considered to be established in New Zealand. To our knowledge, this is the first report of SpLV in tomato.
References: (1) L. Bos et al. Neth. J. Plant Pathol. 86:79, 1980. (2) R. Koeing et al. Arch. Virol. 149:1733, 2004. (3) S. Rozen and H. Skaletsky. Page 365 in: Bioinformatics Methods and Protocols. Humana Press, Totowa, NJ, 2000. (4) Z. Stefenac and M. Wrischer. Acta Bot. Croat. 42:1, 1983.
