First report of Olive latent virus 1 infecting tomato in Greece

In winter 2023, tomato fruit (Solanum lycopersicum cv. Esperia F1 hybrid) from a greenhouse in Ierapetra, Crete, Greece exhibited fruit blotches at a rate of 7%. The plants had previously tested positive by RT-PCR for TFBV (Beris et al., 2023) but a sample was analysed with RNA-Seq to investigate the aetiology of the disease. Total RNA was extracted from leaf and fruit tissue with an RNeasy Plant Mini Kit (Qiagen, Germany) and after ribodepletion, was subjected to RNA-Seq using the Illumina Novaseq 6000 platform. The obtained 30 M, paired-end, 100 bp reads were de novo assembled using Geneious (v. 11.1.5). BLASTn analysis of the resulting contigs revealed the presence of Tomato fruit blotch virus (ToFBV, genus Blunervirus) that was recently detected in Greece (Beris et al., 2023) and associated with fruit blotch disease, and Olive latent virus 1 (OLV-1). The genome of OLV-1 consists of a 3.7 kb, single-stranded, positive sense RNA that contains five open reading frames (ORFs). The nearly complete genome of the Greek isolate (GenBank Accession No. OR498642) contained 3654 nucleotides and had 99.7% nucleotide identity with a Chinese isolate from tobacco (MK376952). A primer pair, OLV-1F (5′-TTATTACTGGGATAGTCGAATT-3′) / OLV-1R (5′-GGAATATAAATAAACCGAAGCT-3′), was designed and used in RT-PCR to amplify a 402-bp fragment of the capsid protein gene. The PCR product was Sanger sequenced and the resulting sequence was identical to that obtained from the RNA-Seq analysis. Eleven tomato samples from the greenhouse in Ierapetra were also tested by RT-PCR for the presence of OLV-1 and two were found to be positive. Since ToFBV is not mechanically transmitted, plant tissue derived from the initial tomato sample was used for the isolation of OLV-1 through sap-inoculation in indicator plants, namely Nicotiana benthamiana, N. tabacum cv. Xanthi NC and Solanum lycopersicum cv. Ace 55 VF. At five days post inoculation (dpi) all plants showed local necrotic lesions (Fig. 1) and the presence of OLV-1 in the symptomatic leaves was confirmed by RT-PCR using the primers described above. Systemic infection was only observed and detected in N. benthamiana plants at 10 dpi. The latter showed severe necrosis of the newly expanded leaves and the plant finally collapsed at 14 dpi. OLV-1 has been reported previously from tomato in Poland (Borodynko et al., 2010) but this is the first report of the virus in Greece. OLV-1 belongs to the Alphanecrovirus genus (family Tombusviridae) and was originally detected in olive trees, while its host range also includes tulips, citrus and tomato (Gallitelli & Savino, 1985; Grieco et al., 1996; Kanematsu et al., 2001; Borodynko et al., 2010). Although the virus is not a direct threat to tomato cultivation, its transmission through soil in the absence of a biological vector, its frequent presence in mixed infections and ability to recombine with other members of the Alphanecrovirus genus to give rise to new viral species (Borodynko et al., 2010), support the need for further investigation of OLV-1 spread, epidemiology and impact on tomato cultivation. This work was funded by the European Union- Next Generation EU, Greece 2.0 National Recovery and Resilience plan (action code TAEDR-0535675).