First report of Tobacco necrosis virus D infecting tomato in Greece

In winter 2023 fruit blotch symptoms (Beris et al., 2023) were observed in tomato samples (Solanum lycopersicum cv. Esperia F1 hybrid) from a greenhouse in Ierapetra (Crete, Greece). To identify the causal agent of the disease, two tomato samples were individually analysed with RNA-Seq. Total RNA was extracted with an RNeasy Plant Mini Kit (Qiagen, Germany) from leaf and fruit tissues and after ribodepletion, it was analysed using an Illumina Novaseq 6000 platform. The analysis of the obtained 30 M, paired-end, 100 bp reads with Geneious (v. 11.1.5) and the subsequent annotation of the resulting contigs with BLASTn revealed the presence of Tomato fruit blotch virus (ToFBV, genus Blunervirus) associated with fruit blotch disease (Beris et al., 2023), Olive latent virus 1 (OLV-1, genus Alphanecrovirus) and Tobacco necrosis virus D (TNV-D) in both samples. The nearly complete genome of the Greek TNV-D isolate (GenBank Accession No. PP580073) consisted of 3757 nucleotides and had 98.4% nucleotide identity with a Dutch isolate from cucumber (ON013905). For the amplification of a 334 bp fragment of the RNA genome, an RT-PCR assay was developed using the TNVD-1F (5′-CTCCTCCAGTTTTCTGGGGCATG-3′) / TNVD-1R (5′-CTGCGACAATGATAATAACAATGATCC-3′) primer pair. The resulting PCR product was Sanger sequenced and the sequence was identical to that obtained from the RNA-Seq. Plant tissue derived from the initial tomato sample was used for the isolation of TNV-D through sap inoculation in indicator plants, namely Nicotiana tabacum cv. Xanthi NC, Solanum lycopersicum cv. Ace 55 VF, Cucumis sativus cv Lungo Verde Degli Ortolani and Cucurbita pepo cv Géode HF1. At five days post inoculation all plants showed local necrotic lesions (Figure 1) and TNV-D presence was further confirmed with RT-PCR using the primer pair described above. Finally, 11 tomato samples from the same greenhouse were analysed with RT-PCR for the presence of TNV-D. In two plants TNV-D was found in mixed infection with ToFBV and OLV-1, in two TNV-D was present along with ToFBV and only one plant was solely infected with TNV-D. To the best of our knowledge this is the first report of TNV-D in Greece and the first nearly complete annotated genome derived from tomato. TNV-D belongs to the Betanecrovirus genus (family Tombusviridae) and was originally described from tobacco (Smith & Bald, 1935). The virus is thought to be spread worldwide, infecting more than 37 botanical families and in nature it is transmitted by the fungus Olpidium brassicae. TNV-D infection results in local necrotic lesions and systemic infection is observed only in a limited number of plant species (Díaz-Cruz et al., 2017). The virus is not a direct threat to tomato cultivation, however its ability to recombine with OLV-1 and to give rise to new viral species (Borodynko et al., 2010), support the need for further investigation of TNV-D 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).