Identification and characterisation of <i>Zucchini yellow fleck virus</i> and a novel <i>Nepovirus</i> from next‐generation sequencing of mixed virus infections in cucumbers (<i>Cucumis sativus</i>) from Crete

Abstract Cucumbers are susceptible to infections with many characterised virus species. In some cases, mixed virus infections occur and produce novel symptoms. In Greece, routine screening is carried out when virus infection is suspected, or novel symptoms are observed in the field. To identify the viruses associated with distinct symptoms observed in samples from commercial cucumber production areas on the island of Crete, Greece, we carried out high‐throughput sequencing (HTS) from a pool of six samples. Following assembly and BLAST analysis, we identified at least seven viruses based on similarity to published sequences. Two of these sequences represented novel, near‐complete genomes of a putative new nepovirus and of zucchini yellow fleck virus (ZYFV). To confirm the HTS results, the six samples were screened for all identified viruses, and their presence was confirmed through Sanger sequencing of PCR products. The full‐length genomes of both the nepovirus and ZYFV were amplified by PCR and confirmed by Sanger sequencing. We have generated the complete genome of a novel nepovirus from cucumber as well as the first complete genome sequence of a cucumber‐infecting ZYFV isolate from Crete. The nepovirus was mechanically transmissible to Nicotiana benthamiana and induced typical cytopathological modifications consistent with virus infection, as revealed by TEM studies. We propose to name this new virus Cucumber nepovirus A (CuNVA).