High Throughput-based Surveillance Reveals a Complex Spanish Citrus Virome Including New Components Related to Tristeza, Impietratura and Yellow Vein Clearing Diseases

Citrus is one of the most important crops cultivated worldwide, representing a strategic source of agricultural income for many countries, including Spain, the main producer within the European Union. The protection of this highly valuable industry against an increasing global movement of pests and pathogens requires effective regulatory measures, including control of plant propagation material, phytosanitary surveillance and risk assessment, based not only on knowledge of the established threats but also on forecasting potential emerging threats that may circulate unnoticed in the production system. In this work high-throughput sequencing (HTS) was used to expand knowledge on established and potential emerging viruses in the Valencian Community, one of the main citrus producer regions in Spain. The results of this study have revealed a much more complex citrus virome than previously reported, including non-regulated citrus yellow vein clearing virus (CYVCV), regulated non-quarantine citrus virus A (CiVA) and the previously undetected T3 genotype of citrus tristeza virus (CTV). Moreover, we report HTS generated contigs of unknown virus-related sequences and discuss their potential as citrus pathogens. HTS detection of CYVCV, CTV-T3 and CiVA in Spanish orchards has been confirmed by conventional RT-PCR and Sanger sequencing. These findings have relevant implications in the development of control and regulatory measures against three important viral diseases, tristeza, impietratura and yellow vein clearing, and demonstrate the added value of HTS-based surveillance to determine emerging components of the citrus virome.