Deep Sequencing of Small RNAs from Citrus Affected by Graft-Transmissible Diseases of Unknown Aetiology Leads to Discovery of Two Novel Viruses

Diseases of unknown aetiology remain difficult or impossible to diagnose. Illumina next generation sequencing (NGS) technology was used to generate sequence datasets from citrus affected by two such diseases: citrus chlorotic dwarf (CCDD) (whitefly-transmitted) and yellow vein clearing disease (YVCD) (aphidtransmitted). Contigs from small RNAs were assembled and used to screen sequence homologies against the virus database in GenBank. DNA fragments from CCDDaffected plants were used to re-construct a circular single-stranded DNA viral genome with homologies to geminiviruses. The genome size and organization of the provisionally named Citrus chlorotic dwarf-associated virus (CCDaV) was shown to be a highly divergent member of the family Geminiviridae. Similarly, the whole genome sequence of a putative filamentous virus associated with CYVCD-affected lemon plants was reconstructed. The genome structure was found to be typical of flexiviruses and sequence data suggested that CYVCV is a new species in the genus Mandarivirus. PCR-based assays for CCDaV and CYVCV were developed that will be an important diagnostic tool for citrus disease management programs in Turkey and other citrus-producing regions.