Characterization of a novel psyllid-transmitted waikavirus in carrots

Carrots collected from the Western Negev region in Israel during the winter of 2019 showed disease symptoms of chlorosis, leaf curling, a loss of apical dominance, and multiple lateral roots that were not associated with known pathogens of the carrot yellows disease. Symptomatic carrots were studied for a possible involvement of plant viruses in disease manifestations using high throughput sequencing analyses. The results revealed the presence of a waikavirus, sharing a ∼70% nucleotide sequence identity with Waikavirus genus members. Virions purified from waikavirus-positive carrots were visualized by transmission electron microscopy, showing icosahedral particle diameter of ∼28 nm. The genome sequence was validated by overlapping amplicons by designed 12 primer sets. A complete genome sequence was achieved by rapid amplification of cDNA ends (RACE) for sequencing the 5' end, and RT-PCR with oligo dT for sequencing the 3' end. The genome encodes a single large ORF, characteristic of waikaviruses. Aligning the waikavirus-deduced amino-acid sequence with other waikavirus species at the Pro-Pol region, a conserved sequence between the putative proteinase and the RNA-dependent RNA polymerase, showed a ∼40% identity, indicating the identification of a new waikavirus species. The amino-acid sequence of the three coat proteins and cleavage sites were experimentally determined by liquid chromatography-mass spectrometry. A phylogenetic analysis based on the Pro-Pol region revealed that the new waikavirus clusters with persimmon waikavirus and actinidia yellowing virus 1. The new waikavirus genome was localized in the phloem of waikavirus-infected carrots. The virus was transmitted to carrot and coriander plants by the psyllid Bactericera trigonica Hodkinson (Hemiptera: Triozidae).