Identification of an emerging cucumber virus in Taiwan using Oxford nanopore sequencing technology.

ABSTRACT

BACKGROUND: In June 2020, severe symptoms of leaf mosaic and fruit malformation were observed on greenhouse-grown cucumber plants in Xizhou Township of Changhua County, Taiwan. An unknown virus, designated CX-2, was isolated from a diseased cucumber sample by single lesion isolation on Chenopodium quinoa leaves. Identification of CX-2 was performed. Moreover, the incidence of cucumber viruses in Taiwan was also investigated. METHODS: Transmission electron microscopy was performed to examine virion morphology. The portable MinION sequencer released by Oxford Nanopore Technologies was used to detect viral sequences in dsRNA of CX-2-infected leaf tissue. The whole genome sequence of CX-2 was completed by Sanger sequencing and analyzed. Reverse transcription-polymerase chain reaction (RT-PCR) with species-specific primers and indirect enzyme-linked immunosorbent assay (ELISA) with anti-coat protein antisera were developed for virus detection in the field [see Additional file 1]. RESULTS: Icosahedral particles about 30 nm in diameter were observed in the crud leaf sap of CX-2-infected C. quinoa plant. The complete genome sequence of CX-2 was determined as 4577 nt long and shared 97.0-97.2% of nucleotide identity with that of two cucumber Bulgarian latent virus (CBLV) isolates in Iran and Bulgaria. Therefore, CX-2 was renamed CBLV-TW. In 2020-2022 field surveys, melon yellow spot virus (MYSV) had the highest detection rate of 74.7%, followed by cucurbit chlorotic yellows virus (CCYV) (32.0%), papaya ringspot virus virus watermelon type (PRSV-W) (10.7%), squash leaf curl Philippines virus (SLCuPV) (9.3%), CBLV (8.0%) and watermelon silver mottle virus (WSMoV) (4.0%). Co-infection of CBLV and MYSV could be detected in field cucumbers. CONCLUSION: The emerging CBLV-TW was identified by nanopore sequencing. Whole genome sequence analysis revealed that CBLV-TW is closely related, but phylogenetically distinct, to two known CBLV isolates in Bulgaria and Iran. Detection methods including RT-PCR and indirect ELISA have been developed to detect CBLV and to investigate cucumber viruses in central Taiwan. The 2020-2022 field survey results showed that MYSV and CCYV were the main threats to cucumbers, with CBLV, SLCuPV and WSMoV were occasionally occurring.