Single-seed detection of an uncharacterised Betaflexiviridae virus in Actinidia

A sensitive conventional RT-PCR assay has been developed for an uncharacterised virus within the family Betaflexiviridae, prompted by detection of the virus in Actinidia seedlings held in post-entry quarantine that had been grown from open-pollinated seed. Infection is asymptomatic and presently assessed as low-to-zero biosecurity risk. The assay targets the RNA-dependent RNA polymerase (RdRP) of the virus, primer sequences ('BetaF2' and 'BetaR2', 278bp amplicon) being based on a partial RdRP sequence for the virus. The assay detects the nine virus isolates found to date, in a range of sample types (fresh leaf, stem cambium, archived RNA). The assay, which includes a multiplexed internal control, has been accredited by International Accreditation New Zealand for Actinidia testing. To support management of the virus, the assay and associated RNA extraction method have been applied to testing dried Actinidia seed directly. They have been found sufficiently sensitive to detect the virus in individual seeds. Initial results show efficient transmission of the virus from infected pollen to the seed.