Development of a multiplex RT-qPCR assay to detect citrus apscaviroids and its use in citrus viroid surveys in Australia

Citrus production in Australia is a significant economic activity occurring in disparate regions. The crop is affected by a range of graft-transmissible pathogens including viroids, which are small, circular, non-coding RNA molecules. To ascertain the levels of infection with viroids in Australian citrus orchards, a survey was undertaken using samples collected between 2020 and 2024. To facilitate diagnosis, an RT-qPCR assay was designed and tested that detects all known citrus-infecting apscaviroids including citrus bent leaf viroid (CBLVd), citrus dwarfing viroid (CDVd), citrus viroid V (CVd-V), citrus viroid VI (CVd-VI), and citrus viroid VII (CVd-VII). Almost 700 samples from throughout the growing areas of Australia were tested using two assays, one newly developed citrus-infecting apscaviroid targeting assay and another targeting non-apscaviroids of citrus. Viroids were detected in ~ 20% of samples as individual or mixed infections. CDVd was the most prevalent viroid and was detected in 14% of samples - both citrus exocortis viroid (CEVd) and hop stunt viroid (HSVd) were found in ~ 5% of samples. citrus bark cracking viroid (CBCVd) was not detected and the remaining citrus-infecting apscaviroids, CBLVd, CVd-V, CVd-VI, CVd-VII, were each found at levels of less than 1%. This new multiplex RT-qPCR assay will allow the Auscitrus budwood scheme and similar schemes elsewhere in the world to more efficiently test propagation material to ensure high health status material is provided to citrus industries.