Development of a Real-Time PCR Assay for the Detection and Identification of Rubus Stunt Phytoplasma in Rubus spp.

Rubus stunt, caused by 'Candidatus Phytoplasma rubi' (Rubus stunt phytoplasma; RSP), is an economically important disease of Rubus. This disease occurs in wild and cultivated Rubus spp. in Europe but has not been reported from North America; however, its major leafhopper vector is well established in western Canada and the U.S.A. RSP has the potential to impact the cane-fruit industry by significantly compromising yields and impacting export potential for Rubus propagation material. To mitigate the risk of this disease entering or establishing, import and export testing of propagation material is a phytosanitary requirement in Canada, the U.S.A., and other countries regulating RSP. In the absence of a specific test for RSP, the current testing scheme involves the use of a generic test to screen for phytoplasmas followed by additional time-consuming procedures to confirm the phytoplasma species. In this study, a real-time PCR assay, targeting a 154-bp region of tuf gene, was developed for sensitive and specific detection of RSP in Rubus spp. The developed assay detected a minimum of five target copies, and no cross-reactivity was observed even with the 'Ca. P. rubi'-related strain associated with blackberry witches' broom, which differs from RSP only by a single nucleotide polymorphism in the target region. Repeatability of the developed assay was checked on two real-time PCR platforms with acceptable results. In conclusion, this real-time PCR assay provides a sensitive and specific detection of RSP for mitigating the introduction and spread of Rubus stunt disease in Rubus spp.