A real-time PCR assay for detection and absolute quantitation of Citrus exocortis viroid in two sensitive and tolerant rootstocks

Citrus exocortis viroid (CEVd) is the causal agent of exocortis disease, the economically important viroid disease of citrus. In this research, CEVd copy number was quantified in sour orange (Citrus aurantium L.) and citrange (Poncirus trifoliata L. Raf. × Citrus sinensis L.) seedlings by absolute real-time PCR. A full-length in vitro transcript of a single-sequence CEVd-S1 variant was synthesized and mechanically inoculated into sour orange and Troyer citrange seedlings as tolerant and sensitive hosts, respectively. CEVd copy number was subsequently monitored at different times after inoculation in the green bark of infected seedlings for up to 16 weeks post inoculation (wpi). CEVd was detected at 3 wpi and totally increased up to 16 wpi. The copy number of CEVd per milligram of bark tissue in both host species was in the range of 103 to 104 at the third and sixth weeks post inoculation, without any significant differences. Although the viroid titer was remarkably higher in citrange (sensitive host) than in sour orange (tolerant host) at 10-16 wpi, we demonstrated that CEVd titer differed in citrus species depending on the time of sampling and host plant species.