| Abstract: | The work described here formed part of the EU SMT DIAGPRO project, to develop diagnostic protocols for 18 regulated pests. The Potato spindle tuber pospiviroid (PSTVd) protocol was developed primarily for testing in vitro‐ and glasshouse‐grown potato plants for the purposes of post‐entry quarantine and the production of pathogen‐tested nuclear stock. After a performance audit of methods used by 12 laboratories in Europe and America by ring testing, four methods were chosen for multilaboratory validation. For most laboratories, the detection limits were 10–20 mg of PSTVd‐infective tissue for R‐PAGE; 0.25–0.5 mg for DIG‐probe; 0.062 mg for RT‐PCR; and 0.0155 mg for TaqMan (this was the lowest weight of infective tissue tested). Some laboratories were able to extend the detection limit to 0.0155 mg for DIG‐probe and RT‐PCR. The DIG‐probe and R‐PAGE are recommended as primary detection methods, with confirmation of viroid presence by any of the four validated detection methods. Specific diagnosis requires the viroid to be sequenced. Other methods may be used for primary detection, providing that they preferably detect all PSTVd isolates and other Pospiviroids that have the potential to infect potato, and detect viroid in at least 1/10 of the tissue weight normally tested per plant. |
