Detection and Quantification of Potato Spindle Tuber Viroid in Wild and Cultivated Potatoes Using a New Validated...

Potato (Solanum tuberosum), as an internationally traded global crop, is exposed to a wide range of diseases that cause economic losses. Economically important pathogens include oomycetes, fungi, viruses, bacteria, and viroids. Potato spindle tuber viroid (PSTVd) is a small, circular, single-stranded RNA with autonomous replication that infects potato, causes tuber deformations, reduces yield, and is readily transmitted from one generation to the next through seed potatoes and botanical zygotic seeds. PSTVd has been controlled effectively in some countries by implementing stringent seed potato certification and field management procedures. PSTVd persists in many other parts of the world, however, and infrequent detections of PSTVd continue to occur in locations where the viroid was thought to be absent. Analytical assays to detect PSTVd have been developed, but these assays have not been validated to establish their level of confidence and fit of purpose. In this manuscript, we describe a reverse-transcription quantitative PCR method that allows for PSTVd diagnostics and quantitative monitoring. The method was validated following the Guidelines for the Validation of Analytical Methods for the Detection of Microbial Pathogens in Foods and Feeds and literature recommendations for a single laboratory (tier 1 validation). The method was used to determine the infection status of 208 potato plants from 10 species that might have been in contact with PSTVd-positive plants in a greenhouse experimental environment. PSTVd abundance at different locations within individual plants was quantified, and these data showed that PSTVd titer can vary widely within the shoot system of a single plant. The validated method can detect and quantify PSTVd in true potato seed, but reliable detection in single seeds depends on viroid amount.