DETECTION OF POSPIVIROIDS IN THE CZECH REPUBLIC AND THEIR DISCRIMINATION BY RESTRICTION ANALYSIS

Abstract

Potato spindle tuber viroid (PSTVd), a member of the genus Pospiviroid, is a quarantine pest in the European Union and its presence in ornamental solanaceous crops represents a serious threat to susceptible vegetable and potato crops. Since the year 2007 the Czech Plant Protection Service (PPS) carries out a survey for PSTVd on ornamental solanaceous species. Within the first year of the survey, several new hosts of PSTVd were identified. Also, a new species of Pospiviroid was detected in the Czech Republic. At the beginning of the survey, a method for detection of PSTVd recommended in the EPPO diagnostic protocol was adopted. Reverse transcription-polymerase chain reaction with a semi-specific primer pair was carried out in the diagnostic laboratory of PPS while the sequencing of PCR products was performed in an external laboratory. Due to this time consuming process, which proved inefficient for the needs of rapid application of phytosanitary measures, the laboratory developed a faster procedure. The entire genomic sequences of all viroids which contained primer identical segments were downloaded from the GenBank database and aligned to search for viroid-specific restriction sites. A combination of four enzymes was chosen to distinguish the detected viroids. Restriction analysis was successfully tested and applied for the PSTVd identification. Later on, after confirmation of other pospiviroids detected for the first time in the Czech Republic (Tomato apical stunt viroid and Chrysanthemum stunt viroid on Solanum jasminoides and Citrus exocortis viroid on Solanum jasminoides and Verbena A— hybrid), the diagnostic method was improved to detect a wide range of Pospiviroid species with the introduction of primers detecting the five most common pospiviroids in the ornamental solanaceous plants in Europe. Similarly, for identification of the amplified PCR products, a new set of restriction enzymes was identified.