A DIAGNOSTIC METHOD FOR THE SIMULTANEOUS DETECTION AND IDENTIFICATION OF POSPIVIROIDS
M. Luigi, E. Costantini, D. Luison, P. Mangiaracina, L. Tomassoli, F. Faggioli
Potato spindle tuber viroid (PSTVd) was the first species of the genus Pospiviroid detected in naturally infected symptomless ornamental plants (Solanum jasminoides) in 2006. Since then, other pospiviroid species were found in several naturally infected solanaceous ornamentals. The combination of latent infection, the widespread occurrence and the possibility of transmission to edible solanaceous crops (tomato and potato) has made the development of accurate and reliable molecular diagnostic detection methods a high priority. The aim of this study was the development of a genus-specific RT-PCR protocol, using universal pospiviroid primers, which would allow also a species-specific identification through restriction fragment length polymorphism (RFLP) analysis. The primers were designed to amplify the majority of the pospiviroid genome (ca. 80%) and the identification of each species using appropriate restriction endonuclease enzymes (AluI and Sau96I). The method was evaluated for specificity using healthy and/or non-target viroid-infected plants and for sensitivity using serial dilutions of total RNA extracted from plants infected by different pospiviroid species. The reliability of the method was successfully ascertained during a survey when numerous plants of the families Solanaceae and Asteraceae were assayed for single and mixed infection of pospiviroid species.