Simultaneous detection of eight pospiviroids infecting ornamentals and vegetables by a single polyprobe

Control measures based on the use of viroid-free propagation material and, when possible, on the interception of infected plants, are needed to constrain the spread of viroids. Effectiveness of these defense strategies requires fast, reliable, sensitive and economically sustainable detection methods. A single molecular probe (polyprobe), composed of assembled sequence fragments derived from several viroid species has the advantage of reducing the costs of simultaneous detection of most viroid species by molecular hybridization. In fact, just one single transcription reaction is needed for synthesizing a polyprobe useful to detect different viroid species. Based on bioinformatics analyses of pospiviroid genomic RNAs, we have developed a polyprobe that can detect at least eight different viroid species belonging to Pospiviroid genus (family Pospiviroidae) (POSPIprobe), including Potato spindle tuber viroid and Chrysanthemum stunt viroid, which are quarantine pests recently intercepted in Italy. The digoxigenin-labelled polyprobe was synthesized by in vitro-transcription using as template a single vector constructed by cloning directionally partial sequences from four different pospiviroids. POSPIprobe sensitivity in dot-blot assays was similar to that obtained using single probes, and its specificity for the eight pospiviroids was further corroborated by Northern blot and tissue printing hybridization. Dot blot assays with the POSPIprobe were validated by simultaneously testing 68 samples from tomato, chrysanthemum, Argyranthemum frutescens and pepper infected by different pospiviroids, which confirmed the high potential of this probe in quarantine, certification and survey programs. Emerging phytosanitary risks due to pospiviroid spread in Europe and effectiveness of control measures will be also discussed.