Journal of Plant Pathology (2014), 96 (1), 151-158 Edizioni ETS Pisa, 2014 Luigi et al. 151 A DIAGNOSTIC METHOD FOR THE SIMULTANEOUS DETECTION AND IDENTIFICATION OF POSPIVIROIDS M. Luigi, E. Costantini, D. Luison, P. Mangiaracina, L. Tomassoli and F. Faggioli Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per la Patologia Vegetale, Via C.G. Bertero 22, 00156 Roma, Italy SUMMARY Viroids are classified into two families, Pospiviroidae [type species Potato spindle tuber viroid (PSTVd)] and Potato spindle tuber viroid (PSTVd) was the first spe- Avsunviroidae, [type species Avocado sunblotch viroid cies of the genus Pospiviroid detected in naturally infected (ASBVd)] (Flores et al., 2003; Owens et al., 2011), that rep- symptomless ornamental plants (Solanum jasminoides) in licate and accumulate in the nucleus and the chloroplast, 2006. Since then, other pospiviroid species were found in respectively. The viroid classification scheme is also sup- several naturally infected solanaceous ornamentals. The ported by other criteria, including the presence of ham- combination of latent infection, the widespread occurrence merhead ribozymes in members of the family Avsunviroi- and the possibility of transmission to edible solanaceous dae and of a central conserved region (CCR) in members crops (tomato and potato) has made the development of the family Pospiviroidae. of accurate and reliable molecular diagnostic detection The family Pospiviroidae includes the genus Pospiviroid methods a high priority. The aim of this study was the composed of ten phylogenetically related species: PSTVd, development of a genus-specific RT-PCR protocol, using Chrysanthemum stunt viroid (CSVd), Citrus exocortis viroid universal pospiviroid primers, which would allow also a (CEVd), Columnea latent viroid (CLVd), Iresine viroid 1 species-specific identification through restriction fragment (IrVd-1), Mexican papita viroid (MPVd), Tomato apical stunt length polymorphism (RFLP) analysis. The primers were viroid (TASVd), Tomato chlorotic dwarf viroid (TCDVd), designed to amplify the majority of the pospiviroid ge- Tomato planta macho viroid (TPMVd) and Pepper chat fru- nome (ca. 80%) and the identification of each species using it viroid (PCFVd) (Verhoeven et al., 2009). These species appropriate restriction endonuclease enzymes (AluI and show a high genomic identity, in some cases higher than Sau96I). The method was evaluated for specificity using 80% (Table 1). Moreover, most pospiviroids are almost healthy and/or non-target viroid-infected plants and for indistinguishable in biological features from one other. sensitivity using serial dilutions of total RNA extracted Most pospiviroids can cause severe diseases and dam- from plants infected by different pospiviroid species. The ages to most species in the family Solanaceae, potato (So- reliability of the method was successfully ascertained dur- lanum tuberosum) and tomato (Solanum lycopersicum) in ing a survey when numerous plants of the families Sola- particular, in which they induce epinasty, stunting, leaf naceae and Asteraceae were assayed for single and mixed distortion or discoloration and necrosis (usually vein ne- infection of pospiviroid species. crosis) (Singh, 1973; Galindo et al., 1982; Owens, 1990; Martinez-Soriano et al., 1996; Singh et al., 1999, 2003). Key words: RT-PCR, RFLP, Pospiviroid, diagnosis, ge- Notwithstanding the above, PSTVd is currently the only nus-specific detection, species-specific detection Solanaceae-infecting pospiviroid with an explicitly regu- lated status in Europe (Council Directive 2000/29/EC), USA and Canada (NAPPO, 2007). INTRODUCTION In 2006, PSTVd was found for the first time in latently infected solanaceous ornamentals. i.e. Solanum jasminoi- Viroids, the smallest pathogens known, are composed des and Brugmansia spp. (Verhoeven et al., 2008). Many of a highly complementary naked single-stranded circular other pospiviroid species were subsequently recovered RNA ranging in size from 246 to 401 nucleotides and do from different symptomless such plants, that represent a not encode any protein. Despite this minimal molecular sanitary hazard, as they constitute a source of inoculum for composition, viroids contain sufficient information to in- other susceptible hosts (Navarro et al., 2009). Simultane- fect host plants, to manipulate their gene expression and ous infections by PSTVd and TASVd, CEVd and TASVd, induce specific diseases (Hadidi et al., 2003). PSTVd and CEVd have recently been detected in S. jasmi- noides and Cestrum auranticum (Luigi et al., 2011; Torchetti et al., 2011). Corresponding author: F. Faggioli Fax: +39.06.82070246 This situation and the recent discovery that CSVd, E-mail: [email protected] another quarantinable pospiviroid, is able to infect 152 Simultaneous detection and identification of pospiviroids Journal of Plant Pathology (2014), 96 (1), 151-158 Table 1. Percentage of nucleotide identity among reference sequences for each pospiviroid species. CSVd CEVd CLVd IrVd MPVd PCFVd PSTVd TASVd TCDVd TPMVd CSVd 100% CEVd 75% 100% CLVd 73% 68% 100% IrVd 68% 70% 62% 100% MPVd 75% 75% 75% 71% 100% PCFVd 69% 71% 67% 74% 73% 100% PSTVd 73% 74% 76% 71% 86% 71% 100% TASVd 81% 83% 76% 67% 79% 74% 75% 100% TCDVd 75% 74% 75% 70% 84% 71% 91% 75% 100% TPMVd 75% 73% 75% 72% 92% 73% 84% 79% 82% 100% Sequences retrieved from the NCBI database were: CSVd [NC_002015], CEVd [NC_001464.1], CLVd [NC_003538.1], IrVd 1[NC_003613.1], MPVd [NC_003637.1], PCFVd [NC_011590.1], PSTVd [NC_002030.1], TASVd [NC_001553.1], TCDVd [NC_000885.1] and TPMVd [NC_001558.1]. In bold are the highest percentages of identity. symptomlessly Argyranthemum frutescens in Italy (Tor- fragment length polymorphism (RFLP) for identification chetti et al., 2012a) and France (Marais et al., 2011), have at the species level. increased the concern for the incumbent phytosanitary risks (EFSA, 2011, 2012), thus calling for the adoption of regulatory measures based on the use of viroid-free propa- MATERIALS AND METHODS gation material and, when possible, on the interception of infected plants. Effectiveness of these defense strategies Source of materials. The CRA-Plant Pathology Re- requires a rapid, efficient and reliable method for pospivi- search Centre (CRA-PAV, Rome) germplasm collection roids generic detection and specific identification. provided S. jasminoides plants infected by CEVd, TASVd In the past, different diagnostic methods have been and PSTVd in single and mixed infection, Chrysanthemum developed either for the genus-specific or species-specific sp. plants infected by CSVd and healthy controls of S. ja- detection of members of the genus Pospiviroid: (i) northern sminoides, tomato and potato. Positive controls infected by blot hybridization (Owens and Diener, 1981; Singh, 1999); CEVd, CLVd, IrVd, MPVd, PCFVd, TASVd, TCDVd and (ii) a single polyprobe for the simultaneous identification TPMVd were kindly supplied by Dr. J. Th. J.Verhoeven of eight viroids (Torchetti et al., 2012b); (iii) RT-PCR and (Dutch Plant Protection Service and Laboratory of Virol- Real time RT-PCR protocols using primers for pospiviroid ogy, Wageningen University) and maintained on tomato generic detection (Bostan et al., 2004; Verhoeven et al., plants in a greenhouse. 2004; Botermans et al., 2013); (iv) specific primer pairs for To test the efficiency and accuracy of the method, 200 species-specific detection (RT-PCR and real-time RT-PCR) plants were collected from different nurseries and exam- (Yang et al., 1992; Hooftman et al., 1996; Spieker, 1996; ined (Table 2). Their sanitary status has previously been Shamloul et al., 1997; Mumford et al., 2000; Boonham et ascertained using specific diagnostic methods (Di Serio, al. 2004; Roenhorst et al., 2005) 2007; Önelge, 1997; Levy and Hadidi, 1992; Tomassoli et This paper reports the development of a genus- and al., 2004; Verhoeven et al., 2010). species-specific diagnostic method for pospiviroids. The method involves RT-PCR amplification of viroidal RNA RNA extraction from pospiviroid-infected and unin- using universal pospiviroid primers for genus detection, fected plants. Total RNA was extracted from each control after which the amplified DNA is analyzed by restriction and test sample using the Spectrum™ Plant Total RNA Kit (Sigma, Germany) according to the manufacturer’s in- structions. RNA was finally eluted with 100 µl of RNase- free water. Primer design and RT-PCR for detecting members of the genus Pospiviroid. Reference sequences of all pospi- viroids were retrieved from the NCBI database (Table 1) and used to perform a multiple alignment for designing an universal genus-specific primer set (Table 3) by selecting the most conserved region among all pospiviroids (Fig. 1). Fig. 1. Alignments of all pospiviroids in the two selected For efficiency testing, selected primers were first used regions for primer design. Left, the reverse primer and in boxes the degenerate nucleotides are highlighted. Right, the in a two-steps, two-tubes RT-PCR (Menzel et al., 2002). forward primer, the letter n highlights the variable positions Specifically, 2 µl of extracted total RNA were added to among different homologous primers. 18 µl of a random reverse transcriptase reaction mixture Journal of Plant Pathology (2014), 96 (1), 151-158 Luigi et al. 153 Table 2. Plant species assayed for possible detection of naturally-infected pospiviroids. Species Viroid detected No. infected plants/ No. analyzed plants Brugmansia sp. 0/8 Cestrum sp.