Spread of potato virus Y NTN in potato cultivars (Solanum tuberosum L.) with different levels of sensitivity Natas ˇa Mehle * , Maja Kovac ˇ, Natas ˇa Petrovic ˇ, Marus ˇa Pompe Novak, S ˇ pela Baebler, Hana Krec ˇic ˇ Stres, Kristina Gruden, Maja Ravnikar Department of Plant Physiology and Biotechnology, National Institute of Biology, Vec ˇna pot 111, 1000 Ljubljana, Slovenia Accepted 20 October 2004 Abstract The aim of this work was to correlate the appearance of the symptoms, multiplication and spread of virus after mechanical inoculation of potato (Solanum tuberosum L.) cultivars showing different levels of susceptibility and sensitivity to Potato virus Y NTN (PVY NTN ). The potato cultivars used were the resistant cultivar Sante and susceptible cultivars Igor, Pentland squire and De ´sire ´e. The spread of the virus PVY NTN in infected plants was monitored using different methods: DAS-ELISA, tissue printing, immuno-serological electron microscopy and real-time PCR. In all three susceptible cultivars, the virus was detected in the inoculated leaves 4–5 days after inoculation. From there virus spread rapidly, first into the stem, then more or less simultaneously to the upper leaves and roots. Real-time PCR was shown to be very sensitive and enabled viral RNA to be detected in non-inoculated leaves of susceptible cultivar Igor earlier than other methods. Therefore, for exact studies of plant–virus interaction, a combination of methods which detect viruses on the basis of their different properties (coat protein, morphology or RNA) should be used to monitor the spread of viruses. q 2004 Elsevier Ltd. All rights reserved. Keywords: Solanum tuberosum L.; Virus spread; PVY NTN ; DAS-ELISA; Tissue printing; Immuno-serological electron microscopy; Real-time PCR 1. Introduction Potato virus Y NTN (PVY NTN ) is a strain belonging to the necrotic group of potato virus Y (PVY) [8], that causes a potato tuber ring necrotic disease in potato [19] The disease, which is tuber-, sap- and aphid-transmitted, is very harmful and has become widespread in Europe [20,21]. Sub- sequently, the virus has spread to other potato growing countries throughout the world [28]. Heretofore, two different PVY NTN isolates have been described, based on the organization of their genome (i.e. the recombinant and the non-recombinant) [3,28]. PVY NTN isolates do not greatly differ serologically from standard PVY N , and can be detected by polyclonal antibodies to PVY and by monoclonal antibodies specific for PVY N . They are distinguished biologically from PVY N by their reactions on test plants and their ability to induce severe necrosis on tubers and foliage of potatoes [21]. A 3-primer PCR, specific to Slovenian isolates of PVY NTN , was developed for large-scale testing of samples [44]. An RT-PCR method, specific to PVY NTN , has been reported [3,23,27,28,43] and a strain-specific restriction cleavage of PCR products has been demonstrated [13,35]. PVY N strains could also be differentiated from PVY NTN by some other PCR-based methods, such as transcript conformation polymorphism (TCP) [36] and a reference transcript annealing procedure [37]. Szemes et al. [40], developed a multiplex AmpliDet RNA assay for simultaneous detection and typing of PVY isolates. A monoclonal antibody with partial specificity for PVY NTN has been described by C ˇ erˇovska ´ [7]. Susceptible potato (Solanum tuberosum L.) cultivars vary from highly sensitive to tolerant in their sensitivity to infection with PVY NTN . Only a few potato cultivars, like cv. Sante, are considered to be resistant to PVY NTN . The resistance of cv. Sante was achieved by classical breeding, by introducing the Ry sto gene from Solanum stoloniferum [15,33]. Resistant lines were also constructed by 0885-5765/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2004.10.005 Physiological and Molecular Plant Pathology 64 (2004) 293–300 www.elsevier.com/locate/pmpp * Corresponding author. Tel.: C386 1 423 3388; fax: C386 1 2573 847. E-mail address: natasa.mehle@nib.si (N. Mehle).