Sensitive detection of potato viruses, PVX, PLRV and PVS, by RT-PCR in potato leaf and tuber

Amultiplex reverse transcription polymerase chain reaction (M-RT-PCR) was developed for the simultaneous detection of three major potato viruses, Potato virus X (PVX), Potato virus S (PVS) and Potato leaf roll virus (PLRV), in potato leaves and tubers (stored and dormant). Primers were designed from the coat protein gene of all three viruses and were able to detect virus in leaf RNA dilutions of 1 : 1024 to 1 : 4096 and from tuber RNA dilutions of 1 : 256 to 1 : 1024. The sensitivity of virus detection in leaves was higher than in tubers. All three viruses were successfully detected from tissues sampled from the tuber cortex, perimedulla and pith, with all PCR bands being strongest from cortex tissues. The reliability of the M-RT-PCR to detect PVX, PVS and PLRV in tubers was determined by testing 95 samples of field, greenhouse and in vitro potato plantlets fromWuhan, China and comparing the results with enzymelinked immunosorbent assays (ELISA). M-RT-PCR detected all infections found by ELISA in leaf and stored tubers. In freshly harvested tubers,M-RT-PCRprovedmore sensitive thanELISA, as indicated by an additional 3.2%of infections detected with the M-RT-PCR method. Additional keywords: cDNA, disease, random primer, survey. Introduction Potato (Solanum tuberosum) is a major food crop worldwide and is susceptible to many plant viruses including Potato virus X (Family Flexiviridae, genus Potexvirus, PVX),Potato virus S (Family Flexiviridae, genus Carlavirus, PVS) and Potato leaf roll virus (FamilyLuteoviridae, genusPolerovirus, PLRV). In China, the largest potato producer in the world (FAO 2006), yields are significantly reduced by 30–50% when potatoes are infectedwith viruses (Sun andYang 2004). The use of certified virus-tested ‘seed’ for planting is the best way to limit the effect of these viruses on potato production (Singh 1998). The development of sensitive methods for detecting viruses is therefore of importance to support seed certification schemes (Singh and Nie 2003). Standardmethods of virus detection includingmechanical inoculation to indicator hosts, electron microscopy, enzymelinked immunosorbent assay (ELISA) and visual inspection are not adequate to reliably detect some potato viruses and might not be suitable for testing tuber samples (Mumford et al. 2000). Increased sensitivity and specificity of detection offered by reverse transcription–polymerase chain reaction (RT-PCR) is an effective method to overcome these problems (Nie and Singh 2002). PCR-based methods for PVY and PLRV detection have been shown to provide a relatively fast alternative to winter grow-out and ELISA post-harvest testing. The detection threshold of PCR-based methods was up to 128 times greater than that of an ELISA (Meunier et al. 2003). Multiplex reverse transcription–polymerase chain reaction (M-RT-PCR) was developed for plant virus detection in 1994 (Bariana et al. 1994) with advantages of the ability to detect lower levels of virus than ELISA and the ability to discriminate two or more targets simultaneously. Recently, surveys of commercial and seed potato fields in Canada used duplex RT-PCR to detect both the common (◦) and tobacco veinal necrosis strains (N/NTN) strains of PVY (Singh et al. 2003). In this study we describe the development and optimisation of an M-RT-PCR system for the detection of PVX, PLRV and PVS from both potato leaves and tubers (freshly harvested and stored at 20–25◦C for 2–4 months). The sensitivity of theM-RT-PCRwasdetermined for different RNA concentrations and compared to ELISA, and the distribution of each virus in potato tubers was investigated. © Australasian Plant Pathology Society 2006 10.1071/DN06017 1833-928X/06/010041 42 Australasian Plant Disease Notes M. Peiman and C. Xie Materials and methods

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