Filling the gaps in diagnostics of Pepino mosaic virus and Potato spindle tuber viroid in water and tomato seeds and leaves

Abstract

Waterborne and seedborne Pepino mosaic virus (PepMV) and Potato spindle tuber viroid (PSTVd) pose serious threats to tomato production due to seed transmission and mechanical transmission, coupled with their long-term stability outside the host plant. Therefore, rapid and sensitive diagnostic procedures are needed to prevent the spread of these quarantine pathogens. In particular, water and seed contamination are difficult to detect and confirm without efficient concentration methods. This study presents procedures that improve detection of PSTVd from tomato seeds and leaf tissue, and PepMV from water and tomato leaf tissue. For efficient concentration of PepMV from water samples, a procedure was optimized using convective interaction media monolithic chromatography columns, which provides concentration by three orders of magnitude. For concentration of PSTVd from seed extracts, an easy-to-use and efficient method was developed based on RNA binding to positively charged anion-exchange resin beads that provides up to 100-fold more sensitive detection in comparison with procedures without a concentration step. This thus allows confirmation of RT-qPCR results with sequencing of RT-PCR products in samples with low viroid levels. In addition, reverse-transcription loop-mediated isothermal amplification assays for detection of PSTVd and PepMV were optimized and adapted to both laboratory and on-site testing requirements. This allows rapid detection of these pathogens in crude leaf homogenates, in under 30 min. These procedures of concentration and detection are shown to be efficient and to fill the gaps in diagnostics of PepMV and PSTVd, especially when these pathogens are present at low levels in difficult matrices such as water and seeds.

Development and validation of a real-time RT-PCR test for screening pepper and tomato seed lots for the presence of pospiviroids

“…However, often tailor-made solutions are needed for the confirmation and identification of pospiviroids in seed samples, e.g. concentration methods (29) nested-RT-PCR, or pooling of PCR-products for further testing. In conclusion, the performance of the PospiSense test, combined with the need of only two parallel reactions and a limited number of probes, shows its perspectives as an alternative test for screening seed lots of solanaceous species.…”

Global Advances in Tomato Virome Research: Current Status and the Impact of High-Throughput Sequencing

“…In order to prevent introduction of viroids, all imported lots of tomato and pepper seeds are tested in Australia (Constable et al, 2019). Since testing of seeds is needed to prevent the introduction of pospiviroids into new areas, many efforts have already been done to optimize diagnostic protocols that enable their detection at low concentration, which is expected in case of seed infestation (Mehle et al, 2017).…”

Molecular Approaches for Low-Cost Point-of-Care Pathogen Detection in Agriculture and Forestry

“…Such protocol can distinguish between living and dead nematodes, by detecting the presence of mRNA encoding an expansin gene as a viability marker (Leal et al, 2014). Other examples of optimized LAMP-based protocols include a tool for the early detection of Heterobasidion irregulare (Sillo et al, 2018); a fast, easy, and in-field deployable method for detection of Pepino mosaic virus and Potato spindle tuber viroid (Mehle et al, 2017); a LAMP protocol starting from crude plant materials for the detection of Xanthomonas fragariae (Getaz et al, 2017); a protocol for the detection of three different phytoplasmas infecting fruit trees (De Jonghe et al, 2017); and an assay for the rapid detection of Pectobacterium atrosepticum (Li et al, 2011). Similar to PCR-based approaches, multiplexing of the samples is possible with LAMP (Denschlag et al, 2014;Yasuhara-Bell et al, 2018;Feng et al, 2019).…”