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

Marleen Botermans,Agata Jodlowska,Mark Kemper,Debora C M Beugelsdijk,Bart P J Geraats,Johanna W Roenhorst,Marinus Hooftman,Marcel Westenberg,Esther J Van Veen,Harrie Koenraadt,Eveline Metz,Jacobus Th J Verhoeven

doi:10.1371/journal.pone.0232502

Abstract

Potato spindle tuber viroid and other pospiviroids can cause serious diseases in potato and tomato crops. Consequently, pospiviroids are regulated in several countries. Since seed transmission is considered as a pathway for the introduction and spread of pospiviroids, some countries demand for the testing of seed lots of solanaceous crops for the presence of pospiviroids. A real-time RT-PCR test, named PospiSense, was developed for testing pepper (Capsicum annuum) and tomato (Solanum lycopersicum) seeds for seven pospiviroid species known to occur naturally in these crops. The test consists of two multiplex reactions running in parallel, PospiSense 1 and PospiSense 2, that target Citrus exocortis viroid (CEVd), Columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd) and tomato planta macho viroid (TPMVd, including the former Mexican papita viroid). Dahlia latent viroid (DLVd) is used as an internal isolation control. Validation of the test showed that for both pepper and tomato seeds the current requirements of a routine screening test are fulfilled, i.e. the ability to detect one infested seed in a sample of c.1000 seeds for each of these seven pospiviroids. Additionally, the PospiSense test performed well in an inter-laboratory comparison, which included two routine seed-testing laboratories, and as such provides a relatively easy alternative to the currently used tests.

Highlights

Pospiviroids are single-stranded circular RNA molecules consisting of around 360 nucleotides
To determine the analytical sensitivity, RNA-extracts of pepper or tomato seeds naturally infested by Columnea latent viroid (CLVd), Potato spindle tuber viroid (PSTVd) or tomato apical stunt viroid (TASVd) were diluted in duplicate in RNA-extracts of non-infested seeds
The newly developed PospiSense test has been shown to fulfil the requirements for routine testing of pepper and tomato seed samples for the seven pospiviroid species known to occur naturally in these crops

Summary

Introduction

Pospiviroids are single-stranded circular RNA molecules consisting of around 360 nucleotides. The genus Pospiviroid is in the family Pospiviroidae, with Potato spindle tuber viroid (PSTVd) being the type species. Most pospiviroids can infect a wide range of plant species, including many solanaceous ornamental and vegetable crops. Infected plants often remain symptomless, PSTVd and some other pospiviroids may cause serious diseases in potato and tomato. Development and validation of a real-time RT-PCR test for screening pepper/tomato seed lots for pospiviroids preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section

Methods

Results

Conclusion