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

PLoS One. 2020 Sep 24;15(9):e0232502. doi: 10.1371/journal.pone.0232502. eCollection 2020.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Agriculture / methods
  • Capsicum / virology*
  • Plant Diseases / virology*
  • RNA, Viral / isolation & purification*
  • Real-Time Polymerase Chain Reaction / methods*
  • Seeds / virology
  • Solanum lycopersicum / virology*
  • Vegetables / virology
  • Viroids / genetics
  • Viroids / isolation & purification*

Substances

  • RNA, Viral

Grants and funding

BASF Vegetable Seeds and Enza Zaden Seed Operations BV provided support in the form of salaries and data collection and analysis for the interlaboratory comparison experiment, for authors B.P.J. Geraats and M. Kemper (BASF Vegetable Seeds), D.C.M. Beugelsdijk (Enza Zaden Seed Operations BV), but did not have any additional role in the study design, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.