Rapid identification of tomato Sw-5 resistance-breaking isolates of Tomato spotted wilt virus using high resolution melting and TaqMan SNP Genotyping assays as allelic discrimination techniques

PLoS One. 2018 Apr 30;13(4):e0196738. doi: 10.1371/journal.pone.0196738. eCollection 2018.

Abstract

In tomato, resistance to Tomato spotted wilt virus (TSWV) is conferred by the dominant gene, designated Sw-5. Virulent Sw-5 resistance breaking (SRB) mutants of TSWV have been reported on Sw-5 tomato cultivars. Two different PCR-based allelic discrimination techniques, namely Custom TaqMan™ SNP Genotyping and high-resolution melting (HRM) assays, were developed and compared for their ability to distinguish between avirulent (Sw-5 non-infecting, SNI) and SRB biotypes. TaqMan assays proved to be more sensitive (threshold of detection in a range of 50-70 TSWV RNA copies) and more reliable than HRM, assigning 25 TSWV isolates to their correct genotype with an accuracy of 100%. Moreover, the TaqMan SNP assays were further improved developing a rapid and simple protocol that included crude leaf extraction for RNA template preparations. On the other hand, HRM assays showed higher levels of sensitivity than TaqMan when used to co-detect both biotypes in different artificial mixtures. These diagnostic assays contributed to gain preliminary information on the epidemiology of TSWV isolates in open field conditions. In fact, the presented data suggest that SRB isolates are present as stable populations established year round, persisting on both winter (globe artichoke) and summer (tomato) crops, in the same cultivated areas of Southern Italy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Biological Assay
  • Disease Resistance / genetics
  • Genes, Plant*
  • Genotype
  • Models, Genetic
  • Mutation
  • Plant Diseases / genetics
  • Plant Diseases / virology*
  • Polymerase Chain Reaction
  • Polymorphism, Single Nucleotide*
  • Solanum lycopersicum / genetics
  • Solanum lycopersicum / virology*
  • Species Specificity
  • Tospovirus / genetics*
  • Virulence

Grants and funding

This work was supported with a grant from the Ministry of Economy and Finance to the CNR, "Integrate knowledge for Sustainability and Innovation of Made in Italy Agro- Food" (CISIA, l. 191/2009), and a grant from the Ministry of Education, University and Research to the Public-Private Laboratory GenoPom "Integrating post-genomic platforms to enhance the tomato production chain" (GenoPOMpro, Cod. PON02_00395_3082360). The funding company SINAGRI s.r.l. did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries to VdR and research materials. The specific roles of these authors are articulated in the ‘author contributions’ section.