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.

Valentina Di Rienzo,Cinzia Montemurro,Fabrizio Cillo,Giovanni Bubici,Shu-Biao Wu

doi:10.1371/journal.pone.0196738

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.

Highlights

Tomato spotted wilt virus (TSWV) [1] is the type member of the plant-infecting genus Tospovirus in the family Bunyaviridae [2]
Leaves were harvested from field crops showing viral symptoms at different locations in Southern Italy, where TSWV outbreaks have occurred on Sw-5-carrying tomato hybrids in recent years
From a selected number of field samples, the cDNA fragment corresponding to the viral NSm gene was RT-polymerase chain reaction (PCR) amplified and sequenced to test consistency between sequence- and Real Time PCR-based methods (i.e. high-resolution melting (HRM) and TaqMan probes)

Summary

Introduction

Tomato spotted wilt virus (TSWV) [1] is the type member of the plant-infecting genus Tospovirus in the family Bunyaviridae [2]. The TSWV genome consists of three negative-sense or ambisense RNA segments denoted as L, M and S. Segment L is a negative sense RNA that encodes the viral component of the RNA dependent RNA polymerase [3]. TSWV SNI and SRB biotypes allelic discrimination 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

Methods

Results

Conclusion