A novel specific duplex real-time RT-PCR method for absolute quantitation of Grapevine Pinot gris virus in plant material and single mites.

Félix Morán,Nikolaos Katis,Antonio Olmos,Varvara Maliogka,Ana B Ruiz-García,Leonidas Lotos,Lukáš Predajňa,Miroslav Glasa

doi:10.1371/journal.pone.0197237

Abstract

Grapevine Pinot gris virus (GPGV) is a widely distributed grapevine pathogen that has been associated to the grapevine leaf mottling and deformation disease. With the aim of better understanding the disease epidemiology and providing efficient control strategies a specific and quantitative duplex TaqMan real-time RT-PCR assay has been developed. This method has allowed reliable quantitation of the GPGV titer ranging from 30 up to 3 x 108 transcript copies, with a detection limit of 70 viral copies in plant material. The assay targets a grapevine internal control that reduces the occurrence of false negative results, thus increasing the diagnostic sensitivity of the technique. Viral isolates both associated and non-associated to symptoms from Greece, Slovakia and Spain have been successfully detected. The method has also been applied to the absolute quantitation of GPGV in its putative transmission vector Colomerus vitis. Moreover, the viral titer present in single mites has been determined. In addition, in the current study a new polymorphism in the GPGV genome responsible for a shorter movement protein has been found. A phylogenetic study based on this genomic region has shown a high variability among Spanish isolates and points to a different evolutionary origin of this new polymorphism. The methodology here developed opens new possibilities for basic and epidemiological studies as well as for the establishment of efficient control strategies.

Highlights

Grapevine Pinot gris virus (GPGV) is a member of the genus Trichovirus, family Betaflexiviridae
In order to decrease the number of putative false negative results, namely positive samples not detected by the method, an internal control of the grapevine phosphoenolpyruvate carboxylase gene (PEP) gene was included in the assay as a duplex RT-PCR reaction
The possible effect of a duplex RT-PCR reaction in GPGV detection technical sensitivity was assessed by the comparison of the signal obtained in a singleplex assay and the signal obtained in a duplex assay in several ten-fold serial dilutions of GPGV infected plant extracts with healthy plant extracts (Fig 1)

Summary

Introduction

Grapevine Pinot gris virus (GPGV) is a member of the genus Trichovirus, family Betaflexiviridae. European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 734736. The research was conducted within the framework of COST Action FA1407. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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