Specific Real-Time PCR for the Detection and Absolute Quantitation of Grapevine Roditis Leaf Discoloration-Associated Virus, an EPPO Alert Pathogen.

Félix Morán,Antonio Olmos,Celia Canales,Ana Belén Ruiz-García,Chrysoula-Lito Sassalou,Varvara I Maliogka

doi:10.3390/plants9091151

Abstract

Grapevine Roditis leaf discoloration-associated virus (GRLDaV) is an emerging grapevine pathogen included in the European and Mediterranean Plant Protection Organization (EPPO) alert list due to its ability to damage grapevine crops and cause production losses. This work aimed to develop a specific and reliable diagnostic tool that would contribute to preventing the spread of this pathogen. Therefore, a TaqMan real-time quantitative PCR was developed. The method was validated according to EPPO guidelines showing a high degree of analytical sensitivity, analytical specificity, selectivity, and repeatability and reproducibility. The sensitivity of this method is much higher than the sensitivity reached by previously reported methods even when tested in crude extracts, which could allow rapid testing by avoiding nucleic acid extraction steps. The method was also able to detect GRLDaV isolates from all the geographic origins reported so far, despite their high degree of genetic diversity. In addition, this new technique has been successfully applied for the quantitative detection of GRLDaV in plant material and two mealybug species, Planococcus citri and Pseudococcus viburni. In conclusion, the methodology developed herein represents a significant contribution to the diagnosis and control of this emerging pathogen in grapevine.

Highlights

Grapevine (Vitis vinifera) is one of the most ancient crops cultivated worldwide for the production of both fresh grapes and wine, which represents an important source of income for many countries.Europe is the leading global wine producer, with about 50% of the world’s vine-growing area, with 3.3 million ha (Food and Agriculture Organization of the United Nations Statistics Division, FAOSTAT, 2018)
We report the development of a specific real-time qPCR method that allows the detection and absolute quantitation of Grapevine Roditis leaf discoloration-associated virus (GRLDaV), a grapevine virus included in the European and Mediterranean Plant Protection Organization (EPPO) alert list as a potential phytosanitary risk for the EPPO region
The GRLDaV genomic sequence shows a high degree of variability, related to its geographical distribution, which might compromise its reliable diagnosis

Summary

Introduction

Grapevine (Vitis vinifera) is one of the most ancient crops cultivated worldwide for the production of both fresh grapes and wine, which represents an important source of income for many countries.Europe is the leading global wine producer, with about 50% of the world’s vine-growing area, with 3.3 million ha (Food and Agriculture Organization of the United Nations Statistics Division, FAOSTAT, 2018). Grapevine (Vitis vinifera) is one of the most ancient crops cultivated worldwide for the production of both fresh grapes and wine, which represents an important source of income for many countries. The grapevine has been described as the crop that hosts the largest number of plant viruses, with more than 85 viral species known to infect this important crop to date [2,3]. Grapevine Roditis leaf discoloration-associated virus (GRLDaV) was identified as a new member of the family Caulimoviridae, genus Badnavirus, infecting grapevine in 2015 [4]. GRLDaV has been associated with a grapevine disease reported in Greece more than thirty years ago, the Roditis leaf discoloration disease (RLD), which produces discolorations and deformations of the leaves, reduction in the number and size of grape berries, and a lower sugar content [5]. No biological vectors have been identified to transmit GRLDaV to date, most badnaviruses are spread by different mealybug species [6]

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