Grapevine Roditis leaf discoloration-associated virus (GRLDaV) was identified as a new member of the family Caulimoviridae, genus Badnavirus, infecting grapevine in 2015 . 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 . The analysis by high throughput sequencing (HTS) of a grapevine sample showing typical RLD symptoms from a Greek vineyard allowed the identification of GRLDaV and the recovery of its full genome . The GRLDaV genome consists of a single molecule of circular double-stranded DNA (between 6988 and 7097 nt in length) that includes four open reading frames (ORFs) and a non-coding region. ORFs 1, 2, and 4 encode for hypothetical proteins of unknown function. ORF3 encodes a large polyprotein containing the movement protein (MP), the coat protein (CP), the protease, the reverse transcriptase (RT), and the RNaseH . GRLDaV has been shown to be mechanically transmissible. Although no biological vectors have been identified to transmit GRLDaV to date, most badnaviruses are spread by different mealybug species .
After its first identification in Greece, GRLDaV has also been reported in Italy , Turkey , and Croatia . The previous evidence of the ability of this virus to damage grapevine crops and to cause losses, along with its emergence in several European countries, point to the need for the control and prevention of its spread. In fact, GRLDaV was included in the European and Mediterranean Plant Protection Organization (EPPO) alert list as a potential phytosanitary risk for the EPPO region in October 2018. Management and control of viral pathogens and prevention of their spread lay on early detection based on specific and reliable diagnostic methods. Quantitative real-time PCR methods have been shown to be powerful tools for viral epidemiological studies and viral detection [10-15]. This work aimed to develop a specific real-time PCR detection method that can perform an absolute quantitation of GRLDaV genetic material in both plant material and mealybugs. Such a technique could be applied to epidemiological studies and would provide a powerful tool to address control strategies designed to prevent GRLDaV spread.
2.1. Detection of GRLDaV by Real-Time qPCR
2.1.1. Design of Real-Time qPCR Primers and Probe
A nucleotide sequence alignment of the GRLDaV complete genomic sequences available in GenBank, from isolates W4 (Crete, Greece), BN (Italy), and VLJ-178 (Croatia) showed a high degree of divergence between them, with an overall nucleotide identity ranging from 84.24% to 89.31%, although the similarity between the sequences was quite variable throughout the genome. With the aim of selecting a target genomic region that could provide specific, broad, and reliable GRLDaV detection, a more in-depth study was conducted in the RNaseH domain inside the ORF4, one of the most conserved genomic regions used for genus Badnavirus specific detection . The sequence variability study in this genomic region led to the selection of a small region of 211 nt (ref-seq genomic positions 5951 to 6161, accession number NC_027131.1), with a nucleotide identity ranging from 85.78% to 93.36% between isolates, as a candidate for primers and probe designing. A larger genomic fragment of 775 nt containing the region of study was amplified by PCR from nine GRLDaV isolates from different geographical origins (Greece and Turkey). The sequences were aligned with the homologous genomic region of the full-length sequences available in the databases from Greece, Italy, and Croatia.
The sequence alignment showed a highly conserved small region that could be a good candidate for the TcqMan probe. Regions surrounding; the alignment site showed some sequence variabflity that was related to the isolates' geographical origins. That variability could be overcome by designing either degenerated peimers or several primers for use; in multiplex PCR. Due to the high number of degenerations fo Ice introduced, thd use of degenecated primers could cceatf preblems related to primer concentration that could compromise detection sensitivity, ae degenerations reduce the final concentration of oach particular sequence. Taking this into account, the multiplexing approach was selected.
Thus, five primers able to cover all the variability observed in the sequence alignment and specifically amplify a region of 196 nt (ref-seq genomic positions 5960 to 6155, accession number NC_027131.1) from all isolates having different geographic origins were designed