Molecular characterisation of Grapevine leafroll-associated virus 3 and implications for diagnostic testing and pathogenicity

Abstract

Grapevine leafroll-associated virus 3 (GLRaV-3) is an economically important virus that is found in all grapevine growing regions worldwide. Currently, the main strategies used to mitigate the negative impacts of GLRaV-3 on the wine industry is to prevent further introduction of GLRaV-3 by using certified planting material and reduce spread of GLRaV-3 in vineyards by roguing infected plants. Both of these approaches require a reliable and sensitive diagnostic tool. However, genetic variability within the virus population can compromise detection. Studies have shown high genetic variability in GLRaV-3 populations from different countries, but little was known about genetic variability of GLRaV-3 in New Zealand. This project examined the genetic variability within the New Zealand GLRaV-3 population to ensure that diagnostic tests would detect all known variants. In addition, knowledge of the genetic variability was used to aid the understanding of the virus biology, epidemiology, and evolution. New Zealand GLRaV-3 variants from phylogenetic groups 1, 2, 3, 5, and 6, plus variants with more than 20% nucleotide identity to previously published GLRaV-3 variants, were identified. Sequences from these and overseas isolates were used to design and develop generic and variant-specific conventional, multiplex, and real-time RT-PCR detection assays. In addition, for the quantification of GLRaV-3 variants, ten host genes were evaluated as reference genes for real-time RT-PCR assays. GLRaV-3 variants from group 1, group 6 (specifically NZ-1), and NZ2 were graft transmitted to eight cultivars. For all cultivars, infections generally resulted in reduced growth of shoots compared to healthy controls, and for red cultivars, infections lead to typical premature leaf reddening. The molecular detection assays were used to study the spatial distribution of specific GLRaV-3 variants within a germplasm block and a commercial field plot, and within individual graft inoculated plants. The GLRaV-3 variants were found to be unevenly distributed within plants, as leaf tested from the basal position of shoots were more likely to yield reliable results, compared to the middle and apical shoot positions, and the spatial patterns were consistent with mealybug transmission within the field plots.