RESUMO
Grapevine leafroll disease (GLRD) is the most globally prevalent and destructive disease complex responsible for significant reductions in grape yield and quality as well as wine production. GLRD is associated with several positive-strand RNA viruses of the family Closteroviridae, designated as grapevine leafroll-associated viruses (GLRaVs). However, the specific etiological role of any of these GLRaVs in GLRD has not been demonstrated. Even though GLRaV-3 is considered the chief GLRD agent, little is known about the molecular, cellular, and pathological properties of this virus. Such a knowledge gap is due to multiple factors, including the unavailability of biologically active virus cDNA clones and the lack of reliable experimental systems for launching grapevine infection using such clones. In this work, we tested four methods for inoculating tissue-cultured grapevine plantlets with cDNA clones of GLRaV-3: (i) vacuum agro-infiltration; (ii) agro-pricking; (iii) agro-drenching; and (iv) agro-injection. We showed that vacuum agro-infiltration was the most effective of these methods. Furthermore, we examined the impacts of different experimental conditions on the survival and infectivity rate of grapevines after infiltration. To verify the infectivity rate for different treatments, we used RT-PCR, RT-qPCR, and Western blotting. We found that humidity plays a critical role in the survival of plantlets after agro-infiltration and that the use of RNA silencing suppressor and dormancy treatment both had strong effects on the infection rates. To our knowledge, the experimental protocol reported herein is the most effective system for launching the infection of grapevine using cDNA clones of grapevine viruses featuring up to a 70% infection rate. This system has strong potential to facilitate grapevine virology research including the fulfillment of Koch's postulates for GLRD and other major virus diseases as well as identifying the molecular, cellular, and pathological properties of GLRaVs and, potentially, other important grapevine viruses.
RESUMO
BACKGROUND: In recent years, the Ontario grape and wine industry has experienced outbreaks of viral diseases across the province. Little is known about the prevalence of viruses and viral diseases in Ontario. Since 2015, we have conducted large-scale surveys for major viruses in commercial wine grapes in order to obtain a comprehensive understanding of the prevalence and severity of viral diseases in Ontario. METHODS: A total of 657 composite leaf samples representing 3285 vines collected from 137 vine blocks of 33 vineyards from three appellations: Niagara Peninsula, Lake Erie North Shore and Prince Edward County. These samples covered six major red cultivars and five major white grape cultivars. Using a multiplex RT-PCR format, we tested these samples for 17 viruses including those involved in all major viral diseases of the grapevine, such as five grapevine leafroll-associated viruses (GLRaV-1, 2, 3, 4, 7), grapevine red blotch virus (GRBV), grapevine Pinot gris virus (GPGV), grapevine rupestris stem sitting-associated virus (GRSPaV), grapevine virus A (GVA), grapevine virus B (GVB), grapevine fleck virus (GFkV), arabis mosaic virus (ArMV), tomato ringspot virus (ToRSV), trapevine fanleaf virus (GFLV), among others. RESULTS: Fourteen of the 17 viruses were detected from these samples and the predominant viruses are GRSPaV, GLRaV-3, GFkV, GPGV and GRBaV with an incidence of 84.0, 47.9, 21.8, 21.6 and 18.3%, respectively. As expected, mixed infections with multiple viruses are common. 95.6% of the samples included in the survey were infected with at least one virus; 67% of the samples with 2-4 viruses and 4.7% of the samples with 5-6 viruses. The major grape cultivars all tested positive for these major viruses. The results also suggested that the use of infected planting material may have been one of the chief factors responsible for the recent outbreaks of viral diseases across the province. CONCLUSIONS: This is the first such comprehensive survey for grapevine viruses in Ontario and one of the most extensive surveys ever conducted in Canada. The recent outbreaks of viral diseases in Ontario vineyards were likely caused by GLRaV-3, GRBV and GPGV. Findings from this survey provides a baseline for the grape and wine industry in developing strategies for managing grapevine viral diseases in Ontario vineyards.
