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A V. californica that tested positive for GLRaV-3.
In an effort to identify possible alternate hosts for the grapevine leafroll- associated viruses and vitiviruses, numerous Vitis and non-Vitis species were surveyed within and surrounding nine vineyards in Napa County, CA, with vines showing typical leafroll disease symptoms. Three riparian areas in Napa and Yolo counties that were not near vineyards but that included a number of endemic Vitis spp. with red leaves were also identified.
Materials and methods
VINEYARDS AND SAMPLE COLLECTION: Nine Napa County vineyards were selected that had V. vinifera with red leaf symptoms typical of leafroll disease and were adjacent to areas with wild Vitis and non-Vitis species. Three riparian areas were also chosen that were not adjacent to vineyards, but had a number of non-cultivated Vitis with red leaves.
Outside the vineyard, Vitis spp. and the most common woody non-Vitis spp. that were present within a 25 m2 area adjacent to symptomatic V. vinifera blocks were collected. Within each vineyard, at least three vines from a block adjacent to non-cultivated Vitis and/or non-Vitis were collected.
Herbaceous plants were collected by establishing three 1 m2 plots located within 10 meters of the symptomatic V. vinifera collected the previous Fall. Within each plot, three replicate samples of all the dicotyledonous species were collected and pooled. Like species were also pooled between plots due to the large number of different species.
VIRUS DETECTION: Vitis and woody samples collected in Fall 2008 were assayed for GLRaV-1, -2, -3, -4, -5, -9, GVA, GVB, and GVD, by both conventional and real-time RT-PCR. Only real-time RT-PCR was used to detect these same viruses in herbaceous samples collected in Spring 2009, and Vitis and non-Vitis samples collected in Fall 2009 and 2010. Real-time RT-PCR assays were run in duplicate or triplicate, and data was analyzed quantitatively by measuring the cycle threshold (CT).
VIRAL DNA SEQUENCES: Total RNA from the samples positive by real-time RT-PCR was re-amplified, also using RT-PCR. Nucleic acid sequences were compared using BLAST from the National Center for Biotechnology Information, and aligned and edited.
VITIS GENOTYPE: The non-cultivated Vitis samples from outside the vineyards were analyzed at 18 Simple Sequence Repeat (SSR) DNA markers. The resulting DNA profiles were compared to over 1,200 unique grape DNA profiles. To identify the species background of the samples, a genetic approach was used to calculate the probability of observing each unique profile from this study and those of five diverse V. vinifera cultivars, based on gene frequencies from a population of 200 random V. vinifera
A hybrid of V. californica x V. vinifera ‘Merlot’ that tested positive for GLRaV-3.
cultivars. The results were expressed as the inverse of these probabilities divided by the average of the probabilities of the five V. vinifera profiles.
Results I: Plant survey
More than 600 plant samples (2008, 2009, 2010) were collected from in and around nine vineyards (Sites 1 to 9) and two riparian areas (Sites 10 and 11) in Napa County. Samples included wild Vitis, non-Vitis representing 83 species, and cultivated V. vinifera from within vineyards.
Results II: Wild Vitis genotypes
Leaf morphology indicated that the majority of the wild Vitis spp. were V. californica or V. californica x V. vinifera hybrids. This was also confirmed by genetic analyses. Leaves of V. californica x V. vinifera hybrids were generally heart-shaped like V. californica, with some lobing like V. vinifera.
DNA fingerprinting indicated 48 unique multilocus genotypes. Four of these were known scion or rootstock cultivars and were excluded from further analyses.
Figure 1. Grouping of Vitis genotypes. Each circle represents a unique genotype; solid circles are genotypes that are positive for GLRaV-2 and -3, GVA and/or GVB. The value shown is the inverse log10 of the probability of the genotypes being V. vinifera divided by the average probability associated with five diverse V. vinifera.
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Graph: Real-time RT-PCR output. Positive results are above the threshold (orange line).