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
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
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
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.
GENOTYPE: The non-cultivated
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
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
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.
Graph: Real-time RT-PCR output. Positive results are above the threshold (orange line).