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Symptom Expression and Detection of Grapevine red blotch virus in Red and White Fruited Grape Varieties

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Abstract

Cycle ΔRn Grapevine red blotch associated virus (GRBaV) is a newly described virus associated with red blotch disease. Due to the similarity of foliar symptoms, the effect of GRBaV infection was confused with leafroll disease and/or other disorders that cause reddening in red-fruited grape varieties. In spite of its name, GRBaV is also detected in white-fruited varieties. Besides the typical foliar symptoms, GRBaV has been reported to affect sugar accumulation in grapevines reducing the Brix values and delaying the harvest of fruit. To determine if the detection of virus is seasonal, our lab tested GRBaV-infected vines throughout the year (2012-2014 fall/winter/spring/summer) using tissue from different sections of the vine. Vine samples were collected from basal and apical sections of the vine; green or lignified canes, cordon and trunk were tested. In addition, inflorescences from infected vines were dissected and tested. The results showed that the detection of GRBaV was equally sensitive throughout the season in all grapevine tissues tested. The presentation will also describe symptoms of white and red-fruited varieties infected with GRBaV.
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Grapevine red blotch associated virus (GRBaV) is a newly described virus associated with red blotch disease. Due to the
similarity of foliar symptoms, the effect of GRBaV infection was confused with leafroll disease and/or other disorders that
cause reddening in red-fruited grape varieties. In spite of its name, GRBaV is also detected in white-fruited varieties.
Besides the typical foliar symptoms, GRBaV has been reported to affect sugar accumulation in grapevines reducing the Brix
values and delaying the harvest of fruit.
To determine if the detection of virus is seasonal, our lab tested GRBaV-infected vines throughout the year (2012-2014
fall/winter/spring/summer) using tissue from different sections of the vine. Vine samples were collected from basal and
apical sections of the vine; green or lignified canes, cordon and trunk were tested. In addition, inflorescences from infected
vines were dissected and tested. The results showed that the detection of GRBaV was equally sensitive throughout the season
in all grapevine tissues tested. The presentation will also describe symptoms of white and red-fruited varieties infected with
GRBaV.
INTRODUCTION
RESULTS AND DISCUSSION
The testing results revealed the presence of GRBaV in every infected tissue sampled regardless of position, age, season
and/or method used (Figs. 2 and 3 and data not shown).
The sensitive q-PCR assay allowed the detection of several fold diluted GRBaV nucleic acid extracts as well as nucleic acid
extracts from composite samples collected in different vineyards (Figs. 4 and 5 and data not shown). Composite sampling can
significantly reduce testing costs allowing the combination of samples collected from different vines.
The addition of q-PCR to our tool kit fulfills Eurofins STA Plant Health Services testing strategy as it allows the application of
different methodologies for the detection of the same pathogen(s).
CONCLUSIONS
The distribution of GRBaV appears to be uniform in the infected grapevines tested
High concentration of virus was detected regardless of the tissue tested: apical shoots, apical and basal leaves, petioles
from basal and apical leaves, leaf blades or veins, lignified and green canes, flowers and fruits, inflorescence rachis, etc.
GRBaV can be readily detected in vineyard composite samples (we suggest up to five vines per sample)
Grapevine red blotch associated virus (GRBaV) is reported to infect vineyards throughout the United States and Canada. In
spite of its name, the virus infects both red- and white-fruited grapevine varieties (Fig. 1). Besides the typical foliar
symptoms, GRBaV has been reported to affect sugar accumulation in grapevines and delayed fruit maturation.
At present, information is lacking on the biology of GRBaV as it relates to distribution in the vine, transmission, and spread.
Prior to this study, the detection of GRBaV was limited to samples collected from dormant and lignified tissues.
Here we report extending the detection of GRBaV to actively growing grapevine tissue. In addition, different polymerase
chain reaction (PCR) techniques were validated to determine the best sampling strategy for optimal GRBaV detection.
Plant Health Services, Eurofins STA Laboratories, 7240 Holsclaw Rd. Gilroy, CA 95020
*E-mail: JuditMonis@EurofinsUS.com
Judit Monis*, Laura Miles, Ngoc Le, and Mitchell Vernon
Symptom Expression and Detection of Grapevine red blotch virus in
Red and White-Fruited Grape Varieties
Figure 1. Grapevine red blotch associated virus symptoms in Vitis vinifera L. (A) cv. Tannat (red
fruited) and (B) cv. Sauvignon Blanc (white fruited).
SAMPLING AND DIAGNOSTIC METHODS
At Eurofins STA Laboratories, HealthCheckTM Panel RB was developed to reliably detect the GRBaV using virus-specific primers
and PCR. More recently, we developed and validated a sensitive assay using quantitative (q) PCR, also known as real-time PCR
for the detection of GRBaV.
Due to seasonal availability, the initial samples used consisted of grapevine lignified tissue (i.e., canes, cordons and trunk).
During the spring and summer seasons, tests were performed with actively growing (green) tissue and compared with lignified
tissues. Vines infected with GRBaV (and uninfected controls) were dissected to test for the presence of GRBaV in different
plant parts: apical and basal leaves, lignified and green canes, petioles from basal and apical leaves, flowers and fruits,
inflorescence rachis, etc. (Fig. 2 and data not shown).
In addition, known GRBaV-infected and uninfected vines were combined to determine the effectiveness of our detection
methods in vineyard composite samples.
Figure 2. Dissection of a grapevine plant. (A) Apical and basal leaves, (B) basal canes with
attached leaves and developing grape clusters, (C) green and lignified canes, and (D) cordon and
trunk sections.
A
A
Figure 3. Detection of Grapevine red blotch associated virus (GRBaV) by (A) q-PCR (one virus-
specific primer set) and (B) using two different primer sets by conventional polymerase chain
reaction (PCR). Seven grapevine nucleic acid extracts were obtained from a mix of leaves from
different positions (lanes 1), apical leaves (lanes 2), young expanded leaves (lanes 3), basal leaves
(lanes 4), petioles from basal leaves (lanes 5), apical internode (lanes 6), and basal internode
(lanes 7). Positive (+), No Evidence (N E) of infection controls, and marker (M) are shown to the
left.The grapevine nucleic acid internal control of amplification is shown at the bottom.
M + N E 1 2 3 4 5 6 7
Internal
control
1000
700
500/525
400
300
200
100
50
B
B
CD
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A B
M 1 2 3 4 5 6 7
1000
700
500/525
400
300
200
100
50
Figure 4. Detection of Grapevine red blotch associated virus (GRBaV) by (A) q-PCR (one virus-
specific primer set) and (B) conventional PCR (two virus-specific primer sets). The assay sensitivity
was appraised using undiluted and six 10-fold serial dilutions of a known GRBaV infected grapevine
nucleic acid extraction (i.e., positive control). The marker (M) is shown to the left.
Figure 5. Detection of Grapevine red blotch associated virus (GRBaV) by q-PCR (one virus-specific
primer set) in composite samples. The proportion of healthy tissue to GRBaV-infected tissue is
shown on the table to the right.
Sample Proportion
Healthy/Positive
S1 5/1
S2 4/2
S3 3/3
S4 2/4
S5 5/0
S6 0/5
S5
S1-3
S4, S6
ABSTRACT
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