Grapevine Leafroll Associated Virus -3 Seasonal Titer and Effects on Pinot Noir Fruit in Oregon
Sarah K. Wright1,, Laurent Deluc, PhD2, Robert R. Martin, PhD3
1BioResource
Research Program, and 2Department of Horticulture, Oregon State University
3USDA-ARS Horticultural Crops Research Unit – Corvallis, OR 97330
Introduction
GLRaV-3 Titer of Middle Leaves
GLRaV-3 Titer of Mature Leaves
12
Gloire
Pommard
10
Pommard
10
Poly. (Gloire)
Poly. (Pommard)
8
Gloire
Gloire
6
4
2
Poly. (Gloire)
Poly. (Pommard)
8
6
4
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Poly. (Pommard)
8
 Sugar content, elasticity and color index properties of four
berry ripening classes at mid-verasion for infected plants all
show variation.
6
4
0
0
20
0
1
2
3
4
Week (June - October)
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
0
20
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Week (June - October)
Week (June – October)
Figure 2. Year 2010 scatter plots of each sample; relative quantification of GLRaV-3 titer is measured as ∆∆Ct values against weeks throughout growing season. Sampling began the first week of June and continued for 20 weeks.
Materials and Methods
GLRaV-3 Titer of Young Leaves
GLRaV-3 Titer of Middle Leaves
12
12
Gloire
 GLRaV-3 RNA was quantified using
One-Step
RT-PCR Kit (Applied Biosystems, USA), with primers we
designed for amplification and the Taqman® probe (Fig. 1). A
qPCR assay for the host RNA of the NADHβ subunit, was
used to monitor RNA extraction, RT, and PCR efficiency and
standardize RNA concentrations.
AgPath-IDTM
 GLRaV-3 titers were compared
to the titer obtained the first
week and to healthy plants.
Poly. (Pommard)
8
6
4
Poly. (Gloire)
Poly. (Pommard)
8
6
4
Poly. (Gloire)
6
2
0
0
0
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0
1
2
3
4
Week (June - October)
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
References
4
2
1
Poly. (Pommard)
8
2
0
Figure 5. 'Pinot Noir' wine grape (Vitis vinifera) with
grapevine leafroll virus (GLRaV-3). Copyright
Melodie Putnam 2008, Oregon State University
Pommard
10
ΔΔCт Values
Poly. (Gloire)
Gloire
Pommard
10
ΔΔCт Values
ΔΔCт Values
 Young, middle aged and mature leaf tissue was collected
weekly during the growing season, from 6 vines each of selfrooted (‘Pommard’ clone) and grafted (clone ‘114’ on ‘Gloire’
rootstock) infected with GLRaV-3 from a commercial vineyard
in the Willamette Valley,
Gloire
Pommard
20
0
1
2
3
4
Week (June - October)
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Week (June - October)
Figure 3. Year 2011 scatter plots of each sample; relative quantification of GLRaV-3 titer is measured as ∆∆Ct values against weeks throughout growing season. Sampling began the first week of June and continued for 20 weeks.
Mean sugar content values in healthy berries converged around 26% by harvest in each mid-verasion class; whereas berry classes
from GLRaV-3 infected plants remained highly variable. Mean elasticity measurements displayed an unusual trend suggesting errors in
recording or measurement. However, the lines for healthy berry classes still converged by the last week more than infected. Mean color
index results indicated that healthy mid-verasion berry classes developed more color by harvest than infected classes, which also show
unevenness near the final week.
Elasticity Measurement Results
J. and Martin, R.R. (2009) Influence of grapevine leafroll
associated viruses (GLRaV-2 and -3) on the fruit composition of
Oregon Vitis vinifera L. cv. Pinot noir: Phenolics. Food chemistry
112: 889-896..
2Singh
Brar H, Cameron I, Swinny E, Singh Z. (2008). Girdling
and grapevine leafroll associated viruses affect berry weight,
colour development and accumulation of anthocyanins in
'Crimson Seedless' grapes during maturation and ripening. Plant
Science 175:885-897..
J, et al. (1995) Proposal of an index for the objective
evaluation of the colour of red table grapes. Food Research
International 28:373-377.
Color Index Results
3.0
Infected GH
26
Infected GS
Infected PS
24
Infected RS
Healthy GH
22
Healthy GS
Healthy PS
20
Healthy RS
Poly. (Infected GH)
18
Poly. (Infected GS)
Poly. (Infected PS)
16
Poly. (Infected RS)
Poly. (Healthy GH)
14
Poly. (Healthy GS)
Poly. (Healthy PS)
12
1
2
3
4
5
Week (mid-verasion – harvest)
Poly. (Healthy RS)
Infected GH
2.5
Infected GS
Infected PS
Infected RS
2.0
Healthy GH
Healthy GS
Healthy PS
1.5
Healthy RS
Poly. (Infected GH)
1.0
Poly. (Infected GS)
Poly. (Infected PS)
Poly. (Infected RS)
0.5
Poly. (Healthy GH)
Poly. (Healthy GS)
Poly. (Healthy PS)
0.0
1
2
3
4
5
Week (mid-verasion – harvest)
Poly. (Healthy RS)
Average Color Index Values
28
Average Elasticities (MPa)
 Samples were measured for elasticity using an adapted
skinfold caliper, color using a chromameter with CIELAB
parameters3 for red grapes, and total soluble solids (brix
degree) using a digital refractometer. Comparisons between
healthy and infected grapes were made for a 5-week span.
Brix Measurement Results
Average Sugar Content (%)
 Each week from mid-véraison to harvest, one berry per class,
per cluster, per plant was sampled, n=144.
1Lee,
3Carreno
Figure 1. Example of qPCR amplification
curve with 10-fold dilutions.
 864 berries representative of four ripening transition classes
were tagged (colored strings) on 3 healthy and 3 GLRaV-3
infected vines; 6 berries per class (n=4) per cluster (n=6) per
plant (n=6).
 Berries of healthy plants
show relative
convergence among
mid-verasion classes
for all properties.
GLRaV-3 Titer of Mature Leaves
12
10
 GLRaV-3 titer in younger leaves builds throughout the
growing season.
Poly. (Gloire)
2
2
0
Pommard
10
 GLRaV-3 virus titer shows a dramatic increase during the
month of June, suggesting transmission by vectors could
occur during most of the season. Week –to-week fluctuations
once maximum titer is reached may be due to environmental
conditions, such as collection on extremely warm days.
 RNA from ndhB in the 2010 ‘Pommard’ mature collection was
not thoroughly extracted during the season.
12
ΔΔCт Values
Detection of GLRaV-3 by ELISA and PCR tests is more reliable
later in the season (September) than early (June). If virus titer is
correlated with ease of detection, the virus may not be
transmitted early in the season, and then pesticide application
during this ‘low titer’ time would be unnecessary.
GLRaV-3 Titer of Young Leaves
12
ΔΔCт Values
GLRaV-3 causes reddening in leaves and uneven ripening of the
fruit that leads to negative effects on phenolic composition, color
development and skin elasticity in grapes as they ripen.1,2 The
impacts of GLRaV-3 on fruit, can lead to major impacts on wine
quality.
Conclusions
The ∆ ∆Ct values in each graph below show relative exponential increase, assuming 100% PCR efficiency, of virus titer compared to
Week 1 samples. The graphs from 2010 contain some samples that did not properly amplify, which are visible in the line breaks. Virus
titers in young leaf tissue from each group increased by as much 2(12) (2(∆∆Ct)), approximately a 4000-fold increase, during the growing
season for both years. In middle aged leaf tissue the virus titer increased by 2(7) during the growing season in both years. With the
Pinot noir clone ‘114’ grafted onto ‘Gloire’ the virus titer in the oldest leaves in 2010 increased by 2(7) , while in self-rooted ‘Pommard’
the RNA was not amplified for ndhB, suggesting problems with the RNA extraction from that tissue. In 2011, the virus titer in the oldest
leaves of both cultivars increased; by 2(6) for ‘114’ on ‘Gloire’, and 2(4) for the ‘Pommard’, respectively, relative to the target sample.
ΔΔCт Values
The Willamette Valley, west of the Cascades in Oregon, is home
to a growing number of vineyards. This region is best known for
producing high quality Pinot noir wines. Grapevine leafroll
associated Virus -3 (GLRaV-3), genus Ampelovirus, family,
Closteroviridae, which is transmitted by mealybugs and scale
insects, is a new threat to this industry. If mealybugs are present,
growers use pesticides to prevent them from spreading the virus.
Results
30.0
Infected GH
Infected GS
Infected PS
25.0
Infected RS
Healthy GH
Healthy GS
20.0
Acknowledgements
Healthy PS
Healthy RS
Poly. (Infected GH)
15.0
Poly. (Infected GS)
Poly. (Infected PS)
Poly. (Infected RS)
10.0
Poly. (Healthy GH)
Poly. (Healthy GS)
Poly. (Healthy PS)
5.0
1
2
3
4
5
Week (mid-verasion – harvest)
Figure 4. Scatter plots of Brix (sugar) analysis, Elasticity, and Color Index. Each factor is compared between healthy and infected plants. Weeks of sampling begin at mid-verasion (1) until harvest (5).
Poly. (Healthy RS)
Thanks to everyone at the USDA-ARS Horticultural Crops
Research Unit. This research received funding assistance from
the E.R. Jackman Internship Support Fund, the American Seed
Trade Association, the Oregon Seed Trade Association, and
SCRI grant 2009-02765.