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An Overview of Grapevine Viruses in
Washington Vineyards
Naidu Rayapati Associate Professor (Virology) Department of Plant Pathology
Irrigated Agriculture Research and Extension Center Washington State University
Prosser, WA 99350 [email protected]
509-786-9215
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Good Fruit Grower May 15, 2015. Vol. 66 (No. 10), pages 10-11.
Estimated economic impact of leafroll in Merlot over a 20-yr period:
A grower with 100 acres: $300,500 to $1,980,000 (or more with higher
crop losses)
A grower experiencing • 10% decrease in yield and
0.4° Brix reduction = $3,005 per acre • 30 percent yield decline and
1.0° Brix decrease = $19,800 per acre
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The highest ranked topic with a score of 4.5 out of 5.0:
• the impact of viticultural practices on wine flavor
The second ranked topics with a score of 4.0 out of 5.0
• grapevine viruses • fermentation practices
Washington State Wine Commission Survey
Source: http://www.goodfruit.com/wine-grape-revelations/ July 5, 2016
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Grape Virology Program
Aligned with industry’s priorities
Grape and wine industry research priority Management of viruses that impact
vine health and fruit quality
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Research on critical topics
• How many viruses are present in WA ? • Impact of viruses. • Spread, with emphasis on leafroll disease.
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Grapevine is a ‘treasure trove’ of viruses
AT LEAST 70 VIRUSES AND VIRUS-LIKE AGENTS
REPORTED WORLDWIDE Martelli GP. 2014. Directory of virus and virus-like diseases of the grapevine
and their agents. J. Plant Pathol. 96 (Suppl. 1):1–136.
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• Conducted surveys in vineyards • Optimized sampling strategies • Tested samples using state-of-
the-art diagnostic methods - PCR technology - Next-generation sequencing
Current status of grapevine viruses
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GLRaV-1 GLRaV-2 GLRaV-3 GLRaV-4
GLRaV-4 (strain -5) GLRaV-4 (strain -9)
GRSPaV GVA GVB GVE GFLV TRSV GFkV
GSyV-1 GRBaV
Leafroll complex
Rugose wood complex
Fanleaf degeneration/decline Fleck
Syrah decline?? Red blotch
Current status of grapevine viruses As of 2016
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Total # of vines tested=2083
4.7 11.0
0.3
50.7
9.6 2.9 1.3
9.5 14.2
4.1 3.5
0.0
10.0
20.0
30.0
40.0
50.0
60.0
Freq
uenc
y (%
)
LR1 LR2 2RG LR3 LR4 LR5 LR9 RSP GVA GVB GFLV
Viruses
Current status of grapevine viruses
Leafroll Rugose wood
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Current status of virus diseases
Established • Leafroll
Emerging • Red blotch • Soil-borne
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Grapevine leafroll disease
Naidu, 2011
Naidu et al., 2014. Plant Disease 98:1172-1185.
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Red grape varieties e.g. Cabernet Sauvignon
White grape varieties e.g. Chardonnay
Wine grape cultivars respond differently to leafroll disease
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Red blotch
Good Fruit Grower. April 1, 2013. Vol. 64 (No. 7), pages 44-46.
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• Green veins • Interveinal reddening • Downward rolling of leaf margins
Leafroll symptoms
cv. Merlot
Red blotch symptoms
• Red veins • Red blotches • Interveinal reddening
cv. Merlot
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Is it leafroll or red blotch?
cv. Cabernet franc
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Difficulty with symptom-based diagnosis
cv. Cabernet Sauvignon cv. Syrah cv. Petite Sirah
Red blotch
Leafroll
Red blotch
Leaf roll
Red blotch
Leafroll
Is it leafroll or red blotch?
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cv. Chardonnay
cv. Semillon
cv. Sauvignon Blonc
Is it leafroll or red blotch?
Difficulty with symptom-based diagnosis
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• Survey conducted in 2014 & 2015 seasons in five AVAs.
• Red-berried cultivars: samples from grapevines exhibiting symptoms of GLD or GRBD and suspected for GLD- or GRBD-like symptoms.
• White-berried cultivars: random samples from grapevines due to the absence of visual symptoms of GLD and GRBD.
Leafroll (GLD) and red blotch (GRBD) in
WA vineyards
http://www.washingtonwine.org
Adiputra et al., unpublished data
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Adiputra et al., unpublished data
~1,500 samples during 2014 and 2015 seasons: 14 Red-fruited + 5 White-fruited Tested by RT-PCR (GLRaV-3) and PCR (GRBaV)
Leafroll and red blotch in WA vineyards
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Fanleaf degeneration/decline
cv. Grenache
cv. Cab franc
cv. Merlot
cv. Grenache
cv. Grenache
cv. Grenache
cv. Merlot
Grapevine fanleaf virus Tobacco ring spot virus
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Fanleaf degeneration/decline
cv. Grenache
cv. Cab franc
cv. Merlot
cv. Grenache
cv. Grenache
cv. Grenache
cv. Merlot
Grapevine fanleaf virus – Xiphinema index absent Tobacco ring spot virus – Xiphinema americanum present
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• Leafroll (GLRaV-3) - predominant & widely distributed • Red blotch (GRBaV) - less predominant compared to GLRaV-3 • Soil-borne viruses (TRSV) • Co-infections can occur
• Symptoms are similar, though not identical, in many red-berried cultivars
• Like leafroll, no apparent symptoms of red blotch in white-berried cultivars
• Symptom-based diagnosis is not reliable and diagnostic assays should be used for reliable ID
Current status of virus diseases
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Issues regarding sampling and virus testing
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Disease Early season Late season (Spring) (Fall)
Fanleaf Yes Yes Leafroll No Yes Red blotch No Yes
When to look for symptoms ?
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Pre-Véraison
Post-Véraison
Leafroll Red blotch When to collect samples ?
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-0.5
0
0.5
1
1.5
2
2.5
3
O.D
5/15 5/25 6/12 6/28 7/18 8/3 8/17 8/31 9/17 9/29 10/12 11/2 +ve -ve control control
Cabernet sauvignon Chardonnay Pinot noir Merlot GLRaV-3 titer (by ELISA)
Sampling dates
No correlation between presence of virus and time of symptom expression
Pre-veraison Post-veraison
When to collect samples ?
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When to collect samples ?
Symptoms on foliage during the season
Canes during winter/dormancy period
show no symptoms
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Collect leaf samples with petioles In
Win
ter
D
urin
g th
e se
ason
Collect cane samples 3-4 nodes length
When to collect samples ?
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What type of test to use?
(+) (-) Samples
Serological assays (ELISA)
Molecular assays (PCR)
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0
0.2
0.4
0.6
0.8
1
1.2
ELISA
RT-PCR
+ - 1 2 3 4 5 6 7 8 9 10 11 12 13 14
+ - 1 2 3 4 5 6 7 8 9 10 11 12 13 14
332 bp
GLRaV-2 isolates
OD
(A40
5)
Limitations of ELISA Variability among viruses (e.g. GLRaV-2 strains)
e.g. Red blotch virus can be tested only by PCR
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What type of samples? Leaf
Cane
Detection by PCR methods
Mr -ve +ve 1 2 3 4 5 6 7 8 9
Samples
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Take-home message • Viruses distributed systemically in different
parts – can use leaves, canes, and roots of an infected grapevine for testing.
• Viruses can be detected through out the
season using leaf (petioles) and cane samples.
• Viruses can be detected using dormant canes
in winter.
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Impact of grapevine viruses
Leafroll Red blotch
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Healthy Infected Healthy Infected
Grapevine leafroll disease Impact on fruit yield
cv. Cabernet Sauvignon cv. Merlot
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0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
2008 2009 2010 2011 2012
Tota
l wei
ght o
f clu
ster
s pe
r vin
e (K
g)
Year
-17.17%
-27.82%
-15.82%
-20.57% -11.89%
Leafroll : Impact on yield (cv. Merlot)
No leafroll Leafroll
Average loss/year ~25%
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cv. Cabernet Sauvignon cv. Pinot noir
Infected Healthy
Infected Healthy
Infected Healthy
Infected Healthy
cv. Cabernet franc cv. Chardonnay
Leafroll disease Impact on fruit quality
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*, p = 0.05-0.01; **, p = 0.01-0.002; ***, p < 0.002
Year Healthy Leafroll Difference 2009 24.83 ±0.09 23.30 ±0.10 - 6.16% 2010 25.03 ± 0.32 23.10 ±0.29 - 7.71% 2011 23.50 ±0.06 22.53 ±0.15 - 4.13% 2012 25.96 ±0.05 24.58 ±0.00 - 5.31%
*** * ** ***
Total soluble solids (OBrix) at harvest (cv. Merlot)
Leafroll : Impact on fruit quality
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PLoS ONE 11 (2016): e0149666
Impacts of leafroll
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“The impacts of leafroll on yield and fruit and wine quality traits were variable between the seasons, with greater impacts observed during a cooler season, suggesting the influence of Genotype (G)-by-Environment (E) interactions on overall impacts of the disease”
Key findings
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Impacts on fruit yield cv. Merlot
Grapevine Red Blotch Disease
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cv. Merlot
Year Healthy Infected 2013 24.18 21.00 -13.15% 2014 24.36 21.46 -11.90% 2015 25.32 22.78 -10.03%
Total soluble solids (°Brix)
Impacts of red blotch Yield
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• Both leafroll and red blotch affect fruit yield and quality in the cv. Merlot examined.
• Impacts on berry sugars and anthocyanins more
pronounced than grape juice pH and TA.
Summary points
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Spread of grapevine viruses
All viruses can be spread via planting materials
No chemicals to cure infection and make a sick plant healthy
Prevention better than cure - use ‘clean’ plants - control vectors
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Virus spread via top-grafting
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The ‘Mantra’ of ‘start clean’ Use virus-tested ‘clean’ plants as
the first line of defense
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Source of planting stock matters
Nurseries
Healthy vineyards
http://cpcnw.wsu.edu
Clean Plant Center
Northwest
Neighbors/ Friends/Your own blocks
Outside the state
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Blocks planted with virus-tested ‘clean’
cuttings
Source of planting stock matters
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5.5 acre block Symptomatic vines – 11.8% GLRaV-3 +ve Cabernet Sauvignon
Spread via compromised planting stock
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11.8% in 2015
1.0% in 2016
Roguing
cv. Cabernet Sauvignon 2015 planting Spread via compromised planting stock
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3.5% in 2015
Roguing
cv. Syrah 2015 planting Spread via compromised planting stock stock
0.05% in 2016
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Test source vines before planting new vineyards
Cuttings with virus
Infected vine
Cuttings with no virus
Healthy vine
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• Do ‘clean’ plantings remain healthy ? (stay free from viral infections ?) • What is the risk of leafroll spread from
neighboring old blocks ?
The ‘Mantra’ of ‘stay clean’
Monitored new plantings for several seasons (time and space)
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Monitor spatial distribution and temporal spread of grapevine leafroll disease
Cab Sauv Syrah
Petite Sirah
2007 2004 2009
CS
(GLD
)
CH (GLD)
Semillon (2007)
CS
(GLD
)
CS
(GLD
) CS (GLD)
CS
(GLD
)
CH (GLD)
Open
ZN (2
009)
MR
(200
7)
Apple
Geographically separate locations, but adjacent to old blocks totally infected with leafroll
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• Monitor blocks for leafroll symptoms during each season.
• Record symptomatic vines in a XY-matrix using the row number and vine position as co-ordinates.
• Confirm symptomatic vines for GLRaV-3.
• Compare the data obtained in previous seasons.
• Spatial and temporal distribution maps for each season using cumulative data of new infections from that season and the preceding seasons.
Monitor spatial distribution and temporal spread of grapevine leafroll disease
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Results cv. Cabernet Sauvignon
• Increased number of symptomatic vines each season • A gradient of infected vines – indication of initial
spread from heavily infected old blocks • Clustering of symptomatic vines with time – indication
of secondary spread
H
I I
I
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• Increased number of symptomatic vines each season. • A gradient of infected vines – indication of initial
spread from heavily infected old blocks. • Clustering of symptomatic vines with time – indication
of secondary spread.
cv. Syrah Results
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cv. Petite Sirah
• Increased number of symptomatic vines each season. • A gradient of infected vines – indication of initial
spread from heavily infected old blocks. • Clustering of symptomatic vines with time – indication
of secondary spread.
H
Results
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Multi-year field studies on the spread of GLD in three wine grape cultivars have provided convincing evidence that: • Young vineyards planted with ‘clean’ virus-tested
planting stock can become infected with GLD. • Rate of spread may depend on site-specific
influences (viz. proximity to infected blocks, weather-driven factors, vector species composition, virus strains, etc.).
Summary points
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Early intervention by roguing Cabernet Sauvignon
Syrah
Petite Sirah
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Blame the bugs?
• Grape mealybugs (Pseudococcus maritimus)
as vectors • Soft scale insects (Parthenolecanium corni) as vectors
Bahder, B. W., Poojari, S., Alabi, O.J., Naidu, R.A., and Walsh, D. B. 2013. Pseudococcus maritimus (Hemiptera: Pseudococcidae) and Parthenolecanium corni (Hemiptera: Coccidae) are capable of transmitting Grapevine leafroll-associated virus 3 between Vitis labruscana and Vitis vinifera. Environmental Entomology 42: 1292-1298.
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Remove symptomatic (infected) vine Remove at least one (or two) non-symptomatic vines on either side
Roguing: Remove infected vines
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Roguing as a post-planting strategy
Roguing (within 3-4 years post-planting) and replanting for reducing leafroll spread in new plantings
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Overall Conclusions
• Virus diseases continue to be a major concern - leafroll viruses - red blotch virus - soil-borne viruses - what else is out there? • Visual symptoms are not reliable - Accurate diagnosis is vital
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• Spread via cuttings used for new plantings • Spread by insect vectors (e.g. mealybugs, scale
insects) from old blocks • Why leafroll 3 is more wide spread and prevalent ? • Vector control strategies – when to spray & what to
spray?
Overall Conclusions
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Technical contributors Dr. Basu Bagewadi Dr. Prashant Swamy Dr. Sridhar Jarugula Ms. Bhanu Donda Ms. Nagateja Natra Mr. Jati Adiputra Mr. Leslie Walker Ms. Lakshmi Movva Many post-docs and graduate students worked earlier
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WSU-CAHNRS Agricultural Research Center Washington Grape and Wine Research Program WSU Viticulture & Enology Program CAHNRS Agricultural Extension WSU New Faculty Seed Grant Program
Washington State Commission on Pesticide Registration
Nursery Research & Grapevine Research Funds Specialty Crop Block Grant Program
Wine Research Advisory Committee of the Washington State Wine Commission
Specialty Crop Research Initiative Northwest Center for Small Fruits Research Viticulture Consortium-West
Thanks to:
Washington Association of Wine Grape Growers
Altria - Chateau Ste. Michelle Wine Estates Washington Wine Industry Foundation