Virus Diseases and Their Management€¦ · Virus Diseases and Their Management Bob Martin USDA-ARS...
Transcript of Virus Diseases and Their Management€¦ · Virus Diseases and Their Management Bob Martin USDA-ARS...
Virus Diseases and Their Management
Bob Martin
USDA-ARS HCRL
3420 NW Orchard Ave
Corvallis, OR 97330
START CLEAN
• Starting with clean planting stock is the single most important component of a virus management plan
• NCPN and Certification Programs as described in previous talk
Stay Clean
This is what you do from planning the field all the way through to field removal, hopefully many years later
Preplant - Site selection and preparation
Cultivar selection
Postplant - Site and vector management
Raspberry Viruses in Upper Midwest
• Tomato ringspot – nematode• Raspberry bushy dwarf – pollen• Blackberry chlorotic ringspot – pollen• Strawberry necrotic shock - pollen• Rubus yellow net – aphid• Raspberry leaf mottle – aphid• Black raspberry necrosis – aphid• Raspberry leaf curl complex – aphid
Preplant Site Selection & Preparation
Site selection – nearby inoculum, drainage
Nematode testing
Treatment for nematodes
Fumigation
Crop Rotation
Fallow
Alternate Crop (Blackberries?)
TRSV Retained on Stylet of Xiphinema
Crop Rotation
..
- - - - +
Cleanfallow
CanolaGrass(weed-free)
Raspberryfollowed byfumigation
Continuousraspberry
Cucumber-ELISA assayfor ToRSV in soil
- 20
0
- 40
- 60
- 80
- 100
Clean fallow
Raspberry-fumigation
Grass (non-host for ToRSV)
Rape (allelopathic)
Raspberry (control)
Duration 18 mo.
30 cm
30
cm
Nematodes on the Move
Cultivar Selection
Vector resistance – aphids, Large raspberry aphid, multiple genes
but also multiple aphid biotypesRecent identification of three
resistant genes in black raspberry being used in breeding programs in the PNW
Virus resistance – RBDVSingle gene for resistance, resistance
breaking strain identified in PNW in 2014
Planting
Start Clean
Don’t take cuttings from the neighbor’s field, even if it looks good
Many of the viruses in Raspberry are symptomless in single infections
Raspberry mosaic disease complex (3-4)
Blackberry yellow vein disease complex (13)
This area RLCV complex
Looking for RLCD Upper Midwest
CABMV, NCMV-1, BRNV, RYNVRLMV
Crumbly Fruit Disease in Red Raspberry in PNW (SCRI)
Disease became much more important since mid 1990s
Much more severe on ‘Meeker’ in northern WA and BC than in OR or southern WA on same cultivar
RBDV had been thought to be the causal agent
Mottle Symptoms in AdvancedSelection in Breeding Plots – PNW
Negative for RBDVExtracted dsRNACloned and SequencedRLMV – a typical ClosterovirusRpLV – a unique ReovirusRYNV – a Badnavirus
Black raspberry necrosis virus
Insect and Virus Monitoring
What is out there?
Pan traps and yellow sticky cards to monitor flying insects
Survey focused on RBDV, RLMV and RpLV in northern WA red raspberry fields
> 1500 RNA extractions for RT-PCR
Characterization of RpLV
Insects reared under 14h light at 23°C
Acquisition and inoculation experiments
Transmission biology
Two potential vectors
Large raspberry aphid Amphorophora agathonica
Brambleberry leafhopperMacropsis fuscula
RpLV Transmission
50 aphids
Fixed AAP of 7 days
5 aphids 15 aphids 25 aphids
When is the Virus Moving
Lightle, unpublished data
Empoasca fabae was sporadic
Raspberry aphid A.
agathonica
predominant insect
Few Macropsis
fuscula
012345678
1-Apr 16-May 30-Jun 14-Aug 28-Sep
Aver
age
# ap
hids
/ 10
le
aves
Harvest clean-up sprays applied
Mixed infections
RBDV RLMV RpLV
RBDV
+
RLMV
RBDV
+
RpLVVirus-free
RLMV
+
RpLV
RBDV
+
RLMV
+
RpLV
Graft Inoculations for Field Trial
Plant Growth Establishment Year
Height
109.03
44.90
65.43 63.83
26.09
48.93
33.49 32.00
0.0020.0040.0060.0080.00
100.00120.00140.00
H D M L DM DL LM DMLTreatments
cm
a b bcd d bc cd cd
All virus infected plants grew poorly the first year, none would have had a commercial harvest, healthy plants had about a 40% crop the first year
Plant Growth (2011)July
Height
55.57
48.5051.27 51.03
30.20
50.7344.60 42.13
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
H D M L DM DL LM DML
Treatments
cm
ca b bb db c
Crumbly FruitFirmness
73.54
58.54 61.38 65.58 61.83
46.38
61.46
49.15
0.000
10.000
20.000
30.000
40.000
50.000
60.000
70.000
80.000
90.000
H D M L DM DL LM DML
Treatments
g
a b bc bcbcc dd
H DML
D
DML
Aphid Resistance for Control of RLMV & RpLV
How does aphid resistance affect feeding behavior?
Are there differences in aphid feeding response to aphid resistance genes from alternate sources?
Aphid Performance on Virus Infected Plants
Aphids lived an average of 27 days on healthy plants, compared to 36 days on virus-infected plants (RpLV, RLMV or RpLV + RLMV)
Aphids produced an average of16 nymphs on healthy plants and an average of 32 nymphs on plants infected with RLMV+RpLV, an average of 26 on plants infected with RLMV or RpLV
Aphids preferred RLMV infected plants over healthy plants and healthy plants over RpLV infected plants
Feed on resistant vs susceptible raspberry
Elapsed Time - 14s
Time (0.4s/ division)
Vo
ltag
e
Aphid feeding behavior
Elapsed Time - 12.4s
Time (0.4s/ division)
Vo
ltag
e
E1: phloem salivation
E2: phloem ingestion
(Lightle et al. 2012, J Econ Ent)
The plant breeder, Chad Finn together with graduate student Michael Dossett, has identified resistance to A. agathonica in populations of wild black raspberry
Aphid resistance in red raspberry, aphids do not ingest from resistant plants once they get to the phloem
Aphid resistance in black raspberry, aphids do appear to recognize when they are in the phloem
Virus Incidence in ‘Meeker’ FieldsNorthern Washington
Field Age
(years)
RLMV
(%)
RpLV
(%)
1 4 01 30 01 10 02 58 21
2 0 02 0 02 6 02 16 03 31 63 6 03 13 03 50 04 19 64 13 05 69 05 90 80
5 100 75
5 44 65 100 17
6 70 256 100 6
6 100 12
7 100 6
8 100 46
Southern Washington/Oregon
Field Age
(years)
RLMV
(%)
RpLV
(%)
1 0 0
5 40 20
6 0 20
7 8 17
8 19 0
8 27 0
Virus Interactions, do mixed infections lead to higher transmission rates?
E2.5ndhB
E12RBDV =1.899.5 = 423
Amp. Cycle
ΔRnCt 14.5 Ct 26
RBDV + RLMV
RBDV
DDM DM DMDMD DD
RBDV ELISA test
D DM
D = RBDV
DM = RBDV + RLMV
Similar results with ELISA and PCR
Conventional RT-PCT at 15
cycles
RBDV Infection via PollinationCultivar # Samples RNA1 RNA2
Meeker (S) 14 14 14Willamette (R) 7 7 7
First identification of resistance breaking strain in U.S., found in several cultivars in the PNWCascade Harvest, Willamette, Heritage
Control of RBDV
For last 20 years, breeders have worked to develop RBDV resistant cultivars
Engineered resistance to RBDV developed in 2005Resistant breaking strain detected in the PNW in
2013, confirmed in 2014.RBDV-infected Boyne, Chief, Cascade Harvest,
Willamette, WSU 836 identified in Washington in 2014
How to Manage RBDV When RBDV-RB Strains Are Present
First, realize very few RBDV resistant cultivars are currently being grown in the PNW
Identify cultivars that do not develop crumbly fruit or only very mild symptoms (Cascade Harvest?)
Identify cultivars where the virus spreads very slowly (Wakefield)
Determine impact of mixed infections in cultivars where RBDV is symptomless on symptom development and on the spread of RBDV
Mixed infections, does this increase rate of spread?
Raspberry
Blackberry
Rubus yellow net virus (RYNV)
• RYNV infects Rubus species and cultivars worldwide. First described in British Columbia, Canada mid 1950s
• Is transmitted in a semi-persistent manner by Amphorophora agathonica in North America and A. rubi in Europe
• RYNV is a component of raspberry mosaic disease (RMD), a virus disease complex that cause serious decline in plant vigor and productivity in red and black raspberries.
Detection
• Graft indexing onto R. occidentalis, ‘Munger’• PCR detection• PCR detection – gave positive reaction for
samples that were negative by graft indexing, (G-PCR+) plants
• After grafting with G- PCR+ sources, the indicators tested negative for RYNV in PCR
• Not graft transmissible rather than symptomless on indicator plant
• PCR testing may stop shipment of plants with virus sequence in the plant DNA
Re-examining Some Old Diseases
In the first edition of the Raspberry and Blackberry Compendium and in Small Fruit Virus Handbook, this was reported to be caused by TRSV in blackberry in NC
Blackberry Infected with TRSV via Nematode Transmission
Even after four years, plants did not develop any symptoms, even though positive for TRSV by ELISA and RT-PCR (Gergerich)
Blackberry Decline SE (SCRI)
Cooperative project: AR, NC, OR, CA, MSSymptomatic and dying (Arkansas and Carolinas)
plants that tested negative for all known viruses of Rubus in ELISA tests and nothing transmitted by sap to herbaceous plants.
Electron microscopy – pinwheel inclusions – Poty?DsRNA – lots of bandsFor a virologistIt was beginning to look a lot like Christmas!
Blackberry Yellow Vein DiseaseSymptoms first observed in South Carolina, by Walker Miller, 2000Samples tested for RBDV, TRSV, ToRSV, SNSV – all were negative,samples with similar symptoms observed in Arkansas in 2002
Viruses isolated from BYVD plantsVirus Genus VectorBCRV Ilar Pollen, SeedBLMaV Emara Eriophyid mites?BPYV Crini WhiteflyBYVaV Crini WhiteflyBVS Unassigned ?BVE Marafi ?BVY Bramby Eriophyid mites?)INSV Tospo ThripsGSyV-1 Marafi ?RBDV Ideao Pollen, SeedRuCV-1 FoveaTRSV Nepo NematodeToRSV Nepo Nematode
Symptoms Similar – Virus Complex Varies
None of the viruses identified to date, cause symptoms in blackberry in single infectionsMore viruses = more severe symptomsNo single virus detected in all symptomatic plantsBYVaV and BCRV are most common
ArkansasBYVaV - CriniBVY – Poty (DAG-, AlkB+)BVX – FlexiBCRV - IlarTRSV – Nepo
Occasionally
CarolinasBYVaVBPYVINSVBCRVTRSV quite commonBVX
RBDV
RLMV
RpLV
RYNV
BRNV
BCRV
SNSV
ToRSV
RLCV
Luteo
Rhabdo
ApMV
ArMV
CMV
CLRV
CRLV
PNRSV**
RLBV
RpRSV
RuCV-1
RVCV
SLRSV
SoMV
TBRV
WLV
Phytoplasma
Blue– Key for RaspberryBlack – Key for Blackberry
Green – absent or minor in North America
Rubus Viruses
RLMV
BCV
RpLV
BRNV
RBDV
BPYV* INSV*
BVE* TRSV
BVS* BCRV*
BVX* BCMV*
BVY* BVZ*
BYVaV,* BVBaV*
GSyV-1*, BlMaV*
Badna*
Systemic Pathogens in Vegetatively Propagated Nursery Crops
• No means to cure virus in nursery or field• No ‘cleansing’ of most viruses that happens with
seed crops• Virus control requires production of healthy plants
and preventing reinfection• Symptomless infections can lead to serious
problems in production fields • Nursery stock can be pathway for viruses to move
long distances
Symptomless virus in planting stock, even without disease could erupt into a disaster at any time
Strawberries in eastern Canada – 2012/13
Strawberries in CA in 2002-2003
Blackberries in the southeastern US
Villaricca volcano, Pucon Chile, hiked up in Jan 2014
Villaricca volcano,Pucon Chile, Feb 18, 2015
G1 – Fully tested - Start CleanControl based on BMPs, with audit sampling
focused on high risk viruses to monitor for virus intrusion (Isolation from Production Fields
If virus detected, identify weak link in BMPs, modify to correct problem
If new virus identified, retest the G1 plantsIn Strawberry and Rubus most viruses are
symptomless in single infections, visual inspections are not adequate
Virus Control - Nursery
Disease Control – Production FieldsIsolation from inoculumWhich viruses occur in your area?Which viruses are important for disease?Of these, which are easiest to control?Can virus or vector resistance be deployed?In production fields, often don’t need to control all viruses. Example, RpLV is transmitted inefficiently and easiest to control of the aphid-borne viruses in PNW.
START CLEAN
• Starting with clean planting stock is the single most important component of a virus management plan
• Therefore, nursery systems are critical to developing and maintaining a successful berry industry
The Importance of Proper Identification
Questions
Wind River Range, Wyoming, 2013