Virus Diseases and Their Management

Bob Martin

USDA-ARS HCRL

3420 NW Orchard Ave

Corvallis, OR 97330

Bob.martin@ars.usda.gov

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