Beet curly top virus and other viruses of concern

Robert L. Gilbertson Department of Plant Pathology University of California Davis

A diversity of viruses affect processing tomatoes in California

• Beet curly top virus (BCTV) • Alfalfa mosaic virus (AMV) • Cucumber mosaic virus (CMV) • Tobacco etch virus (TEV) Potato virus Y (PVY) • Tomato mosaic virus (ToMV) Mechanically transmitted • Tomato spotted wilt virus (TSWV) • Tomato necrotic spot virusE (ToNSV) • Tomato yellow leaf curl virusE (TYLCV) • Tomato necrotic stunt virusRE (ToNSV) Whitefly-transmitted

Aphid- transmitted

Whitefly-transmitted

Thrips-transmitted Thrips-associated

Leafhopper-transmitted

Viruses continue to be a threat to processing tomato production in California

• Considerable progress has been made on identifying, understanding and managing these viruses • New tools for detection, prediction and management • Viruses change in terms of their importance and prevalence -Existing viruses -Beet curly top virus (BCTV) -Tomato spotted wilt virus (TSWV) -Alfalfa mosaic virus (AMV) -New and emerging viruses -Tomato necrotic spot virus (ToNSV) -Tomato yellow leaf curl virus (TYLCV) -Tomato necrotic dwarf virus (ToNDV)

Tomato spotted wilt virus (TSWV)

• Bronzing, necrosis, ringspots (fruits) • Symptoms vary depending on variety and plant age

Tomato spotted wilt virus (TSWV)

Prevalence of Virus Diseases in Processing Tomatoes of California

Tomato spotted wilt virus (TSWV)-biology

• Virus: homogenous in tomato in CA, resistance-breaking strains exist in other parts of the world; and new highly virulent tospoviruses have been introduced into Florida • Symptoms maybe confused with curly top early in the season • Detection: ELISA, PCR, immunostrips • Host range: wide, includes many weeds and crops • Insect vector: various species of thrips, F. occidentalis in CA • Mode of transmission: persistent propagative, must be acquired by larval stages • Sources of inoculum: -weeds/bridge crops -thrips • Means of survival: -perennial weeds/bridge crops -thrips (in soil)

Tospovirus Transmission

Cycle

Pupal Stages Do Not Feed

ACQUISITION BY LARVAE IS CRUCIAL

Egg

1st instar

2nd instar

VIRUS PASSAGE

Only adults that acquire as larvae can

transmit.

TRANSMISSION

Photos by J.K. Clark

VIRUS PASSAGE

Tomato spotted wilt virus-management

• Apply risk index to predict potential for TSW • Inoculum levels are low at the beginning of the season • Thrips and virus-free transplants • Resistant (Sw-5) varieties and field placement • Remove or rogue plants early in season (up to 30 d) • Monitor and manage thrips populations-spray early and according to a predictive model to delay the development of virus-carrying adults • Weed management (before, during and after the growing season) • Effective sanitation following harvest (all crops) • Minimize any overlap between bridge crops

An IPM program has been developed for thrips and TSWV in processing tomatoes in California

Tomato spotted wilt virus-current situation

• Current TSWV issues -High incidences of TSWV in late-planted fields in Fresno County -Would risk index have predicted these outbreaks? -Increased incidence of TSW symptoms in varieties with the Sw-5 gene-possibility of the emergence of resistance-breaking TSWV strains or the new species (Tomato chlorotic spot virus and Groudnut ringspot virus) -Rapid molecular assays are available to detect resistance breaking TSWV strains and new species • Application of these tools could improve TSWV management

• Plants are stunted and have up- or down curled leaves with dull green-yellow color and purpling of the veins • Plants infected at a young age often die • Plants infected later produce small fruits that ripen prematurely • No necrosis in leaves or fruits • Early in the season, curly top symptoms can be confused with those of tomato spotted wilt

Curly top disease is a potentially devastating disease of tomato

Curly top disease

Prevalence of Virus Diseases in Processing Tomatoes of California

Curly top is caused by Beet curly top virus (BCTV)

• A geminivirus with a very wide host range (>300 species of plants) including crops and weeds • Some plants show severe symptoms when infected (e.g., tomatoes), whereas others show no symptoms (many species of weeds) • In nature, BCTV is only transmitted by the beet leafhopper, not by contact, other insects or via seed • Exists as a complex of strains, which can be differentiated by their genetic sequences and biological properties

Beet curly top virus

Symptoms of BCTV infection in tomato

BCTV strains

BCTV-Logan BCTV-CFH BCTV-Worland

BCTV

BSCTV

BMCTV

G. Curtovirus Species

G. Curtovirus G. Curtovirus BCTV strains

Other species: SpCTV PeCTV PepYDV

BCTV-CA/Logan BCTV-Svr (BSCTV) BCTV-Wor (BMCTV) BCTV-Mld (BMCTV) BCTV-PeCT BCTV-SpCT BCTV-PeYD BCTV-CO** BCTV-LH71**

2001 2014

Classification of Beet curly top viruses

Curly top disease cycle: Dependent on a migratory insect

Fall: adult leafhoppers migrate for overwintering

in the foothills

Spring: Viruliferous adult leafhoppers migrate to the

valley floor and feed

Multiple generations on the valley floor

Winter/early spring: Adults overwinter

and breed on annual and perennial plants

Beet curly top virus-management

• Monitor for adult beet leafhopper populations and for levels of BCTV in leafhoppers and manage by targeted spraying (CTVCP) • No currently available resistant varieties • Apply systemic neonicotinoid insecticides at transplanting (sugar beets, tomatoes) • Field placement (avoid fields adjacent to the foothills) • Heavy plant populations • Weed management on the valley floor?

Curly top outbreak of 2013 • Losses of ~1 million tons (~$100 million) • Associated with high beet leafhopper populations with high BCTV titers • Appearance of curly top in new locations, e.g., San Joaquin County and in new crops, e.g., melons • Emergence of new strains of BCTV more virulent to tomato

Tomato field with 90% curly top in Fresno Co.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 + -

High BCTV titers in most of the leafhopper samples

Rapid and Specific Detection of BCTV by the Polymerase Chain Reaction (PCR) Provides New Opportunities for Improved Disease Management

• Detection in plants -rapid detection -ID viruses/strains -ID host plants • Detection in the beet leafhopper vector -predictor of curly top severity -target areas for spraying -ID prevalent viruses/strains

Add 300ul STE buffer and grind

Spin 5’ at 13,000 rpm

Phenol/Chloroform extraction twice

Precipitation of nucleic acid

PCR reaction

PCR Detection of BCTV in beet leafhoppers

Monthly leafhopper collections from CDFA CTVCP personnel

Curly top outbreaks are correlated with high populations of virus-carrying leafhoppers early in the growing season

• Using the PCR detection method and leafhoppers provided by the Curly Top Virus Control Board (CTVCB), a study was conducted to ID factors associated with curly top outbreaks in tomato • The highest levels of curly top in tomato were correlated with high populations of virus-carrying leafhoppers early in the growing season (i.e., February-April)

Monitoring BCTV in beet leafhoppers from October 2014 to July 2015 in the Central Valley of California (sweep net samples)

Large numbers of beet leafhoppers observed in winter and spring of 2015 High virus titer detected in these leafhoppers

Disease incidence in 2015: Moderate*

0%10%20%30%40%50%60%70%80%90%

100%

Oct Nov Dec Jan Feb Feb(IV)

Mar Apr May Jun(IV)

Jul

PCR Positive Percentages of Sweep LH Samples

Strong % Medium % Weak % (IV=Imperial Valley)

BCTV in reservoir hosts • Foothills samples: over 246 weeds tested and 79% were positive for BCTV • Weeds with the highest infection rates included Atriplex, filaree, goosefoot, London rocket, peppergrass, plantago, Russian thistle and shepherd’s purse • Most had no symptoms and low virus titers, but ~15% (29/246) had high virus titers • Similar results were obtained with weeds from Valley floor in the winter and during the growing season: >50% positive for BCTV (~400 total weeds tested), most with low titer but a low percentage with high titer • Alfalfa and safflower are poor hosts of the beet leafhopper and BCTV • Big question: Can beet leafhoppers acquire BCTV from low titer weed reservoir hosts?

Appearance of New BCTV Strains in the Central Valley

• Characterization of BCTV strains associated with the 2013 outbreak has revealed emergence of two new recombinant strains: BCTV-CO and BCTV-LH71 • In 2014 and 2015 , BCTV-CO and -LH71 were the predominant strains in leafhoppers, tomatoes and other crops, and weeds; the only exception was in sugar beets in Imperial Valley where BCTV-Srv (CFH) was predominant • BCTV-SpCT was detected in tomatoes and peppers for the first time • These results suggest a shift in predominant BCTV strains in the Central Valley and more diversity in the California BCTV population • These strains are highly virulent in tomato, though this alone is unlikely to be the main reason for the 2013 outbreak • These new strains are being used for screening for disease resistance

New BCTV strains associated with the 2013 outbreak

BCTV-Wor BCTV-LH71** BCTV-Svr BCTV-CO**

Sugar beet

Tomato

Mild phenotype strains Severe phenotype strains

N. benthamiana

1st

Tomato

2nd

3rd

Agrobacterium-mediated inoculation for screening tomato tomato varieties and germplasm for resistance to BCTV

48 hr old liquid culture of Agrobacterium tumefaciens containing the viral DNA

BCTV Observe symptom development (up to 35 dpi)

Check for virus infection (by PCR) (21-28 dpi)

Ty genes confer resistance to BCTV • The Ty genes (Ty 1-6) are a series of genes from wild tomato species that confer resistance to whitefly- transmitted TYLCV • Commercial tomato varieties with Ty genes and resistance to TYLCV are now available • Some breeding lines from the World Vegetable Center (WVC) with different compliments of Ty genes showed high levels of resistance when agroinoculated with BCTV

Tomato yellow leaf curl virus (TYLCV)

Control Line 15 (1.2) Line 11 (3.2)

Some WVC breeding lines are showing promising levels of curly top resistance and the level of resistance

varies among strains

Results of screening of tomato lines from the World Vegetable Center with different compliments of Ty genes for resistance to Beet curly

top virus-CFH strain

Results of screening of tomato lines from the World Vegetable Center with different compliments of Ty genes for resistance to Beet curly

top virus-LH71 strain

Advancement and characterization of a breeding line (line 20) with curly top resistance

Advancement and characterization of a breeding line (line 20) with curly top resistance

Line Disease Rating % Symptoms____________ 20-59-12-10 11/25 (0) (44) No symptoms 7/25 (1) (28) Mild, delayed 2/25 (2) (12) Up curl, mild yellowing 4/25 (4) (16) Severe symptoms 20-59-4-4-9 9/25 (0) (36) No symptoms 10/25 (1) (40) Mild, delayed 4/25 (2) (16) Up curl, mild yellowing 2/25 (4) (8) Severe symptoms 20-59-9-4-8 12/25 (0) (48) No symptoms 8/25 (1) (32) Mild, delayed 4/25 (2) (16) Up curl, mild yellowing 1/25 (3) (4) Severe symptoms ________________________________________________________

-The F5 generation of the most promising lines have high levels of resistance in ~80% of plants and this has been associated with the Ty-1 gene

Advancement and characterization of a breeding line (line 20) with curly top resistance: High levels of resistance in F5 lines

Genetics of Resistance in Line 20 Cross Rating/No. plants % plants with rating Line 20-59-12-10 X M-82 1 (4) 20 2 (15) 75 3 (1) 5 Line 20-59-4-4 X M-82 1 (1) 25 2 (14) 70 3 (1) 5 Line 20-59-12-10 X E-6203 1 (8) 40 2 (6) 30 3 (6) 30 Line 20-59-4-4 X E-6203 1 (3) 15 2 (7) 35 3 (10) 50 Male M-82 4 (10) ---- Male E-6203 4 (10) ---- 20-59-4-4 0 (7), 1(1), 2(2) ---- 20-59-12-10 0 (9), 1 (2) ----________

-BCTV resistance in line 20 is heritable, but progeny showed ratings in between the S and R parents, indicating that more than one gene is involved

Feeding deterrent and preference studies -We have been investigating feeding deterrents and preferences of the beet leafhopper -We have developed a ‘choice’ system where we allow beet leafhoppers to chose between treated and untreated plants and between plant species -Deterrents tested include extracts of tobacco plants, orange oil, pepper powder and neem oil -Host preference studies are being performed with various crop plants grown on the valley floor

LHs

Treated tomato plant

Untreated tomato plant

25 Non-viruliferous adult leafhoppers per cage were starved for two hours

Non-viruliferous leafhopper colony

Starved LHs were released in the middle of each cage

Orange Oil Treated

Water Treated

Water Treated

Orange Oil Treated

Experimental design for assessment of feeding deterrents for leafhopper

Time course reading on numbers of leafhoppers feeding on each plant

% le

afho

pper

s fou

nd o

n di

ffere

nt tr

eate

d pl

ants

Orange oil-treated vs. Water-treated

Results of feeding deterrents and host preference for beet leafhoppers

Shepherd’s purse vs. tomato

Chenopodium vs. tomato Chenopodium vs. alfalfa

A diversity of viruses affect processing tomatoes in California

• Beet curly top virus (BCTV) • Alfalfa mosaic virus (AMV) • Cucumber mosaic virus (CMV) • Tobacco etch virus (TEV) Potato virus Y (PVY) • Tomato mosaic virus (ToMV) Mechanically transmitted • Tomato spotted wilt virus (TSWV) • Tomato necrotic spot virusE (ToNSV) • Tomato yellow leaf curl virusE (TYLCV) • Tomato necrotic stunt virusRE (ToNSV) Whitefly-transmitted

Aphid- transmitted

Whitefly-transmitted

Thrips-transmitted Thrips-associated

Leafhopper-transmitted

Unusual virus-like disease symptoms were observed in a late-planted tomato field with high populations of whiteflies

in Kern County in October 2015

• Unusual virus-like symptoms were observed in a late season experimental tomato plot in Kern County that was planted to assess the effect of a repellent on beet leafhoppers and Beet curly top virus • Many of the plants in the field were stunted; had distorted growth; curling, crumpling and necrosis of leaves and necrosis of stems and petioles • These symptoms were most similar to those caused by Tomato spotted wilt virus (TSWV) • However, immunostrip tests performed on

these plants for TSWV were negative • Plants in this plot had

high populations of whiteflies (B. tabaci)

Appearance of a new tomato-infecting virus in 2015

Results of tests for other viruses

Tomato spotted wilt virus RT-PCR test

Torradovirus RT-PCR test

• RT-PCR tests for TSWV infection were mostly negative • RT-PCR tests for Alfalfa mosaic virus (AMV) and Tomato

necrotic spot virus (ToNSV), two other viruses that induce necrosis in tomato were negative

• However, RT-PCR tests for another type of virus that induces necrosis in tomato, torradovirus, were POSITIVE!

• Torradoviruses are a relatively new group of single-stranded RNA viruses with spherical virions and a bipartite genome • They primarily cause diseases in tomato, including leaf curling and necrosis of stem and petioles, especially young growth • The symptoms are similar to those caused by tospoviruses (e.g., TSWV) • The first torradovirus was characterized from tomatoes with necrosis

symptoms in Spain in 1999 and was named Tomato torrado virus (ToTV), from which the group (genus) gets its name

• Additional species appeared in Mexico (Tomato marchitez virus [ToMarV]) and Central America (Tomato chocolate spot virus [ToChSV]) in the early 2000s

• Outbreaks of these viruses always were associated with whiteflies, including Bemisia tabaci and Trialeurodes vaporariorum, and economic losses were experienced in some situations (e.g., in susceptible varieties and under high whitefly pressure)

• ToTV is transmitted by both of these species of whiteflies and in a semi-persistent manner

What are torradoviruses?

RNA 2

RNA 1 5 ' 3’ Pro Hel RdRp AAAA

UTR UTR

ORF1

7.2 kb

ORF2 5 ' 3’

ORF1

MP Vp35 Vp26 Vp24 AAAA UTR UTR 4.9 kb

Torradoviruses can be detected by RT-PCR with degenerate primers for RNA-1 and RNA-2

Target sites for the degenerate torradovirus primers

-RT-PCR analyses were performed with degenerate primers for portions of torradovirus RNA-1 and RNA-2

• The expected size ~500 bp DNA fragments were PCR- amplified with the RNA-1 and RNA-2 primers from representative samples from the Kern County tomato field • Sequence analysis of the RNA-1 fragments revealed ~94% identity with Tomato necrotic dwarf virus (ToNDV), a provisional species of torradovirus • Similar results were obtained for the RNA-2 fragments • ToNDV is a whitefly-transmitted virus that was reported from southern California in 1980s

RT-PCR results with torradovirus primers

Results with torradovirus RNA-1 primers

500

Torradovirus symptoms in tomatoes in the Kern County plot

• In the 1980’s a disease called tomato necrotic dwarf appeared on tomatoes in the Imperial Valley

• The disease spread rapidly and caused major losses to tomato production • In fact, this disease was one of the factors that drove tomato production

out of the Imperial Valley • The virus involved was whitefly-transmitted and was named Tomato

necrotic dwarf virus (ToNDV), but the properties of the virus were unclear

Tomato necrotic dwarf disease

Pictures: G. Holmes, California Polytechnic State University at San Luis Obispo, 1995

http://www.forestryimages.org

• An isolate of ToNDV (ToNDV-R)

from the Imperial Valley had been maintained by whitefly transmission at the

USDA-ARS in the laboratory of Dr. William Wintermantel • Molecular characterization of ToNDV-R revealed that it was a torradovirus • Sequence comparisons revealed it was most closely related to ToMarV from Mexico, but most likely was divergent enough to be considered a distinct torradovirus species • ToNDV has not been observed in California since the 1980’s

ToNDV is a torradovirus!

Conclusions and future directions

• We have documented an outbreak of a tomato-infecting torradovirus in Kern County, California in a late season tomato plot

• The Kern County torradovirus is most likely a strain of Tomato necrotic dwarf virus (ToNDV-K)

• The symptoms induced by ToNDV-K included stunting and distorted growth, leaf curling, crumpling and necrosis, and necrosis of stems and shoots

• The complete sequence of ToNDV-K RNA-1 and RNA-2 are being determined to understand the relationship with other torradoviruses

• Determine the host range of ToNDV-K by sap-inoculation and/or whitefly

transmission • Survey production areas in the Central Valley to determine the prevalence of

ToNDV-K in processing tomatoes and identify potential inoculum sources

Acknowledgements UC Davis • Dr. Li Fang Chen • Dr. Ozgur Batuman • Dr. Maria Rojas • Ms. Maria Hernandez • Numerous outstanding undergraduate students

UCCE • Tom Turini, Farm Advisor, Fresno County • Joe Nunez, Farm Advisor, Kern County

USDA-ARS • William Wintermantel

CDFA-CTVCB • Patrick Akers • Jennifer Willems and Lauren Murphy

California Processing Tomato Growers