Beet curly top virus and other viruses of concern. Robert L. Gilbertson Department of Plant Pathology University of California Davis

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1 Beet curly top virus and other viruses of concern Robert L. Gilbertson Department of Plant Pathology University of California Davis

2 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) Tomato spotted wilt virus (TSWV) Tomato necrotic spot virus E (ToNSV) Tomato yellow leaf curl virus E (TYLCV) Tomato necrotic stunt virus RE (ToNSV) Leafhopper-transmitted Aphidtransmitted Mechanically transmitted Thrips-transmitted Thrips-associated Whitefly-transmitted Whitefly-transmitted

3 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)

4 Tomato spotted wilt virus (TSWV) Bronzing, necrosis, ringspots (fruits) Symptoms vary depending on variety and plant age

5 Prevalence of Virus Diseases in Processing Tomatoes of California Tomato spotted wilt virus (TSWV)

6 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)

7 TRANSMISSION ACQUISITION BY LARVAE IS CRUCIAL Egg Tospovirus Transmission Cycle 1 st instar 2 nd instar VIRUS PASSAGE VIRUS PASSAGE Only adults that acquire as larvae can transmit. Pupal Stages Do Not Feed Photos by J.K. Clark

8 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

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

10 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

11 Curly top disease is a potentially devastating disease of tomato 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

12 Prevalence of Virus Diseases in Processing Tomatoes of California Curly top disease

13 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

14 Classification of Beet curly top viruses G. Curtovirus G. Curtovirus G. Curtovirus BCTV strains Species BCTV strains BCTV-Logan BCTV-CFH BCTV-Worland BCTV BSCTV BMCTV 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**

15 Curly top disease cycle: Dependent on a migratory insect Spring: Viruliferous adult leafhoppers migrate to the valley floor and feed Winter/early spring: Adults overwinter and breed on annual and perennial plants Fall: adult leafhoppers migrate for overwintering in the foothills Multiple generations on the valley floor

16 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?

17 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 High BCTV titers in most of the leafhopper samples

18 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

19 PCR Detection of BCTV in beet leafhoppers Monthly leafhopper collections from CDFA CTVCP personnel Add 300ul STE buffer and grind Spin 5 at 13,000 rpm Phenol/Chloroform extraction twice PCR reaction Precipitation of nucleic acid

20 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)

21 Monitoring BCTV in beet leafhoppers from October 2014 to July 2015 in the Central Valley of California (sweep net samples) PCR Positive Percentages of Sweep LH Samples 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Oct Nov Dec Jan Feb Feb (IV) Mar Apr May Jun (IV) Strong % Medium % Weak % (IV=Imperial Valley) Jul Large numbers of beet leafhoppers observed in winter and spring of 2015 High virus titer detected in these leafhoppers Disease incidence in 2015: Moderate*

22 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?

23 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

24 New BCTV strains associated with the 2013 outbreak Mild phenotype strains Severe phenotype strains BCTV-Wor BCTV-CO** BCTV-Svr BCTV-LH71** Sugar beet Tomato N. benthamiana

25 Agrobacterium-mediated inoculation for screening tomato tomato varieties and germplasm for resistance to BCTV BCTV 3rd Observe symptom development (up to 35 dpi) 2nd 48 hr old liquid culture of Agrobacterium tumefaciens containing the viral DNA 1st Tomato Check for virus infection (by PCR) (21-28 dpi)

26 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 whiteflytransmitted TYLCV Commercial tomato varieties Tomato yellow leaf curl virus with Ty genes and resistance to TYLCV (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

27 Some WVC breeding lines are showing promising levels of curly top resistance and the level of resistance varies among strains Line 11 (3.2) Line 15 (1.2) Control

28 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

29 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

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

31 Advancement and characterization of a breeding line (line 20) with curly top resistance Line Disease Rating % Symptoms /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 /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 /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

32 Advancement and characterization of a breeding line (line 20) with curly top resistance: High levels of resistance in F 5 lines

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

34 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 Treated tomato plant LHs Untreated tomato plant

35 Experimental design for assessment of feeding deterrents for leafhopper Non-viruliferous leafhopper colony 25 Non-viruliferous adult leafhoppers per cage were starved for two hours Starved LHs were released in the middle of each cage Water Treated Orange Oil Treated Water Treated Orange Oil Treated Time course reading on numbers of leafhoppers feeding on each plant

36 Results of feeding deterrents and host preference for beet leafhoppers Orange oil-treated vs. Water-treated Shepherd s purse vs. tomato % leafhoppers found on different treated plants Chenopodium vs. tomato Chenopodium vs. alfalfa

37 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) Tomato spotted wilt virus (TSWV) Tomato necrotic spot virus E (ToNSV) Tomato yellow leaf curl virus E (TYLCV) Tomato necrotic stunt virus RE (ToNSV) Leafhopper-transmitted Aphidtransmitted Mechanically transmitted Thrips-transmitted Thrips-associated Whitefly-transmitted Whitefly-transmitted

38 Unusual virus-like disease symptoms were observed in a late-planted tomato field with high populations of whiteflies in Kern County in October 2015

39 Appearance of a new tomato-infecting virus in 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)

40 Results of tests for other viruses 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! Tomato spotted wilt virus RT-PCR test Torradovirus RT-PCR test

41 What are torradoviruses? 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 semipersistent manner

42 Torradoviruses can be detected by RT-PCR with degenerate primers for RNA-1 and RNA-2 5 ' 3 Hel Pro RdRp RNA 1 AAAA 7.2 kb UTR ORF1 UTR ORF1 Target sites for the degenerate torradovirus primers RNA kb ORF2 5 ' 3 UTR MP Vp35 Vp26 Vp24 AAAA UTR -RT-PCR analyses were performed with degenerate primers for portions of torradovirus RNA-1 and RNA-2

43 RT-PCR results with torradovirus primers The expected size ~500 bp DNA fragments were PCRamplified with the RNA 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 Results with torradovirus RNA-1 primers Torradovirus symptoms in tomatoes in the Kern County plot

44 Tomato necrotic dwarf disease Pictures: G. Holmes, California Polytechnic State University at San Luis Obispo, 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

45 ToNDV is a torradovirus! 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

46 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

47 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