Location: Crop Improvement and Protection Research
2019 Annual Report
Objectives
Plant viruses and their vectors cause millions of dollars in losses to vegetable and cucurbit production each year through decreased yield, quality, and plant longevity, as well as the need for regular pesticide application. Molecular characterization of viruses affecting these crops, as well as how they interact with insect vectors, is critical in order to gain an understanding of factors contributing to transmission, disease development, and for the development of accurate and specific diagnostic tools for pathogen identification in crop plants, as well as for development of novel means of virus and vector control. This research will lead to new approaches to reduce vector populations and the ability of vectors to transmit viruses to crop plants, benefitting U.S. industry, growers, and improving food quality for consumers.
Objective 1: Identify and compare gene expression changes in insect vectors of plant viruses, such as whiteflies and leafhoppers, and use the information to develop new strategies, such as RNAi, to reduce vector populations and interfere with virus transmission in vegetable and cucurbit crops.
• Subobjective 1A: Compare the effect of virus infection of a host plant on feeding behavior and gene expression in whitefly. (Wintermantel)
• Subobjective 1B: Develop strategies for RNAi based control of whitefly in melon and tomato. (Wintermantel)
• Subobjective 1C: Compare the effect of Beet curly top virus (BCTV) infection of a tomato host plant on gene expression in the beet leafhopper (Circulifer tenellus). (Wintermantel and Vacant)
• Subobjective 1D: Develop strategies for RNAi based control of the beet leafhopper. (Wintermantel and Vacant)
Objective 2: Identify environmental, physiological, and biological influences leading to development of tombusvirus-induced disease symptoms in lettuce, and use this information to develop crop management recommendations, such as soil fertility regimes, to reduce frequency of disease development.
• Subobjective 2A: Conduct RNA sequencing (RNA-seq) of lettuce plants with dieback disease symptoms to determine the presence of additional biotic agents potentially contributing to disease development. (Wintermantel)
• Subobjective 2B: Compare soil treatments to induce lettuce dieback symptoms under controlled conditions, and to understand abiotic factors contributing to disease development in the field. (Wintermantel)
Objective 3: Biologically and molecularly characterize a new torradovirus from California and determine its relationship to other tomato-infecting torradoviruses.
• Subobjective 3A: Develop an infectious clone of Tomato necrotic dwarf virus (ToNDV) for use in host range evaluation and further virus characterization. (Wintermantel)
• Subobjective 3B: Evaluate the host range of Tomato necrotic dwarf virus (ToNDV) and differences in vector transmissibility among isolates from tomato and weed hosts. (Wintermantel)
Objective 4. Identify whitefly proteins that interact with virus proteins during transmission of a semipersistently transmitted virus using virus overlay assays and co-precipitation of proteins. (Vacant)
Approach
1A: Electrical penetration graphing (EPG) will be used to determine whitefly vector feeding patterns in healthy and virus-infected host plants, followed by RNA-sequencing to determine gene expression differences associated with feeding behavior differences between virus-infected and healthy host plants.
1B: Develop strategies for control of whitefly in melon and tomato using RNA interference. Transgenic plants will be developed expressing selected constructs shown to induce RNA interference in preliminary studies, accompanied by development of methods for inducing resistance in non-transformed plants. This should reduce whitefly feeding damage and whitefly-transmitted viruses impact agriculture both in the U.S. and in the developing world.
1C: Comparative gene expression (RNA sequencing) analysis will be used to determine differences in gene expression associated with feeding on either healthy host plants or those infected with the persistent circulative beet curly top virus (BCTV). Results will be compared with previous studies to determine common gene expression changes associated with virus transmission.
1D: Develop strategies for control of leafhopper in tomato using RNA interference and related methods. Transgenic plants will be developed expressing selected constructs shown to induce RNA interference in preliminary studies, accompanied by development of methods for inducing resistance in non-transformed plants. This should reduce transmission of BCTV in tomato and can be applied to several other at-risk hosts.
2A. Conduct RNA sequencing (RNA-seq) of lettuce plants with dieback disease symptoms to determine the presence of additional biotic agents potentially contributing to disease development.
2B. Compare soil treatments under controlled conditions to identify environmental influences on development of lettuce dieback disease symptoms, and to understand abiotic factors contributing to disease development in the field. Results will improve management recommendations to reduce losses in lettuce.
3A. Develop an infectious clone of the Tomato necrotic dwarf virus (ToNDV) for use in host range evaluations and further characterization of ToNDV, its interactions with host plants and vectors, as well as with other members of the genus, Torradovirus.
3B. Evaluate the host range of Tomato necrotic dwarf virus (ToNDV) and compare differences in vector transmissibility among isolates from tomato and weed hosts, as this will provide important information on virus epidemiology and risk to tomato and other crop hosts in California and the West.
4. Identify whitefly proteins that interact with proteins associated with virus particles during transmission of a semipersistently transmitted virus. Dual binding methods will be used including, but not limited to virus overlay assays and co-immunoprecipitation of proteins.
Progress Report
This report documents progress for project 2038-22000-018-00D, which started in August 2018 and continues research from project 2038-22000-017-00D, "Biology, Epidemiology and Management of Vector-Borne Viruses of Sugarbeet and Vegetable Crops."
Plant viruses and their vectors cause millions of dollars in losses to vegetable and cucurbit production each year through decreased yield, quality, and plant longevity, as well as the need for regular pesticide application. Research conducted at ARS in Salinas, California, through Sub-objective 1A, evaluated feeding behavior of whiteflies (Bemisia tabaci MEAM1) on tomato leaves infected with the semipersistently transmitted crinivirus, tomato chlorosis virus (ToCV) as well as on healthy tomato leaves using electrical penetration graphs, and was used to optimize methods for additional continuing research to determine how feeding behavior of whiteflies influences gene expression in the whitefly. This follows previous work by scientists at ARS in Salinas that demonstrated extensive differences in whitefly gene expression due to infection of tomato plants by ToCV. In addition, two dsRNA constructs were used in plant uptake studies to evaluate the potential to kill whiteflies using dsRNA delivered transiently to plants under Sub-objective 1B. The constructs used were the two best constructs based on previous work, which identified six double-stranded DNA constructs that induced whitefly mortality when acquired by whiteflies during in vitro feeding assays. Studies on tomato show promise for control of whitefly when constructs are delivered directly to plants; however, performance on an additional plant, melon, will require further research. Studies will continue as transient delivery offers an alternative to using genetically modified plants expressing the constructs. Related studies have led to the development of dsRNA constructs for control of beet leafhopper (Circulifer tenellus) the vector of beet curly top virus (BCTV) through Sub-objective 1D, and these are being evaluated jointly by scientists at ARS in Salinas, California, and collaborators at the University of California, Davis. In further efforts to understand how beet curly top virus may influence its vector insect, studies were conducted through Sub-objective 1C to compare the transcriptomes of leafhoppers fed on healthy tomato or sugarbeet plants in comparison with those fed on BCTV, respectively. Although all feeding experiments and transcriptome studies have been completed, data analysis is still in progress. Results will be compared with previous studies by ARS in Salinas, California, and Charleston, South Carolina, that examined the influence of a related virus, tomato yellow leaf curl virus, on its whitefly vector.
Lettuce dieback causes severe losses to lettuce production in the western U.S. where approximately 80% of U.S. lettuce production occurs, often with complete loss of crop. Two related and highly stable soil-borne tombusviruses are known to cause the disease, but in recent years disease symptoms have been increasingly observed in plants that are not infected by either virus, suggesting an additional virus may be involved. Diseased lettuce samples were collected and confirmed to contain a transmissible virus, but not the tombusviruses as a component of Sub-objective 2A. Total RNA extracted and used in RNA sequencing and small RNA analysis and subsequent results led to identification and partial sequence of an unknown virus that shows a high correlation with its presence in diseased lettuce and absence in healthy lettuce, suggesting this may be the missing causative agent of lettuce dieback disease. Further validation studies are in progress.
Additional research is working toward development of clones for agroinoculation of tomato necrotic dwarf virus (ToNDV), a torradovirus from California that threatens tomato production through Sub-objective 3A. The clones will facilitate more efficient evaluation of differences in host range and potentially transmission among isolates of this torradovirus through Sub-objective 3B. Both segments of the bipartite genome of the a ToNDV isolate were amplified and cloned into plasmid vectors and used to inoculate tomato and weed host plants, which became infected, confirming infectivity. Research is in progress to move the full-length clones into agrobacterium vectors to improve efficiency of transmission. Additionally, studies identified six melon breeding lines with a high degree of resistance to cucumber mosaic virus, and also identified a strain of beet necrotic yellow vein virus that compromised resistance induced not only by the widely used Rz1 gene, but also appears to compromise resistance to an additional resistance gene as well. This work was completed by ARS scientists at Salinas in collaboration with a beet sugar processing company and scientists at ARS in Fargo, North Dakota. Together, this research will lead to new approaches to reduce insect vector populations and the ability of vectors to transmit viruses to crop plants, improved management of resistance to Beet necrotic yellow vein virus (BNYVV), sources of resistance to Cucumber mosaic virus (CMV), and better control of virus diseases affecting tomato. This will benefit U.S. industry, growers, and improve food quality for consumers.
Accomplishments
1. Identified a new virus associated with lettuce dieback disease. Lettuce dieback causes necrosis, stunting and death of lettuce in the western U.S. where approximately 80% of U.S. lettuce production occurs, often with complete loss of crop. Two related and highly stable soil-borne tombusviruses are known to cause the disease, but in recent years disease symptoms have been increasingly observed in plants that are not infected by either virus, suggesting an additional virus may be involved. Diseased lettuce samples were collected previously from throughout the Salinas Valley region by a researcher in Salinas, California, with assistance from University of California Cooperative Extension. RNA sequencing and small RNA analysis of the samples led to identification and partial sequence of an unknown virus that is distantly related to a recently characterized member of the Phenuiviridae. Archived samples and additional field samples were subsequently evaluated for presence of the new virus, tentatively named lettuce dieback associated virus (LDaV), and show a high correlation between presence of the virus and disease. Characterization of the relationship between LDaV and disease development will clarify its importance in disease development and epidemiological importance for lettuce production, leading to improved knowledge of factors contributing to disease and benefitting the lettuce industry.
2. Identified Cucurbit chlorotic yellows virus infecting melons in California. The whitefly-transmitted virus, cucurbit yellow stunting disorder virus (CYSDV), causes severe yellowing, reduces fruit sugars, and results in yield loss of cucurbit crops, and severely impacts production of melon and watermelon in the low desert of California and Arizona. Researchers in Salinas, California, identified sources of resistance to this virus that will aid in the management of CYSDV. However, in summer 2018, a related virus, Cucurbit chlorotic yellows virus (CCYV) was found by the Salinas, California, researchers to be co-infecting melons with CYSDV from this region, its first identification in the Western Hemisphere. CCYV causes symptoms nearly identical to those of CYSDV, and preliminary studies suggest it is not controlled by resistance to CYSDV. New epidemiological studies are evaluating performance of CYSDV-resistant melon against CCYV and are examining potential weed and alternate crop reservoirs. This work will lead to new knowledge of how resistant cucurbit germplasm responds to this newly introduced virus, its potential for long-term impact on cucurbit production in the region, and improved management strategies for both whitefly-transmitted viruses.
3. Identified melon germplasm with strong resistance to cucumber mosaic virus. Cucumber mosaic virus (CMV) is an aphid-transmitted virus that has re-emerged as a serious threat to melon production in the western U.S. Cornell University released 25 melon breeding lines in the 1990s with resistance to CMV and one or two other aphid-borne viruses, but commercial melon cultivars with resistance to CMV are still not available for western growers. Researchers at ARS in Salinas, California, in collaboration with Cornell University, identified six breeding lines among the 25 that exhibited strong resistance to CMV and characterized their horticultural qualities for their use in development of CMV-resistant, western U.S. shipper type melons. Advancement of this material and introgression of resistance into commercial melon varieties will greatly improve control of CMV in the western U.S., and also offer protection from this virus in other areas of the world where the virus threatens production.
Review Publications
Nansen, C., Stewart, A.N., Gutierrez, T.A.M., Wintermantel, W.M., McRoberts, N., Gilbertson, R.L. 2019. Proximal remote sensing to differentiate nonviruliferous and viruliferous insect vectors – proof of concept and importance of input data robustness. Plant Pathology. 68(4)746-754. https://doi.org/10.1111/ppa.12984.
Wintermantel, W.M., Hladky, L.L., Fashing, P.L., Ando, K., McCreight, J.D. 2019. First report of Cucurbit chlorotic yellows virus infecting melon in the New World. Plant Disease. 103(4):778. https://doi.org/10.1094/PDIS-08-18-1390-PDN.
Farina, A.E., Rezende, J.A.M., Wintermantel, W.M. 2019. Expanding knowledge of the host range of Tomato chlorosis virus and host plant preference of Bemisia tabaci MEAM1. Plant Disease. 103(6):1132-1137. https://doi.org/10.1094/PDIS-11-18-1941-RE.
Wintermantel, W.M. 2018. Integration of omics approaches toward understanding whitefly transmission of viruses. Advances in Virus Research. 102:199-223.
Rojas, M.R., Macedo, M.A., Maliano, M.R., Soto-Aguilar, M., Souza, J.O., Briddon, R.W., Kenyon, L.A., Rivera Bustamante, R.F., Zerbini, M.F., Adkins, S.T., Legg, J.P., Kvarnheden, A., Wintermantel, W.M., Sudarshana, M.R., Peterschmitt, M., Lapidot, M., Martin, D.P., Moriones, E., Inoue-Nagata, A.K., Gilbertson, R.L. 2018. World management of geminiviruses. Annual Review of Phytopathology. 56:637-677. https://doi.org/10.1146/annurev-phyto-080615-100327.
Shamimuzzaman, M., Hasegawa, D.K., Chen, W., Simmons, A.M., Fei, Z., Ling, K. 2019. Genome-wide profiling of piRNAs in the whitefly, Bemisia tabaci reveals cluster distribution and potential association with begomovirus transmission. PLoS One. 14(3):e0213149. https://doi.org/10.1371/Journal.pone.0213149.
Keinath, A.P., Ling, K., Adkins, S.T., Hasegawa, D.K., Simmons, A.M., Hoak, S., Mellinger, C., Kousik, C.S. 2018. First report of cucurbit leaf crumple virus infecting three cucurbit crops in South Carolina. Plant Health Progress. 19:322-323. https://doi.org/10.1094/PHP-07-18-0039-BR.
