Location: Crop Improvement and Protection Research
2025 Annual Report
Objectives
Plant viruses and their vectors cause millions of dollars in losses to vegetable production each year through decreased yield, quality, and plant longevity, as well as the need for regular pesticide application. Research is necessary to understand factors driving the emergence of and changes in the prevalence of new viruses, as well as to develop more environmentally friendly control methods. This research will lead to safer and more sustainable practices for management of vector populations, reduce transmission of viruses to crop plants, benefit the U.S. vegetable industry and growers, and improve food quality for consumers.
Objective 1: Characterize the epidemiology of and interactions between insects and insect-transmitted viruses to understand their distribution and threat to vegetable crop production.
Sub-objective 1.A: Conduct field surveys to identify the host range for thrips-transmitted tospoviruses affecting lettuce production in California and Arizona.
Sub-objective 1.B: Characterize the genetic variation of Impatiens necrotic spot virus (INSV) isolates using RNA sequencing.
Sub-objective 1.C: Evaluate soil-borne organisms as potential vectors of lettuce dieback associated virus (LDaV).
Sub-objective 1.D: Compare competitive binding of cucurbit chlorotic yellows virus (CCYV) and cucurbit yellow stunting disorder virus (CYSDV) (both genus Crinivirus, Closteroviridae) in whitefly vectors and how this relates to preferential transmission of one virus over the other.
Objective 2: Develop resources to identify and monitor vegetable crops for introduction or emergence of novel viruses and vectors.
Sub-objective 2.A: Develop monitoring strategies for identifying thrips vector species and thrips-transmitted viruses using genetic markers.
Sub-objective 2.B: Develop and validate molecular methods for detection of torradoviruses that infect vegetable crops.
Objective 3: Develop biotechnology tools, such as RNA interference to create new tools for managing insects and insect-transmitted viruses affecting vegetables.
Sub-objective 3.A: Develop RNA interference (RNAi) technologies for managing insects and insect-transmitted viruses, including thrips, whiteflies, and leafhoppers.
Sub-objective 3.B: Evaluate precision-spray technologies for managing insects and insect-transmitted viruses.
Sub-objective 3.C: Evaluate plant immune priming agents for managing insect-transmitted viruses.
Approach
1A. Field surveys will be conducted to determine the presence of impatiens necrotic spot virus (INSV) in symptomatic and asymptomatic plant species that are common in lettuce production areas. Symptomatic and asymptomatic plants will be sampled across four locations including different habitats and tested for presence of INSV to identify alternate hosts.
1B. Full genomes of current and archived isolates of INSV samples from lettuce will be determined using Oxford Nanopore or Illumina sequencing methods. Based on the outcomes of these studies, genetic markers will be identified for any unique INSV isolates and PCR primers will be developed to amplify the genetic region. This will allow more rapid determination of the prevalence of different virus variants.
1C. Soil-borne root-associated organisms will be isolated from soil and virus-free isolates characterized and propagated. This virus-free culture will be exposed to virus infected lettuce and other host plants in an attempt to determine if the soil-borne organisms can acquire the virus. Lettuce will be grown in these soils and tested for virus incidence to determine if the soil-borne organism can transmit the virus to lettuce.
1D. Confocal microscopy and fluorescent in situ hybridization will be used to compare competitive differential binding of two closely related whitefly-transmitted viruses. This will contribute to determining what influences differential transmission and virus species dominance in agricultural ecosystems.
2A. Available genetic information will be used to design primers to differentiate thrips species common in the Salinas Valley from one another, and these will be used to identify thrips species and their host range among regional plants.
2B. Multiplex (multiple primers in a single reaction) and virus-specific primers will be designed against known torradovirus sequences and specificity confirmed against target and non-target virus isolates. Methods will aid in identification of torradoviruses in imported plant material.
3A. Genetic targets to western flower thrips (Frankliniella occidentalis), and beet leafhopper (Circulifer tenellus) will be identified for suppression using RNAi strategies. In vitro testing will be used to evaluate effectiveness of RNAi against insect pest targets and against non-target insects. Methods will lead to novel methods to suppress populations of these important virus vectors.
3B. Field trials will be conducted to evaluate precision spray technologies to optimize performance of reduced pesticide application methods for control of thrips on commercial lettuce. Results will be compared directly with conventional approaches.
3C. Commercially available agents that activate plant defenses against pathogens will be evaluated to determine their efficacy in protecting lettuce from impatiens necrotic spot virus (INSV), an important thrips-transmitted virus threatening lettuce production. Agents with high level performance will be combined with reduced pesticide application programs to enhance control of INSV in lettuce.
Progress Report
This report documents progress for project 2038-22000-020-00D, which started in August 2022 and continues research from project 2038-22000-018-00D, "Epidemiology, Vector-Host Plant Interactions, and Biology of Vegetable and Cucurbit Viruses."
In support of Sub-objective 1A, ARS scientists in Salinas, California, in partnership with the University of California Cooperative Extension, Grower-Shipper Association of Central California, and Monterey County Agricultural Commissioner’s Office, have conducted winter field surveys to identify weed species that serve as hosts for impatiens necrotic spot virus (INSV) in the Salinas Valley. The findings provide new knowledge on plant species and specific locations to direct weed management efforts to mitigate the impact of the virus during the lettuce season. Additional INSV susceptibility tests are being conducted on beneficial plant species that provide various ecosystem services, including cover crops, pollinator and beneficial insect habitat, and erosion management. The findings provide knowledge on ‘INSV safe’ plants that can be integrated into various on and off farm purposes, including weed replacement strategies to minimize the impact of INSV. A manuscript describing the work will be submitted in FY26.
In support of Sub-objective 1B, ARS scientists in Salinas, California, in partnership with Washington State University and University of California, Davis, have obtained high-quality genome assemblies for 159 INSV isolates from California, Arizona, Mexico, and other parts of North America. This information will provide a greater understanding of the variation and evolution of the virus, as it has emerged as a major threat to lettuce production in western regions of North America. The virus can also infect numerous other vegetables, fruits, and ornamental crops. A manuscript describing the work will be submitted in FY26.
Supporting Sub-objective 1C, ARS scientists in Salinas, California, have begun isolating organisms from the root zone of lettuce plants exhibiting symptoms of lettuce dieback disease and infected with lettuce dieback associated virus (LDaV) to identify potential vector organisms that may transmit this newly identified virus in the field. LDaV was initially described from lettuce with symptoms of lettuce dieback disease and has an infection pattern in fields that is suggestive of a virus transmitted by a soil-borne organism. Knowledge of soil-borne organisms associated with lettuce production and taxonomic relationships between LDaV and other viruses may offer indications of potential vector organisms. The first step of this process is to isolate potential vector organisms and establish pure cultures. Identifying a vector that transmits LDaV is an important step in developing effective management strategies and will be of great value both field management and breeding programs evaluating plants for resistance.
In a project related to Sub-objective 1C, ARS scientists in Salinas, California, cloned the genome of LDaV. Studies are in progress to determine infectivity of the clones and to establish an agroinoculation system that will allow rapid delivery of the infectious virus to plants. This will facilitate more efficient laboratory and greenhouse inoculations that will enhance the ability of the seed industry to advance lettuce breeding lines with resistance to LDaV. Additionally, the ARS scientists cloned the LDaV coat protein gene, expressed the coat protein (outer shell of virus particle), and are in the process of developing an antiserum that can be used for detection of LDaV from lettuce. The antiserum will need to be purified and evaluated, but when complete, should be a cost-effective method for detection of LDaV from infected lettuce in both the field and laboratory. A reliable antiserum-based detection system should improve availability and reduce costs associated with the identification of lettuce dieback disease in lettuce fields.
Under Sub-objective 1E, ARS scientists in Salinas, California in collaboration with scientists at Mississippi State University identified a new orthotospovirus, melon severe mosaic virus (MSMV), in pumpkin plants collected from Mississippi in 2022 and exhibiting severe leaf symptoms including mosaic patterns and frilled leaf edges. This is the second detection in the United States but predates the initial finding in Arkansas by one year, making this the earliest detection of the virus in the United States. MSMV was also identified in Mississippi in 2024 demonstrating its continued presence in the state. The ARS scientists developed methods for molecular-based detection of MSMV, facilitating more rapid detection and differentiation of the virus from other viruses that may cause similar symptoms not only in Mississippi, but in other locations where cucurbit crops are grown. A manuscript describing this work has been submitted for publication.
Under Sub-objective 1E, ARS scientists in collaboration with scientists from the University of Arizona and the University of California, Riverside, have been monitoring melon and watermelon crops, as well as regional weeds throughout the Sonoran Desert production region of California and Arizona for the presence of watermelon chlorotic stunt virus (WmCSV), a newly emergent virus first identified by ARS scientists in Salinas, California, in 2023 that causes disease on watermelon and melon plants. Surveys are evaluating weeds and crops that may be hosts of the virus, and studies are examining if some of these hosts may be efficient overwintering sources of WmCSV from which the sweetpotato whitefly (Bemisia tabaci) vector can acquire the virus and transmit it to melon and watermelon crops. A peer-reviewed manuscript describing the first detection of WmCSV infecting cucurbit crops in the Western Hemisphere, specifically in Arizona and California, was recently published.
In support of Sub-objective 2A, ARS scientists in Salinas, California, in partnership with the University of California, Davis, have developed new trapping methods for thrips. The traps improve the capture efficiency of thrips while reducing the bycatch of non-target insects, including pollinators and other beneficial insects. Thrips that are captured from the new traps are processed for quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to identify vector species and determine the presence of thrips-transmitted viruses including INSV. Together, the new surveillance tools enable the detection of plant viruses from thrips vectors to improve the monitoring of economically important thrips vectors and viruses that impact vegetables in the region. A patent application for the invention has been submitted for the technology.
For Sub-objective 2B, ARS scientists collaborated with scientists at USDA-APHIS plant protection and quarantine for validation of methods for detection of tomato torrado virus (ToTV) developed previously through a collaboration with researchers at ARS in Corvallis, Oregon, and the University of Minnesota. ToTV and the related virus, tomato marchitez virus (ToMarV) cause severe necrotic symptoms tomato leaves and fruit and can lead to loss of yield and severely curtail tomato seed production. The methods developed by ARS differentiate ToTV from other torradovirus species and have been shown to detect all variants of ToTV for which sequences were available in Genbank and for which targets were tested. Research to complete methods for detection and differentiation of ToTV and the closely related torradoviruses are continuing. These methods will facilitate interception of ToTV and other torradoviruses on plant material imported into the U.S. from other countries, protecting American tomato production.
In support of Sub-objective 3A, ARS scientists in Salinas, California, in partnership with a commercial party, are developing RNA interference (RNAi) technologies to manage thrips and INSV. The active materials of RNAi, referred to as double-stranded RNA (dsRNA) were delivered to lettuce plants and successfully distributed throughout the plants. The dsRNAs remained in the plants for several weeks, demonstrating a persistent delivery strategy for RNAi technologies. Studies are underway to test the efficacy of the RNAi technologies for managing INSV, while longer term research continues to explore RNAi for control of other insect vectors and viruses, including whitefly and beet leafhopper. These studies are expected to lead to improved control of insect pests and virus vectors, reducing their impact on crop production in the United States.
For Sub-objective 3B, ARS scientists in partnership with University of California, Davis, evaluated the utility of precision-spray technologies for managing aphids and thrips in lettuce. Precision spray technologies have the potential to reduce the total volume of pesticide use per application by 90%, which could have tremendous benefits for human and environmental health and cost savings. Several field trials were completed, which demonstrated precision spray technologies provided greater residual efficacy than conventional application methods. A manuscript describing the work has been submitted for publication.
In support of Sub-objective 3C, ARS scientists in Salinas, California, in partnership with the University of California, Riverside, are evaluating the effects of several plant immune priming agents for managing INSV in lettuce crops. Several field and greenhouse trials have been performed, demonstrating promising utility for the immune priming agents in minimizing INSV infection severity. The outcomes of the work will provide alternative strategies for managing thrips-transmitted viruses in lettuce crops.
Accomplishments
1. Harnessing insect vectors to predict virus outbreaks in California lettuce. The Salinas Valley of California grows over 70% of all lettuce in the United States with an estimated crop value of 1.2 billion U.S. dollars annually. However, plant viruses transmitted by insects have caused hundreds of millions of dollars in losses in recent years. An ARS scientist in Salinas, California, in partnership with the University of California, Davis, developed a new insect trap that improves the capture rate of target insect species, while allowing downstream diagnostics to screen the insects for plant pathogenic viruses. The trap also minimizes capturing pollinators and other beneficial insects. This surveillance tool provides new, more efficient and effective strategies for predicting virus outbreaks in lettuce crops and can potentially be used in other cropping systems where insect-transmitted viruses are an issue.
2. Nothing but the pest: precision spray technologies improve the management of insects in California lettuce. The Salinas Valley of California grows over 70% of all lettuce in the United States. However, lettuce production is threatened by several important insect pests, particularly in organic systems where management options are limited. An ARS scientist in Salinas, California, in partnership with the University of California, Davis, used automated spray technologies that apply insecticides precisely to lettuce plants. The technology maintains insecticidal efficacy and can reduce pesticide inputs by 90%, compared to traditional application methods, while reducing the risks for human and environmental harm, and providing cost savings.
3. A new virus affecting cucurbits in the United States. Melon severe mosaic virus (MSMV) is a recently identified orthotospovirus previously characterized from cucurbit crops in Mexico. The identification of MSMV in Mississippi in 2022 and again in 2024 through a collaboration between scientists at ARS in Salinas, California, and Mississippi State University and a separate detection in Arkansas by others in 2023 raises concern that this emerging virus could establish and impact cucurbit production in the United States. Although there is no known vector identified for MSMV, its taxonomic classification as an orthotospovirus suggests it is likely transmitted by one or more species of thrips. This would make MSMV the first thrips-transmitted virus infecting cucurbit crops in the United States, which is of concern for cucurbit growers who already have extensive concerns with viruses transmitted by whiteflies and aphids.
4. A new tool to advance development of virus resistance in blackberry breeding programs. Blackberry yellow vein associated virus (BYVaV) is a pathogen transmitted by the greenhouse whitefly (Trialeurodes vaporariorum) and is found throughout many blackberry producing regions of the United States. The virus can cause serious losses resulting from interactions with other viruses when present together in blackberry plants. BYVaV was cloned into an Agrobacterium vector for easy delivery to plants by collaborators at the University of Arkansas. Researchers at ARS in Salinas, California, demonstrated that the cloned virus could be transmitted from plant-to-plant by greenhouse whitefly, producing symptoms typical of infection by the virus and confirming whitefly transmissibility of the cloned virus. The use of the clone is important to screen plant materials for BYVaV resistance in breeding programs for blackberry and other Rubus crops.
Review Publications
Mejia Quevedo, I., Hladky, L.L., Tlapal Bolanos, B., Hasegawa, D.K. 2024. First report of Impatiens necrotic spot virus infecting lettuce in Mexico. Plant Disease. 108(11):3423. https://doi.org/10.1094/PDIS-06-24-1191-PDN.
Zhang, S., Hladky, L.L., Hasegawa, D.K. 2024. Rapid detection of Impatiens necrotic spot virus from thrips vectors using reverse transcription-recombinase polymerase amplification. Scientific Reports. 14. Article 21946. https://doi.org/10.1038/s41598-024-73078-4.
McCoy, A.G., Martin, F.N., Matson, M.E., Koike, S., Miles, L.A., Hasegawa, D.K., Camelo, V., Miles, T.D., Chilvers, M.I. 2025. Development and application of species-specific qPCR and RPA assays for the lettuce wilt pathogen, Globisporangium uncinulatum (syn. Pythium uncinulatum). PhytoFrontiers. 5(2):209-219. https://doi.org/10.1094/PHYTOFR-10-24-0112-FI.
Sierra-Mejia, A., Villamor, D.E.V., Rocha, A., Wintermantel, W.M., Tzanetakis, I.E. 2024. Engineering a robust infectious clone and gene silencing vector from blackberry yellow vein associated virus. Virus Research. 350. Article 199488. https://doi.org/10.1016/j.virusres.2024.199488.
de los Angeles Mora-Ugalde, M., Miranda-Campana, O.A., Ramirez-Soto, M., Cruz-Jaramillo, J.L., de los Reyes Bolaños, I.E., Tovar-Pedraza, J.M., Wintermantel, W.M., Diaz-Lara, A. 2024. High throughput sequencing reveals mixed infections of impatiens necrotic spot virus and tomato brown rugose fruit virus in bell pepper crops in Mexico. Journal of Plant Pathology. 107:711–714. https://doi.org/10.1007/s42161-024-01806-w.
Alves de Macedo, M., Melgarejo, T., Vasquez-Mayorga, M., Cespedes, M., Rojas, M., Turini, T., Batuman, O., Wintermantel, W.M., Gilbertson, R.L. 2024. Squash vein yellowing virus from California emerged in the Middle East via intragenic and intergeneric recombination events in the hypervariable potyvirus P1 and ipomovirus P1a genes. Journal of General Virology. 105(10). Article 002033. https://doi.org/10.1099/jgv.0.002033.
Wintermantel, W.M., Tian, T., Chen, C., Winarto, N., Szumski, S., Hladky, L.L., Gurung, S., Palumbo, J.C. 2024. Emergence of watermelon chlorotic stunt virus in melon and watermelon in the southwestern United States. Plant Disease. 108(12):3664. https://doi.org/10.1094/PDIS-05-24-1009-PDN.