Location: Crop Improvement and Protection Research
2024 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 2038-22000-020-000D, titled, “Biological and Biotechnological Approaches for Management of Insect Vectors and Vector-borne Viruses Affecting Vegetable Crops”, which started in September 2022.
In support of Sub-objective 1A, ARS scientists in Salinas, California, have conducted field surveys to identify plant species that can serve as hosts for impatiens necrotic spot virus (INSV) in the Salinas Valley. The findings provide new knowledge of the host range of the virus and will guide management strategies to minimize the impact of INSV. ARS scientists have also partnered with the Autonomous University of Chapingo, Mexico, to provide the first documentation of INSV affecting lettuce in central Mexico. In 2023, a total of 22,092 hectares of lettuce were planted in Mexico, with a total production of 523,739 tons. Lettuce samples were collected in several states of central Mexico and sent to Salinas, California, for diagnostic testing, which identified INSV as the dominant thrips-transmitted Tospovirus present. Additional efforts have begun to understand the host range for INSV, as well as tomato spotted wilt virus (TSWV), a second Tospovirus, that has been known to affect a wide range of commodities in the region, including lettuce, spinach, broad beans, peas, and chrysanthemums. The work has identified several important commodities and weed species that are infected by the two viruses and advances the knowledge on the epidemiology of the diseases in Mexico. Studies are ongoing to provide additional scientific support to improve the management of thrips and thrips-transmitted viruses affecting lettuce and other major commodities grown in Mexico.
For Sub-objective 1B, ARS scientists have sequenced portions of INSV genomes to better understand the variation and evolution of the virus. To provide a greater understanding of the genomic diversity of INSV isolates, researchers have partnered with Washington State University and University of California, Davis, to conduct whole genome sequencing. Historical samples of INSV from the Salinas Valley, as well as samples from across the United States and international locations have been collected and submitted for whole genome sequencing. The work provides a better understanding of virus evolution and diversity from the largest and most economically important regions where production of lettuce and other INSV-susceptible vegetable and horticultural crops occurs.
Supporting Sub-objective 1C, ARS scientists in Salinas, California, have collected soil from the root zone of symptomatic lettuce plants infected with lettuce dieback associated virus (LDaV). LDaV was previously determined to be the true causative agent of lettuce dieback disease by ARS scientists in Salinas, California. Soilborne organisms associated with roots and the root zone of infected plants are being isolated for evaluation as potential vectors of this newly characterized virus in the family Phenuiviridae. This effort is time consuming and challenging, but it is important toward understanding the transmission of this soil-borne virus that has likely caused disease on lettuce for nearly a century known as both lettuce dieback and previously as brown blight.
For Sub-objective 1D, ARS scientists previously determined that competition occurs between two closely related criniviruses, cucurbit yellow stunting disorder virus (CYSDV) and cucurbit chlorotic yellows virus (CCYV), and that timing of infection, likely driven by whitefly transmission of these viruses from non-crop host plants in the spring and from melon in the summer/fall seasons influence virus dominance in each season. Results showed that CCYV is the dominant yellowing virus during the fall, whereas CYSDV is the dominant virus in the fall. This may have substantial impact on decisions for advancement of melon breeding lines. In efforts to understand how these viruses interact in melon and watermelon, molecular probes were developed and validated for detection of each virus in plant tissue. Efforts to characterize virus accumulation and host plant distribution for each virus will continue and should clarify how these related viruses and other virus that occur together in the yellowing virus complex establish infection and influence disease development. Ultimately this may lead to identification of novel approaches to reduce virus accumulation in plants and limit spread.
Under Sub-objective 1E, ARS scientists identified a new begomovirus, watermelon chlorotic stunt virus (WmCSV), infecting watermelon and melon in the Arizona and southern California desert production region. Research led by ARS in Salinas, California, involved collaborators at the California Department of Food and Agriculture, the University of Arizona at Yuma, Arizona, and the Imperial County, California Agriculture Commissioner’s Office. This is the first identification of WmCSV infecting cucurbit crops in the Western Hemisphere. Subsequent studies indicate the emergence of WmCSV may be altering the seasonal dynamics of CYSDV and CCYV in the region.
In support of Sub-objective 2A, ARS researchers developed molecular tools to improve the detection of INSV from thrips vectors. The molecular tool, referred to as reverse-transcription recombinase polymerase amplification (RT-RPA), allows for the rapid detection of INSV from thrips vectors in 20 minutes, and provides similar levels of sensitivity as other molecular tools at a lower cost. Additional diagnostic tools are also being developed that allow for the simultaneous detection of key thrips species, including western flower thrips (Frankliniella occidentalis), the primary vector for INSV, as well as viruses. Together, the two molecular tools enable flexibility in the detection of target pests and pathogens to meet various surveillance applications and provide improved diagnostics of economically important thrips vectors and viruses that impact vegetable production.
For Sub-objective 2B, ARS researchers in Salinas, California, in collaboration with researchers at ARS in Corvallis, Oregon, and the University of Minnesota, developed and validated methods for detection of the torradovirus, tomato torrado virus (ToTV). The methods 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. Additional multiplex RT-PCR primers were also developed for detection of tomato marchitez virus and its subspecies, including tomato necrotic dwarf virus, tomato chocolate spot virus, and tomato chocolate virus. Optimization of the latter is continuing. Methods for detection of torradoviruses that infect non-tomato crops and other species are more challenging as isolates are more difficult to obtain and viruses species do not have a broad range of isolates available; however, progress has been made and is continuing toward development of methods for detection of this broader group of viruses as well.
In support of Sub-objective 3A, ARS scientists are developing RNA interference (RNAi) technology to manage thrips and INSV. Genetic targets to thrips and INSV have been identified and are being tested for their efficacy in lettuce. For the first time, a lettuce field trial was conducted to test the efficacy of RNAi for managing INSV. The active materials of RNAi, referred to as double-stranded RNA (dsRNA) were delivered to the roots of lettuce plants and was successfully absorbed and distributed throughout the plants. Furthermore, the dsRNAs remained in the plants for several weeks, demonstrating a persistent delivery strategy for RNAi technologies. Longer term research continues to explore RNAi for control of other sap-feeding insects that transmit plants 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 Salinas, California, are evaluating 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. Several field trials were completed, which demonstrated that the precision spray technologies did provide greater residual efficacy than conventional application methods. Additional studies are ongoing to exploit the full potential for these emerging technologies as they are integrated into pest management practices.
In support of Sub-objective 3C, ARS scientists are evaluating the effects of several plant immune priming agents for managing INSV in lettuce crops. One field trial was conducted and demonstrate promising utility for minimizing INSV infection severity. Additional greenhouse studies are in progress to further optimize the timing of application and delivery dose for several immune priming products, which are already commercially available and approved for use in lettuce crops. The outcomes of the work will provide alternative strategies for managing thrips-transmitted viruses in lettuce crops.
Accomplishments
1. First report of Impatiens necrotic spot virus affecting lettuce in Mexico. Annually, Mexico produces over 500,000 tons of lettuce. In recent years, high levels of disease incidence have occurred, with plants showing symptoms of virus infection. ARS scientists in Salinas, California, have worked collaboratively with the Autonomous University of Chapingo, Mexico, to determine Impatiens necrotic spot virus (INSV) as the major pathogen resulting that has resulted in economic losses in lettuce. INSV is transmitted by a small insect, which is ubiquitous in the region. This finding has provided new knowledge on the causative pathogen and provides a baseline for developing management strategies for INSV and its insect vector.
2. Identification of watermelon chlorotic stunt virus for the first time in the Western Hemisphere. Unusual symptoms were observed in melon and watermelon plants from Yuma County, Arizona, and Imperial County, California, during Fall 2023. Samples were sent to ARS scientists in Salinas, California, who identified watermelon chlorotic stunt virus (WmCSV) as the causal agent of the symptoms and sequenced the genome of this virus for comparison to other global isolates. This is the first identification of WmCSV infecting cucurbit plants in the Western Hemisphere. This virus can cause significant yield loss in watermelon and is contributing to a virus yellowing complex on melon in the southwestern United States. ARS scientists in Salinas, California, have notified the cucurbit industry of the threat posed by this virus in efforts to keep it from being transported to other regions of the United States and are evaluating its epidemiological impact on cucurbit production.
3. Development of a new method for detecting Impatiens necrotic spot virus from its insect vector. Impatiens necrotic spot virus (INSV) is transmitted by small insects, called thrips. In recent years, INSV has caused hundreds of millions of dollars in losses to the lettuce industry in the Salinas Valley of California, which accounts for over 70% of all production in the United States. Due to the ubiquitous nature of thrips, it is difficult to predict when and where viral outbreaks will occur. ARS scientists in Salinas, California, developed a cheap and rapid tool to identify thrips populations that are infected with INSV. The new tool provides a new surveillance tool for identifying INSV by screening their insect vectors, and thus, new strategies for predicting virus outbreaks in lettuce crops.
4. Understanding the genetic diversity of viruses affecting vegetable and horticultural crops. Impatiens necrotic spot virus (INSV) is a global pathogen that affects vegetable and horticultural crops, including lettuce production in the California. ARS researchers in Salinas, California, and scientists at Washington State University and the University of California, Davis, are sequencing the genomes of INSV from samples collected throughout the Unites States and internationally. The work will provide a greater understanding of the diversity of the virus across regions where it is economically important for vegetable and horticultural crops.
5. Exploring new strategies for managing insect-transmitted viruses affecting lettuce. Impatiens necrotic spot virus (INSV) is a major challenge for lettuce production in California. INSV is transmitted by the small insect, western flower thrips and new strategies to improve the management of the virus and vector are urgently needed. ARS researchers in Salinas, California, are working with scientists at the University of California, Davis, to evaluate the utility of precision spray technologies for managing thrips, as well as researchers at the University of California, Riverside, to test immune priming products for managing INSV. Outcomes from both projects will provide additional tactics for managing thrips and INSV.
6. Development of molecular diagnostics for interception and identification of tomato torrado virus. Tomato torrado virus has emerged as a concern for tomato production in many parts of the world, but it has not been identified to date in the United States. USDA-APHIS needed a reliable method to identify low levels of ToTV on diverse plant materials. Therefore, ARS scientists in Salinas, California, and Corvallis, Oregon, developed a multiplex real-time (quantitative) diagnostic system for detection and quantification of tomato torrado virus (ToTV) from plant samples. The method targets two different genes of ToTV and includes an internal control for assay validation. The assay is specific for ToTV and showed no cross reactivity with other related torradovirus species. The methods have been provided to USDA-APHIS for use in interception of infected plant materials.
Review Publications
Lee, H., Wintermantel, W.M., Trumble, J.T., Nansen, C. 2024. Timing matters: Remotely sensed vegetation greenness can predict insect vector migration and therefore outbreaks of curly top disease. Journal of Pest Science. https://doi.org/10.1007/s10340-024-01771-4.
Pitter, P.L., Mondal, S., Chang, P.G., Morgan, L.M., Aikman, S., Wintermantel, W.M., Tennant, P.F. 2024. First report of cucurbit yellow stunting disorder virus infecting cucurbit crops in Jamaica. Plant Disease. 108(4):1120. https://doi.org/10.1094/PDIS-08-23-1551-PDN.
Jeger, M.J., Fereres, A., Malmstrom, C.E., Mauck, K.E., Wintermantel, W.M. 2023. Epidemiology and management of plant viruses under a changing climate. Phytopathology. 113(9):1620-1621. https://doi.org/10.1094/PHYTO-07-23-0262-V.
Nemchinov, L.G., Postnikova, O.A., Wintermantel, W.M., Palumbo, J.C., Grinstead, S.C. 2023. Alfalfa vein mottling virus, a novel potyvirid infecting Medicago sativa L. Virology Journal. 20:284. https://doi.org/10.1186/s12985-023-02250-5.
Weiland, J.J., Wyatt, N.A., Camelo, V., Spanner, R., Hladky, L.L., Ramachandran, V., Secor, G., Martin, F.N., Wintermantel, W.M., Bolton, M.D. 2024. Beet soil-borne virus is a helper virus for the novel Beta vulgaris satellite virus 1A. Phytopathology. 114(5):1126-1136.