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Research Project: Biological and Biotechnological Approaches for Management of Insect Vectors and Vector-borne Viruses Affecting Vegetable Crops

Location: Crop Improvement and Protection Research

2023 Annual Report

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.

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, which started in August 2022 and continues research from project 2038-22000-018-000D, "Epidemiology, Vector-Host Plant Interactions, and Biology of Vegetable and Cucurbit Viruses". In support of Sub-objective 1A, research in Salinas, California, has been conducted to understand the host range for impatiens necrotic spot virus (INSV), which has emerged as a primary problem in lettuce production since 2019, resulting in millions of dollars in losses. The virus has also been detected in Imperial County, California, and in Arizona, where winter lettuce production occurs. Research conducted in Salinas, California, surveyed over 3,000 plants in the Salinas Valley, as well as Southern California and Arizona to identify numerous weeds that are hosts for INSV. The work has led to recommendations for managing specific weed species throughout the Salinas Valley to minimize the impact of INSV on lettuce production. In support of Sub-objective 1B, researchers in Salinas, California, have sequenced portions of INSV genomes to better understand the variation and evolution of the virus. Historical samples of INSV from the Salinas Valley are being sequenced, as well as samples from across the United States and international locations to identify differences among isolates. 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. In support of Sub-objective 1B, scientists in Salinas, California, and Corvallis, Oregon, developed a diagnostic method for reliable detection of tomato torrado virus (ToTV; genus Torradovirus) using RT-PCR (reverse transcription-polymerase chain reaction), a reliable and efficient molecular diagnostic method, that can be used for interception of the virus at ports-of-entry or by diagnostic laboratories to identify ToTV from infected plant material. Additional studies are continuing toward development of a diagnostic system that can detect all members of the torradovirus genus because many have the potential to threaten American agriculture if introduced into areas where affected crops, such as lettuce or tomato, are produced. In support of Sub-objective 1B, scientists in Salinas, California, are developing a serological detection method for a newly described virus that causes lettuce dieback disease. Lettuce dieback causes necrosis and wilting of lettuce plants, preventing marketability. The disease is regularly associated with soils having poor drainage or in areas where flooding has recently occurred. Recently, scientists at ARS in Salinas, California, identified and characterized a new virus that causes lettuce dieback disease and have provisionally named this virus, lettuce dieback associated virus (LDaV). Ongoing research is developing antiserum for routine detection of this virus using an expressed protein approach. Availability of antiserum will make detection readily available to seed companies and diagnostic labs serving the lettuce industry. In support of Sub-objective 2A, researchers in Salinas, California, have developed molecular tools to improve the detection of key thrips species, including western flower thrips (Frankliniella occidentalis), the primary vector for INSV. The molecular tools enable the simultaneous detection of several thrips species and thrips-transmitted viruses, and enhances our ability to detect economically important thrips vectors and viruses that impact vegetable production. In support of Sub-objective 2C, researchers in Salinas, California, evaluated the competitiveness of two closely related whitefly-transmitted viruses, cucurbit yellow stunting disorder virus (CYSDV) and cucurbit chlorotic yellows virus (CCYV), during mixed infections to determine how relative time of infection influences which virus is most abundant or dominates infection of melon (Cucumis melo) plants. Ongoing studies are examining how this impacts which virus is most efficiently transmitted to new melon plants. The outcome of these studies will contribute to understanding the epidemiology of these viruses in melon production regions and may in part explain differential seasonal prevalence of these viruses in western and southwestern U.S. melon production regions. In support of Sub-objective 2C, scientists in Salinas, California, are studying the host range of cucurbit chlorotic yellows virus (CCYV) in the melon production regions of the western and southwestern United States. CCYV is an emerging virus, first identified by ARS researchers in Salinas, California, in 2018. The virus has now spread to many areas of the country including Texas and many southeastern states where cucurbit crops such as melon, watermelon, squash and pumpkin, are produced. Recent studies by scientists in Salinas, California, have demonstrated differences in abundance of CCYV between spring and fall melon seasons in the Southwest, and results suggest this may be influenced by abundance of non-cucurbit host plants. Results of these ongoing studies should lead to better management of weeds and other plants that may harbor CCYV during the winter months when cucurbit crops are not grown. In support of Sub-objective 3A, researchers in Salinas, California, are developing RNA interference (RNAi) technology to manage thrips and INSV. Genetic targets in thrips and INSV have been identified and are being tested for their efficacy in lettuce. Similar RNAi technologies to manage diamondback moth in cole crops were also performed in one field trial in the Salinas Valley. 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. In support of Sub-objective 3B, researchers 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. Additional studies are in progress to determine the efficacy and full potential for these emerging technologies as they are integrated into pest management practices. In support of Sub-objective 3C, researchers in Salinas, California, are evaluating the effects of several plant immune priming agents for managing INSV in lettuce crops. One field trial has been conducted and demonstrate promise for minimizing INSV infection severity. Additional greenhouse and field 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.

1. Improved weed management to combat an insect-transmitted virus affecting lettuce. Annually, lettuce production in the Salinas Valley of California accounts for over 70 percent of the total production in the United States. However, since 2019, the insect-transmitted impatiens necrotic spot virus (INSV) has severely impacted lettuce production and in 2022, losses were reported at over $150 million. Due to limited insecticides that can manage thrips and no existing viable methods to manage the virus, ARS researchers in Salinas, California, identified important weeds and geographic locations that can serve as virus reservoirs during the winter months, that can bridge the gap during the off-season when lettuce is not grown. This has resulted in improved weed management as part of a cooperative effort by growers, industry association partners, and county-wide programs.

2. Development of detection methods for identification of Tomato torrado virus and other members of the Torradovirus genus. Tomato torrado virus (ToTV) is a virus of concern for tomato production, causing fruit and leaf necrosis and loss of crop and seed production. The virus is not present in the United States but can be introduced to new areas in plant material; therefore, development of methods for detection of virus at ports of entry is critical. ARS scientists in Salinas, California, and Corvallis, Oregon, designed methods to detect all genomic variants of ToTV currently known and focused on detection of genes encoding the virus coat protein. Methods did not cross-react with related members of the genus torradovirus based on computer alignment evaluations (in silico testing) using the genomic sequence of 35 torradovirus species available in the public genome database. This method is being delivered to USDA Animal and Plant Health Inspection Service for use in identification of ToTV in imported plant material to protect U.S. tomato fruit and seed production.

3. Competitive accumulation of criniviruses in melon plants during co-infection. The whitefly-transmitted and closely related viruses, cucurbit chlorotic yellows virus (CCYV) and cucurbit yellow stunting disorder virus (CYSDV), frequently infect the same cucurbit plants, but differ in abundance between spring and fall melon growing seasons. The seasonal differences in virus prevalence suggested differences in competitive accumulation of each virus in melon plants. To understand the basis for this, melon plants were singly inoculated with either CCYV or CYSDV, or simultaneously co-inoculated with both CCYV and CYSDV in standardized experiments conducted by ARS researchers in Salinas, California. Results showed that the timing of infection influenced which virus dominates the infection, with earlier infection generally leading to diminished titer of the later-infecting virus, but that in certain situations, early infection can lead to complete exclusion of infection by the second virus. Results add to the growing body of knowledge necessary to understand what determines which of the many viruses now present in U.S. cucurbit production are economic threats. The information contributes to efforts toward advancement of resistance to viruses in melon and disease management.

4. Detection methods for identification of impatiens necrotic spot virus and thrips vector species. Impatiens necrotic spot virus (INSV) is a primary concern for lettuce production in California. INSV is transmitted by the insect, western flower thrips, which can be challenging to identify due to their small size (1-2 mm). ARS researchers in Salinas, California, have developed molecular tools to improve the detection of western flower thrips and identification of thrips populations that are actively vectoring INSV. The diagnostic tools enhance the ability of researchers, diagnostic labs, and extension personnel to detect economically important thrips vectors and associated viruses that impact vegetable production.

5. Understanding the genetic diversity of viruses affecting vegetable and horticultural crops. Impatiens necrotic spot virus (INSV) affects vegetable and horticultural crops globally, including lettuce production in the California. ARS researchers in Salinas, California, and scientists at Washington State University, sequenced the several genomes of INSV from samples collected throughout the Unites States. These results demonstrate that INSV isolates currently impacting lettuce production in the western United States are very similar to historical isolates, indicating that the current epidemic is not driven by a change in the virus population itself, but rather by other causes.

Review Publications
Hasegawa, D.K., Del Pozo-Valdivia, A.I. 2023. Epidemiology and economic impact of Impatiens necrotic spot virus: A resurging pathogen affecting lettuce in the Salinas Valley of California. Plant Disease. 107(4):1192-1201.
Yun, S., Jang, H., Ahn, S., Price, B.E., Hasegawa, D.K., Choi, M.Y. 2023. Identification and characterisation of PRXamide peptides in the western flower thrips, Frankliniella occidentalis. Insect Molecular Biology.
Simko, I., Hasegawa, D.K., Peng, H., Zhao, R.B. 2023. Genetic and physiological determinants of lettuce partial resistance to Impatiens necrotic spot virus. Frontiers in Plant Science. 14. Article 1163683.
Mondal, S., Hladky, L.L., Wintermantel, W.M. 2023. Differential seasonal prevalence of yellowing viruses infecting melon crops in southern California and Arizona determined by multiplex RT-PCR and RT-qPCR. Plant Disease.
Vicentin, E., Mituti, T., Nogueira, A., Fecury Moura, M., Bello, V., Ribeiro-Junior, M.R., Wintermantel, W.M., Fiallo-Olive, E., Navas-Castillo, J., Krausse-Sakate, R., Rezende, J. 2022. Differential reaction of sweet pepper to infection with the crinivirus tomato chlorosis virus probably depends on the viral variant. Plant Pathology. 71(6):1313-1322.
Mondal, S., Wintermantel, W.M., Gray, S.M. 2023. Infection dynamics of potato virus Y isolate combinations in three potato cultivars. Plant Disease. 107(1):157-166.
Egel, D.S., Adkins, S.T., Wintermantel, W.M., Keinath, A.P., D'Arcangelo, K.N., Parada-Rojas, C.H., Rennberger, G., Toporek, S.M., Hausbeck, M.K., Quesada-Ocampo, L.M. 2022. Diseases of cucumbers, melons, pumpkins, squash, and watermelons. In: Elmer, W.H., McGrath, M., McGovern, R.J., editors. Handbook of Vegetable and Herb Diseases. Cham, Switzerland: Springer International Publishing. p. 1-105.