Research Project: Epidemiology, Vector-Host Plant Interactions, and Biology of Vegetable and Cucurbit Viruses

Location: Crop Improvement and Protection Research

2022 Annual Report

Objectives
Plant viruses and their vectors cause millions of dollars in losses to vegetable and cucurbit production each year. 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. • Subobjective 1B: Develop strategies for RNAi based control of whitefly in melon and tomato. • 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). • Subobjective 1D: Develop strategies for RNAi based control of the beet leafhopper. • Subobjective 1E: Develop strategies to characterize emerging insect pests and viruses in vegetable crops. 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. • 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. 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. • Subobjective 3B: Evaluate the host range of Tomato necrotic dwarf virus (ToNDV) and differences in vector transmissibility among isolates from tomato and weed hosts. 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.

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. 1E: Develop protocols for studying emerging insect pests and pathogens, insect performance and pathogen transmission in vegetable crops using in-field and laboratory based strategies. Molecular and genetic-based detection tools including, qPCR and ELISA will also be developed to monitor emerging insect pests and pathogens. 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 is the final report for project 2038-22000-018-000D, Epidemiology, Vector-Host Plant Interactions, and Biology of Vegetable and Cucurbit Viruses, which concluded in September 2022 and has been replaced by project 2038-22000-020-000D, Biological and biotechnological approaches for management of insect vectors and vector borne viruses affecting vegetable crops. For additional information, see the new project report. 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. In support of Sub-objective 1A, ARS researchers at Salinas, California, in collaboration with scientists at the University of California, Riverside, compared gene expression in whiteflies (Bemisia tabaci) on melon plants with and without infection by cucurbit yellow stunting disorder virus (CYSDV), in response to feeding behavior. Original plans were to use a different virus, tomato chlorosis virus (ToCV), for these studies, but CYSDV offered greater potential for application of results toward management of a critical pathogen affecting American agriculture, and additional parallel studies with a related emergent virus. This research follows previous work by ARS scientists in Salinas, California, that demonstrated extensive differences in whitefly gene expression when feeding on virus-infected melon plants compared to healthy melon plants. Virus infection of host plants versus healthy plants resulted in limited differences in whitefly gene expression; however, highly significant differences were found in response to switching from one type of host plant to another, and virus infection of the melon host plants clearly influenced the timing at which the expression changes occurred, suggesting that virus infection of the host plant may ease the transition of the whitefly from one type of host plant to another. This will lead to further research toward understanding how changing whitefly feeding host plants and virus infection of these hosts influences feeding behavior and virus acquisition. New detection methods were developed by ARS scientists to identify and quantify whitefly-transmitted viruses causing yellowing disease and severe losses for U.S. cucurbit crops. These diagnostic tools are being used by ARS, university and industry researchers in the Southeast and Southwest United States to identify the presence of whitefly-transmitted viruses in melon, watermelon, and other cucurbit crops, as well as in at least one other country. In support of Sub-objective 1B, ARS researchers evaluated double stranded RNA (dsRNA) constructs in transient delivery treatments to evaluate their potential to kill whiteflies (Bemisia tabaci) using dsRNA delivered to plants without genetic modification of the plants. Transgenic tomatoes expressing selected constructs are being evaluated for performance against whiteflies and show promise for control of whitefly. Related studies developed dsRNA constructs for control of beet leafhopper (Circulifer tenellus) the vector of beet curly top virus (BCTV). DNA sequencing confirmed integrity of the constructs. Studies are continuing toward development of reliable delivery methods for transient application of dsRNA for control of BCTV and whiteflies. In support of Sub-objectives 1C and 1D, the genome of the beet leafhopper (Circulifer tenellus) was fully sequenced through a collaboration between ARS scientists in Salinas and researchers at the University of California, Davis. Assembly of the 1GB genome is nearly complete and annotation of the genome is in progress and will facilitate completion of studies comparing differences in gene expression in beet leafhopper resulting from feeding on tomato and sugar beet plants with and without infection by beet curly top virus, an economically important virus affecting production of tomato, pepper, sugar beet and other crops. Additionally, ARS researchers compared the transcriptomes of leafhoppers fed on healthy tomato and sugarbeet plants with those fed on BCTV-infected plants of the two hosts. The availability of the beet leafhopper genome sequence will facilitate opportunities to understand leafhopper genetics, interspecies relationships, and development of novel control methods targeting the leafhopper, which are being conducted in support of Sub-objective 1D. In support of Sub-objective 1E, multiple projects are studying emerging insect pests and pathogens and developing management strategies. ARS researchers studied the epidemiology of thrips vectors (Frankliniella occidentalis) and a thrips-transmitted virus, impatiens necrotic spot virus (INSV), which is the most significant threat to lettuce production in the western United States, resulting in millions of dollars in losses, and have also detected INSV in low desert regions of Arizona and California, where winter lettuce production occurs. Due to the lack of efficacious pesticides to manage thrips, a lack of genetic-based resistance to the virus in commercial lettuce cultivars, and a large host range of plants that can support thrips and INSV, there is a need for new strategies to manage thrips and INSV. Research conducted in Salinas, California, has identified numerous weeds that can be a host for INSV, and has led to recommendations for managing specific weed species throughout the Salinas Valley. Similar research has identified weed hosts for INSV in Southern California and Arizona. Field surveys are examining the diversity of thrips species that are present in the Salinas Valley. Three field trials were conducted to test 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 massive benefits on human and environmental health. Trials were completed to assess the efficacy of predatory insects delivered by drones to manage aphids and thrips in organic commercial lettuce fields, and to test the effect of roguing as a cultural strategy to manage INSV infections in lettuce. Research to develop RNA interference (RNAi) technology for managing thrips and INSV has also been conducted (modified approach for Sub-objective 1B) and will continue to advance with future objectives. Similar RNAi technologies to manage diamondback moth in cole crops were also performed in one field trial in the Salinas Valley. One-field trial was also completed to test semiochemical lures as a method for mass capturing thrips in commercial lettuce production settings. Several key insect pests of lettuce and other leafy vegetables, including aphids, thrips, and diamondback moth, were monitored on the Central Coast of California, with 21 traps throughout the Salinas Valley with results reported weekly to stakeholders. Lettuce dieback causes severe losses to lettuce production in western U.S. regions where approximately 80% of U.S. lettuce production occurs, often with complete loss of crop. Two related and highly stable soil-borne tombusviruses were believed to cause the disease, but in recent years disease symptoms have been increasingly observed in plants not infected by either virus, suggesting an additional virus is involved. As a component of Sub-objective 2A, diseased lettuce samples were collected and confirmed to contain a transmissible virus, but not the anticipated tombusviruses. Total RNA was extracted and used in RNA sequencing and small RNA analysis for identification of unknown viruses. Results led to identification and partial sequence of a previously unknown virus that shows a high correlation with its presence in diseased lettuce and absence in healthy lettuce. The new virus is provisionally named lettuce dieback associated virus (LDaV), and using high throughput sequencing the ARS research team has sequenced the genome of this virus and determined it is likely the lone member of a new virus genus. Detection methods were developed to LDaV, and are now in use by industry for detection of the virus in commercial fields (Sub-objective 2A). Studies under Sub-objective 2B demonstrated the virus is transmissible mechanically to the highly susceptible experimental host plant, Nicotiana benthamiana, but is poorly transmissible mechanically to other plants, including lettuce; however, limited transmissions have induced dieback-like symptoms in controlled environment testing. The virus is likely to be obligately transmitted by a soil-borne organism in nature, which fits the pattern of virus distribution in fields. Research has been initiated toward development of experiments to evaluate the potential of soil-borne organisms for transmission of LDaV to lettuce. Tomato necrotic dwarf virus (ToNDV) is a whitefly-transmitted virus of tomato that causes severe stunting, limited fruit and seed production in tomato. Studies by ARS scientists determined the sequence of this virus. As a component of Sub-objective 3A, infectious clones were developed for inoculation of ToNDV to tomato and other plants without the need for transmission by whitefly vectors. The infectious clones have led to more efficient evaluation of differences in host range among isolates of ToNDV and other torradoviruses through Sub-objective 3B. Furthermore, the clones can be used to establish infections in tomato and other plants, which reduces the need to maintain the virus in infected plant material. Researchers are evaluating differences in host range and potentially transmission among isolates of this torradovirus, including those from tomato and a divergent isolate obtained from a wild species of Datura collected from the California desert. In parallel research, methods for detection of a wide range of torradoviruses are under development by the ARS scientists in collaboration with USDA Animal and Plant Inspection Service.

Accomplishments
1. Improved weed management to combat an insect-transmitted virus affecting lettuce. Lettuce production in the Salinas Valley of California accounts for over 50% of the total annual production in the United States. However, since 2018, impatiens necrotic spot virus (INSV), a thrips-transmitted virus, has severely impacted lettuce production. In 2021, over 750 fields reported disease incidence, with crop losses exceeding $100 million. Due to higher pest management costs associated with efforts to manage the insect, total farming costs were estimated to increase 10-15% in a survey conducted during 2020. Due to limited insecticides that can manage thrips and no existing methods for managing the virus, ARS researchers in Salinas, California, identified weeds and geographical locations that serve as virus reservoirs during the winter months, when lettuce is not grown. These findings are being used by growers for improved weed management, as well as by the California Department of Transportation, which is managing weeds along major highways throughout the Salinas Valley.

2. Virus infection of host plants may assist whiteflies in adaptation to new feeding hosts. Whitefly (Bemisia tabaci) is a virus vector that feeds on a wide range of host plants. Previous studies by ARS researchers in Salinas, California, have: 1) identified differences in gene expression within whitefly in response to the length of time whiteflies fed on virus-infected versus virus-free host plants in two biological systems, melon and tomato; and 2) that foregut-borne viruses altered whitefly gene expression differences to a greater extent relative to viruses that infiltrate the body cavity and salivary glands of the insect. Follow-up studies conducted in collaboration with the University of California, Riverside, examined gene expression in whiteflies after feeding on virus-free and healthy melon, as well as the influence of switching host plants (virus non-host to virus host) on whitefly gene expression. Virus infection of host plants versus healthy plants resulted in limited differences in whitefly gene expression; however, highly significant differences were found in response to switching from one type of host plant to another. Virus infection of the melon host plants clearly influenced the timing at which expression changes occurred, suggesting that virus infection of the host plant may ease the transition of the whitefly from one type of host plant to another. This has stimulated further research toward understanding how changing whitefly feeding host plants and virus infection of these hosts influences feeding behavior and virus acquisition.

3. Differential seasonal prevalence of yellowing viruses infecting melon crops in southern California and Arizona determined using a multi-virus detection method. Viruses transmitted by the whitefly (Bemisia tabaci) are an increasing threat to cucurbit production in the southwestern United States, and mixed virus infections challenge efforts at disease management and the development of resistant varieties. A method for detecting and quantifying multiple viruses was developed by ARS researchers in Salinas, California, and used to determine the prevalence and distribution of these viruses in melon samples collected from fields in the Sonoran Desert melon production region of California and Arizona, from 2019 through 2021. Results demonstrated that cucurbit yellow stunting disorder virus (CYSDV) is the predominant virus during the fall season, whereas cucurbit chlorotic yellows virus (CCYV) was by far the most prevalent virus during spring seasons. Comparative evaluations demonstrated differences in competitive accumulation of CCYV and CYSDV within melon as well as association with two other viruses in mixed infections within the region. This study also provided the first official report of squash vein yellowing virus in Arizona and provides a new diagnostic resource for virus detection and quantification that can be used by researchers and diagnostic labs.

Review Publications
Hunter, W.B., Wintermantel, W.M. 2021. Optimizing efficient RNAi-mediated control of Hemipteran pests (psyllids, leafhoppers, whitefly): Modified pyrimidines in dsRNA triggers. Plants. 10(9). Article 1782. https://doi.org/10.3390/plants10091782.
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. https://doi.org/10.1111/ppa.13572.
Hasegawa, D.K., Hladky, L.L., Wintermantel, W.M., Putman, A., Barman, A., Slinski, S., Palumbo, J., Poudel-Ward, B. 2022. First report of impatiens necrotic spot virus infecting lettuce in Arizona and southern desert regions of California. Plant Disease. https://doi.org/10.1094/PDIS-09-21-2118-PDN.