Location: Crop Improvement and Protection Research
2020 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
In support of Sub-objective 1A, ARS researchers evaluated gene expression in whiteflies (Bemisia tabaci MEAM1) in association with feeding behavior on healthy melon leaves and melon leaves infected with the whitefly-transmitted crinivirus, Cucurbit yellow stunting disorder virus (CYSDV). This involved a minor change from original plans following the emergence of new virus closely related to CYSDV, causing severe economic concerns for melon production in the Southwest and the need to establish a system to understand competitive interactions between these viruses and how this may impact virus epidemiology and crop disease. Original plans intended to use a different virus for these studies, but CYSDV offers more potential for application of results toward management of a critical pathogen affecting American agriculture. This research follows previous preliminary work that demonstrated extensive differences in whitefly gene expression when feeding on virus-infected tomato and melon. New detection methods were developed to identify and quantify whitefly-transmitted viruses affecting U.S. cucurbit crops and are being used in Salinas and the southeastern United States to identify the presence of whitefly-transmitted viruses in melon and watermelon, as well as identify alternate hosts of recently introduced whitefly-transmitted viruses of cucurbit crops in the desert southwest United States.
In support of Sub-objective 1B, ARS researchers evaluated double stranded RNA (dsRNA) constructs in transient delivery treatments to evaluate the potential to kill whiteflies 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, and results are in progress.
In support of Sub-objective 1C, 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. All experiments and transcriptome sequencing were completed. Analysis of gene expression is pending completion of the genome sequence of the beet leafhopper. This will facilitate opportunities to understand leafhopper genetics, interspecies relationships, and development of novel control targeting the leafhopper, as described in Sub-objective 1D.
New research was initiated under Objective 1 to characterize emerging insect pests and pathogens: thrips vector (Frankliniella occidentalis) and a thrips-transmitted virus, Impatiens necrotic spot virus (INSV), which has become an increasing problem in lettuce production in California over the past several years. Due to the lack of efficacious pesticides to manage thrips, no sources of genetic resistance to the virus in lettuce, and large host range of plants that can support thrips and INSV, there is a need for alternative strategies to manage thrips and INSV. ARS documented the timing of thrips and INSV infection in commercial lettuce fields and their impact on the market quality of lettuce. The data led to recommendations for making pesticide applications that coincides with thrips infestations. Field surveys were also initiated to identify weed host reservoirs for INSV during the winter months when lettuce planted during the winter for early-Spring harvests is emerging. The data led to recommendations for managing specific weeds throughout the Salinas Valley as a means to reduce thrips habitat as INSV reservoirs. Research will continue to identify the impact of weeds on INSV incidence and thrips abundance in lettuce production. Insect colonies to propagate thrips under controlled laboratory and greenhouse settings have also been established to further characterize timing of symptom development of thrips transmission of INSV to lettuce, which will complement ARS field-based epidemiology studies to understand the impact of thrips and INSV on market quality of lettuce. Research to develop RNA interference (RNAi) technology for managing thrips and INSV have also been initiated. Genetic targets have been identified and dsRNA is being synthesized to conduct laboratory and greenhouse studies testing the efficacy of RNAi technology in managing thrips and INSV in lettuce production systems. In addition, ARS initiated research on a lettuce aphid (LA; Nasonovia ribisnigri) biotype Nr:1 that emerged in Salinas Valley in 2018 and is problematic because of its ability to overcome resistance to biotype Nr:0. LA bioassays have been developed to assess the performance and survival of field-collected aphids on LA biotype Nr:0-resistant lettuce cultivars. Results are confirming presence of the new aphid biotype Nr:1 in the Salinas Valley and research will continue to document its distribution.
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. In support of Sub-objective 2A, ARS researchers identified a previously unknown virus, tentatively named lettuce dieback associated virus (LDaV), and have now sequenced the genome of this virus and confirmed a close association between presence of the virus and lettuce dieback disease. Studies are in progress under Sub-objective 2B to determine whether or not LDaV incites disease when inoculated to lettuce after isolation from an alternate host. Fields known to have a history of lettuce dieback disease have been identified in the Salinas Valley, and soils are being collected for use in controlled experiments to determine associations of soil characteristics with LDaV infection, as was previously shown for tombusvirus infection of lettuce.
In support of Sub-objective 3A, ARS researchers developed clones of tomato necrotic dwarf virus (ToNDV), a torradovirus from California that threatens tomato production. Clones of the virus' two genomic RNAs were infectious when inoculated to plants together using rub-inoculation, but ‘agro-inoculation clones’ will allow a more efficient and dose-regulated mode of inoculation for evaluation of differences in host range and potentially transmission among isolates of this torradovirus. This work supports Sub-objective 3B.
Accomplishments
1. New detection methods to identify and quantify whitefly-transmitted viruses affecting U.S. cucurbit crops. In recent years, numerous whitefly-transmitted viruses have emerged in the major U.S. cucurbit production regions, leading to reduced yields and in some areas, significant reductions in crop production. Many of these viruses produce similar symptoms on infected plants, making resistance evaluations difficult. Alternate host plants that can harbor these viruses are not known for two of these viruses, cucurbit chlorotic yellows virus and squash vein yellowing virus. Methods were developed by ARS in Salinas, California, to allow efficient identification of four different viruses that commonly co-infect cucurbit crops in the United States, as well as a separate system to quantify differences in accumulation among the four viruses. These methods are actively being used by ARS in Salinas and other laboratories throughout the United States to determine virus distribution and regional alternate host plants, as well as for evaluation of plants for virus resistance.
2. Improved weed management to combat an insect-transmitted virus affecting lettuce. Lettuce production in the Salinas Valley of California accounts for over 50% of annual U.S. lettuce production. However, over the past several years, there have been increasing reports of a thrips-transmitted virus, impatiens necrotic spot virus, severely impacting lettuce production, and in 2019, several growers reported up to 100% crop losses due to this virus. Due to limited insecticides that can manage thrips and no existing methods for managing the virus, ARS researchers in Salinas, California, identified important weeds that can serve as virus reservoirs during the winter months, and thus, bridge the gap during the off-season when lettuce is not grown. This has resulted in improved weed management by growers, as well as the California Department of Transportation, which is managing weeds along major highways throughout the Salinas Valley.
3. Genetic characterization of lettuce dieback associated virus isolates from coastal and desert regions. Lettuce dieback causes necrosis, stunting and death of lettuce in western U.S. lettuce production regions, where approximately 80% of U.S. lettuce production occurs, often with complete loss of crop and severe economic losses to producers. 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. Indeed, a new, previously unknown virus distantly related to a recently characterized member of the Phenuiviridae, was identified by ARS researchers in Salinas, California, using high throughput sequencing from lettuce plants exhibiting dieback symptoms. The complete genome of the virus, tentatively named lettuce dieback associated virus (LDaV), has now been determined, and routine detection methods developed and used to confirm correlation with presence of the disease. This information, along with new detection methods, will clarify the importance of LDaV in disease development, its epidemiological importance for lettuce production, and determine performance of resistant varieties against the new virus.
4. Determined influence of feeding on virus-infected plant sap and virus retention in the whitefly vector on insect gene expression. Whitefly-transmitted viruses, and the whiteflies that transmit them, devastate production of vegetable and other crops in the United States and throughout the world. There is growing evidence that the acquisition of viruses by their insect vectors can significantly alter the physiology, behavior, and survival of the insect, and this could have important implications for control of both viruses and their insect vectors. Prior research by ARS in Salinas, California, found that whiteflies undergo significant changes in gene expression at 72 hours following acquisition of Cucurbit yellow stunting disorder virus (CYSDV), a foregut-borne virus, to a greater extent relative to whiteflies acquiring a virus that has a much more intimate association with the whiteflies. Experiments were conducted by scientists at ARS in Salinas and the University of California, Riverside, to compare changes in gene expression in whitefly vectors in response to feeding on plants infected with CYSDV at multiple time points, as well as due to retention of virions in the whitefly over time. Results will determine physiological responses in the whitefly to virus acquisition and retention and should lead to opportunities to control whiteflies and virus transmission by targeting insect physiology.
5. Sequenced the genome of the beet leafhopper. The beet leafhopper has been established in the United States for over a century and transmits beet curly top virus (BCTV) resulting in economic losses and reduced yields for numerous crops throughout the western United States, including tomato, pepper, sugar beet, bean, and other crops. In an effort to identify new strategies for control of the beet leafhopper and reduce spread of BCTV, ARS researchers in Salinas, California, and collaborators at the University of California, Davis, conducted experiments to sequence the genome of the beet leafhopper (Circulifer tenellus). This information will facilitate ongoing analysis of gene expression in the beet leafhopper, lead to identification of additional gene targets for potential control of leafhoppers, and provide information for comparative studies with other hemipteran insect pests.
Review Publications
Chen, W., Wosula, E.N., Hasegawa, D.K., Casinga, C., Shirima, R.R., Fiaboe, K.K., Hanna, R., Fosto, A., Goergen, G., Tamò, M., Mahuku, G., Tripathi, L., Mware, B., Kumar, L.P., Ntawuruhunga, P., Moyo, C., Yomeni, M., Boahen, S., Edet, M., Awoyale, W., Wintermantel, W.M., Ling, K., Legg, J.P., Fei, Z. 2019. Genome of the African cassava whitefly Bemisia tabaci and distribution and genetic diversity of cassava-colonizing whiteflies in Africa. Insect Biochemistry and Molecular Biology. 110:112-120. https://doi.org/10.1016/J.Ibmb.2019.05.003.
Maliogka, V.I., Wintermantel, W.M., Orfanidou, C.G., Katis, N.I. 2020. Criniviruses infecting vegetable crops. In: Poltronieri, P., Hong, Y., editors. Applied Plant Biotechnology for Improving Resistance to Biotic Stress. San Diego, CA: Elsevier. p. 251-289.
