Location: Crop Improvement and Protection Research
2021 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 at Salinas, California, in collaboration with scientists at the University of California, Riverside, examined feeding behavior of whiteflies (Bemisia tabaci), the vector of the crinivirus, cucurbit yellow stunting disorder virus (CYSDV), on melon plants infected with CYSDV and virus-free melon plants. In association with this, samples have been collected for studies on gene expression in whitefly in response to feeding behavior. 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., 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.
In support of Sub-objective 1C, ARS researchers compared the transcriptomes of leafhoppers fed on healthy tomato and sugarbeet plants with those fed on beet curly top virus (BCTV)-infected plants of these two hosts. All experiments and transcriptome sequencing have been completed; however, completion of the analysis of gene expression is pending completion of the genome sequence of the beet leafhopper, which is in the process of assembly in studies conducted through a collaboration with scientists at the University of California, Davis. Completion of gene expression studies and the leafhopper genome will facilitate opportunities to understand leafhopper genetics, interspecies relationships, and development of novel control targeting the leafhopper. This work is in support of Sub-objective 1D.
In support of Sub-objective 1E, multiple projects have been initiated to characterize emerging insect pests and pathogens. Research has been initiated to understand the epidemiology of thrips vectors (Frankliniella occidentalis) and a thrips-transmitted virus, impatiens necrotic spot virus (INSV), which has emerged as one of the greatest threats to western U.S. lettuce production during the past few years. The virus has also been detected in regions where lettuce is produced during the winter, including Imperial County, California, and Arizona, by ARS scientists in Salinas. Due to the lack of efficacious pesticides to manage thrips, a lack of genetic resistance to the virus in lettuce cultivars that are commercially available, and a large host range of plants that can support thrips and INSV, there is a need for alternative strategies to manage thrips and INSV. Research conducted by the ARS scientists has identified numerous weed hosts that serve as infection reservoirs for INSV. They developed associated recommendations for managing specific weed species throughout the Salinas Valley as a means to reduce INSV reservoirs for thrips to acquire the virus from and transmit it to lettuce crops. Research will continue to identify important habitats and locations that support these weed species in the Salinas Valley, as well as winter production regions in Southern California and Arizona. Molecular detection tools were also developed to simultaneously detect INSV and tomato spotted wilt virus (TSWV), as well as a related thrips-transmitted virus that affects lettuce.
The emergence of thrips and INSV as the preeminent problem for California lettuce production has led to initiation of research by ARS scientists to develop RNA interference (RNAi) technology for managing thrips and INSV as an extension of Sub-objectives 1B and 1D. Genetic targets have been identified and double-stranded RNA (dsRNA) has been synthesized to conduct laboratory and greenhouse studies testing the efficacy of RNAi technology in managing thrips and INSV in lettuce production systems. Similar RNAi technologies to manage diamondback moth in cole crops were also conducted as field trials in the Salinas Valley. Additionally, in support of Sub-objective 1E, research has begun to test precision spray technologies for managing insect pests 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 to human and environmental health. However, there is needed work to determine the efficacy and full potential for these emerging technologies as they are integrated into pest management practices.
In support of Sub-objective 2B, ARS scientists have examined rub inoculation of the newly identified causative agent of lettuce dieback disease, lettuce dieback associated virus (LDaV), to test plants. These studies demonstrated that although LDaV is transmissible by rub inoculation to the highly susceptible experimental host plant, Nicotiana benthamiana, it is poorly transmissible by this method to other plants, including lettuce. This strongly suggests 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 and N. benthamiana, and will include development of pure culture specimens of potential soil-borne vector organisms. To date, one organism has been isolated in pure culture for these studies, while another is in progress.
In support of Sub-objective 3B, ARS researchers are using clones of tomato necrotic dwarf virus (ToNDV), a torradovirus from California that threatens tomato production, to establish infections in tomato and other plants. This work is evaluating differences in host range and potential transmission among isolates of this torradovirus. In parallel research, methods for detection of a wide range of tomato-infecting torradoviruses are under development by ARS scientists to improve efficiency of detection for not only ToNDV, but for other torradoviruses affecting tomato, as well as those impacting other vegetable crops, including lettuce and cucurbit crops.
Accomplishments
1. Improved weed management to combat an insect-transmitted virus affecting lettuce. Annually, lettuce production in the Salinas Valley of California accounts for over 50% of the total production in the United States. However, over the past several years, an insect-transmitted virus has severely impacted lettuce production and in 2020, crop losses exceeded $50 million. Due to higher pest management costs associated with efforts to manage the insect, total farming costs were estimated to increase 10-15% in 2020. Due to limited insecticides that can manage thrips and no existing methods for managing the virus, ARS researchers in Salinas, California, identified important weeds and geographical locations 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 by the California Department of Transportation, which is managing weeds along major highways throughout the Salinas Valley.
2. Cucurbit chlorotic yellows virus was identified for the first time in Alabama and Georgia. The whitefly-transmitted virus, cucurbit chlorotic yellows virus (CCYV), was first identified in the United States in California and Arizona in 2018 and results in reduced plant vigor and low fruit sugar, impacting marketability of melons. Research by ARS scientists in Salinas, California, in collaboration with the University of Georgia, Auburn University, and others, confirmed the presence of CCYV in both Alabama and Georgia, and identified the related virus, cucurbit yellow stunting disorder virus (CYSDV), for the first time in Alabama as well. These results are of concern to the cucurbit industry, as infection can impact yields of all cucurbit crops if plants become infected early in the season.
3. Emergence of whitefly-transmitted yellowing viruses of cucurbit crops in the Central Valley of California. Cucurbit chlorotic yellows virus (CCYV) and cucurbit yellow stunting disorder virus (CYSDV) have caused severe losses for summer and fall production of cucurbits in the U.S. Southwestern low desert production region, but had not been identified previously in the Central Valley of California, where over half of U.S. cantaloupe production occurs. ARS scientists in Salinas, California, identified both viruses from melon plants in Fresno County, California, during the fall of 2020 using a multiplex virus detection system developed by the ARS laboratory. These viruses have the potential to cause severe losses, and it is important for the cucurbit industry that continued monitoring occur to determine prevalence and to develop strategies to reduce impact.
4. Identification of new viruses affecting agricultural crops in South Korea. ARS scientists from Salinas, California, collaborating with scientists from the Korean Rural Development Administration and Chungbuk University (South Korea), identified two viruses in new host plants in South Korea. Tomato aspermy virus (TAV) was identified for the first time on the Korean herbal and medicinal plant, Chrysanthemum zawadskii, and cucurbit chlorotic yellows virus (CCYV) was identified for the first time in cucumber in Korea. The identification of TAV may impact trade of C. zawadskii, and is of concern for the medicinal plant industry in Korea, whereas CCYV has the potential to cause severe losses, particularly for greenhouse production of cucurbits.
Review Publications
Mondal, S., Wintermantel, W.M., Gray, S.M. 2021. Virus and helper component interactions favour the transmission of recombinant potato virus Y strains. Journal of General Virology. 102(6). https://doi.org/10.1099/jgv.0.001620.
Esquivel-Farina, A., Rezende, J.A.M., Wintermantel, W.M., Jenkins Hladky, L.L., Bampi, D. 2021. Natural infection rate of known Tomato chlorosis virus-susceptible hosts and the influence of the host plant on the virus relationship with Bemisia tabaci MEAM1. Plant Disease. https://doi.org/10.1094/PDIS-08-20-1642-RE.
Kwak, H.-R., Kim, J.-G., Kim, J.-E., Byun, H.-S., Choi, H.-S., Wintermantel, W.M., Kim, M. 2021. First report of Tomato aspermy virus in Chrysanthemum zawadskii var. latilobum in Korea. Journal of Plant Pathology. https://doi.org/10.1007/s42161-021-00797-2.
Kwak, H.-R., Byun, H.-S., Choi, H.-S., Han, J.-W., Kim, C.-S., Wintermantel, W.M., Kim, J.-E., Kim, M. 2021. First report of cucurbit chlorotic yellows virus infecting cucumber in South Korea. Plant Disease. https://doi.org/10.1094/PDIS-10-20-2254-PDN.
Mondal, S., Jenkins Hladky, L.L., Fashing, P.L., McCreight, J.D., Turini, T.A., Wintermantel, W.M. 2021. First report of cucurbit yellow stunting disorder virus and cucurbit chlorotic yellows virus in melon in the Central Valley of California. Plant Disease. https://doi.org/10.1094/PDIS-01-21-0184-PDN.
Mondal, S., Jenkins Hladky, L.L., Melanson, R.A., Singh, R., Sikora, E., Wintermantel, W.M. 2021. First report of cucurbit yellow stunting disorder virus and cucurbit chlorotic yellows virus in cucurbit crops in Alabama. Plant Disease. https://doi.org/10.1094/PDIS-05-21-0922-PDN.
Kavalappara, S.R., Milner, H., Konakalla, N.C., Morgan, K., Sparks, A.N., McGregor, C., Culbreath, A.K., Wintermantel, W.M., Bag, S. 2021. High throughput sequencing-aided survey reveals widespread mixed infections of whitefly-transmitted viruses in cucurbits in Georgia, USA. Viruses. 13(6). Article 988. https://doi.org/10.3390/v13060988.
Tamang, P., Ando, K., Wintermantel, W.M., McCreight, J.D. 2021. QTL mapping of Cucurbit yellow stunting disorder virus resistance in melon accession PI 313970. HortScience. 56(4):424-430. https://doi.org/10.21273/HORTSCI15495-20.
Kavalappara, S.R., Milner, H., Sparks, A., McGregor, C., Wintermantel, W.M., Bag, S. 2021. First report of cucurbit chlorotic yellows virus in association with other whitefly-transmitted viruses in yellow squash (Cucurbita pepo) in Georgia, U.S.A. Plant Disease. https://doi.org/10.1094/PDIS-11-20-2429-PDN.
