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ARS Home » Pacific West Area » Salinas, California » Crop Improvement and Protection Research » Research » Research Project #423228

Research Project: Biology, Epidemiology and Management of Vector-Borne Viruses of Sugarbeet and Vegetable Crops

Location: Crop Improvement and Protection Research

2017 Annual Report

1a. Objectives (from AD-416):
Objective 1: Identify specific genes associated with Beet necrotic yellow vein virus infection of sugarbeet that contribute to development of rhizomania disease and the ability of the virus to overcome resistance for use as potential targets for induced resistance. This will involve comparisons with other soil-borne pathogens using in-house funds. Completion within 5 years. Objective 2: Determine environmental and epidemiological factors contributing to the ability of sugarbeet and vegetable viruses to emerge and establish over competing viruses, to provide effective disease management recommendations and prolong the durability of resistance sources. Specifically: 2.A. Determine the effect of variation among Polymyxa betae isolates on prevalence and dominance of soil-borne viruses affecting sugarbeet, including evaluation of virus competitiveness through collaborative studies involving this project using both in-house funds and those of a local collaborator in NP308. Completion within 5 years. 2.B. Assess accumulation of CYSDV in different host plants in relation to transmission and in development of host resistance using both in-house funds and collaboration with ARS Salinas vegetable breeding program (NP301). Completion within 5 years. 2.C. Identification of factors influencing emergence and dominance of existing and new curtoviruses in North America through analysis of competitive virus accumulation in host plants. Research will involve in-house funds, with completion within 3 years. Objective 3: Determine environmental and cultural factors contributing to the ability of viruses to induce disease to facilitate breeding efforts for resistance to soil-borne and insect-transmitted viruses affecting lettuce. Completion of both subobjectives within 5 years using both in-house funds and collaboration with Salinas vegetable project (NP301). 3.A. Develop methods for greenhouse-based evaluation of lettuce for resistance to soilborne tombusviruses through identification of environmental factors influencing disease development, and application of this knowledge to germplasm evaluation using controlled environments. 3.B. Identify sources of tospovirus resistance through evaluation of lettuce and Lactuca germplasm using mechanical transmission and viruliferous thrips under greenhouse conditions, for further development by breeders. Objective 4: Determine biological and ecological relationships among vectors and their host plants, the pathogens they transmit, and the environment, and develop novel intervention and management strategies for control of vector-borne diseases of vegetables, through the use of traditional, molecular biology, and bioinformatics approaches.

1b. Approach (from AD-416):
Objective 1: Some defense genes will be common to general sugarbeet or plant defense against pathogens. Determine similarities and differences among pathogens for gene expression between infected and pathogen free host plants based on results of studies currently concluding. Results will be compared with others including BNYVV and other pathogens through parallel studies. Objective 2: 2.A. Isolates of the plasmodiophorid vector of BNYVV, Polymyxa betae, differ for BNYVV transmission to sugarbeet. This is associated with increased presence of resistance-breaking forms of BNYVV. Single spore isolates of P. betae will be tested for differences in efficiency of BNYVV transmission and effect of vector isolate on virus competitiveness with virus titer. 2.B. Resistance from the exotic melon (Cucumis melo) accession PI 313970 can enhance resistance to CYSDV in cultivated melon, and provide high levels of resistance when combined with resistance source TGR-1551. Exotic melon accessions will be evaluated in replicated field plantings and studies will examine transmission efficiency of CYSDV from resistant and susceptible melons in comparison with virus concentration. 2.C. Individual curtoviruses accumulate to higher or lower titers during single and mixed infections, and this varies by host plant. This influences virus dominance in the field. Curtoviruses will be transmitted by beet leafhoppers from single and mixed infections with qPCR used for virus titer determination. If needed Agro-based delivery of virus isolates to specific hosts, or leafhopper membrane feeding studies could be used for virus delivery. Objective 3: 3.A. Long-day or high temperature treatment will induce development of tombusvirus symptoms on susceptible lettuce and can be used for selecting resistant and susceptible varieties. Growth chamber experiments will be used to determine optimal environmental conditions (light, temp, soil moisture etc.) for tombusvirus infection of lettuce using mechanical transmission experiments. Chamber and soil moisture and nutrition conditions can be modified as needed. 3.B. Resistance to Impatiens necrotic spot virus (INSV) and Tomato spotted wilt virus (TSWV) exists in wild or cultivated Lactuca germplasm and can be identified through greenhouse evaluation. Transmission of INSV and TSWV to Lactuca germplasm sources will be conducted using thrips vectors in the greenhouse. Virus detection will be performed using standard ELISA. If necessary, virus can be mechanically transmitted directly to lettuce from select hosts. Objective 4: Use a combination of bioinformatics analysis of insects with related applied and molecular entomological approaches to examine how insect vectors respond biologically and biochemically to environmental parameters. This will include but is not limited to the responses of whiteflies and leafhoppers to specific host plants, the presence or absence of plant viruses in host plants, and pesticides applied to host plants. Knowledge gained through these studies will be used to develop novel methods for vector population control through both biotechnology-based and genetics approaches.

3. Progress Report:
This is the final report for project 2038-22000-013-00D, which expired on April 24, 2017 and has been replaced by bridging project 2038-22000-017-00D while a new project plan is developed to address changes in agricultural production within the Pacific West Area. Semi-persistent whitefly-transmitted viruses result in significantly decreased agricultural productivity worldwide through reduced yields and plant longevity, yet very little information exists on the specific virus–insect interactions that determine whether a virus can be transmitted by a whitefly or how the insect is influenced by the presence of the virus in a host plant. A complex collaborative project entitled, “Genomics-guided RNAi solutions for whitefly management in cassava and world food crops” (2038-22000-017-03A) is focused on control of whitefly, a damaging insect pest of agricultural and horticultural crops throughout the world, and a vector of numerous plant viruses. Through this project the genome sequence of the whitefly, Bemisia tabaci, was completed for the first time by scientists at ARS in Salinas, California and Charleston, South Carolina, in collaboration with scientists at the Boyce Thompson Institute, Ithaca, New York. B. tabaci is one of the most important vectors of plant viruses in the world, and completion of the genome sequence facilitates additional studies toward understanding the biology of this insect, how viruses are transmitted, and new research toward control of the whitefly. In addition to the genome, ARS scientists in Salinas sequenced the transcriptome (RNA used for gene expression) of the whitefly in response to transmission of two important and related whitefly-transmitted viruses, Tomato chlorosis virus and Cucurbit yellow stunting disorder virus (both are genus Crinivirus, family Closteroviridae) and identified over 1000 and 250 differentially expressed genes, respectively, in response to virus infection of the source plant on which the whitefly fed. This information, in support of Objective 4, is being used to understand how these viruses interact with the whitefly vector and for development of RNA interference (RNAi), a method for specific elimination of the whitefly vector (and not other insects) in vegetable crops and cassava, an important food staple in the developing world through collaborations in Africa. During the course of this project more than 35 constructs were tested for whitefly control, 10 have been delivered to collaborators in Africa to evaluate control of whitefly on cassava, and an invention disclosure was filed on the six most effective constructs to facilitate commercial licensing. Cucurbit yellow stunting disorder virus (CYSDV) impacts production of melon and other cucurbit crops in the southwestern and southeastern U.S., and Texas. In support of Subobjective 2B, we first identified this virus as it emerged in 2006, and through this project thoroughly characterized the epidemiology of the virus in the southwestern U.S., and collaborated with the ARS Vegetable Breeding Program in Salinas to identify new and independent sources of resistance in melon to both CYSDV and its whitefly vector, B. tabaci. In addition to steady advancement of resistant material benefitting virus and vector control globally, studies demonstrated how not only virus titer in a host, but also other physical and biological features of host plants influence the survival and transmission of CYSDV. This has implications for management of not only CYSDV, but also other insect transmitted viruses throughout the world. Combining epidemiologically based control with improved virus and vector resistance should ultimately lead to an effective management strategy. New variants of another insect transmitted virus, Beet curly top virus (BCTV) have been emerging and replacing traditional BCTV strains in several locations in California’s San Joaquin Valley since 2013, and similar but distinct variants have been identified in the Mountain West. We have an ongoing collaboration with the sugarbeet and tomato industries to examine competitiveness of these new variants to determine their potential for long-term persistence in the field. Studies under Subobjective 2C have shown preferential adaptation of some strains to crop and weed hosts. Further studies involved direct interaction with companies to improve their ability to evaluate virus resistance, and partnering with ARS in Kimberly, Idaho, and the University of California, Davis to develop reliable methods to differentiate virus strains, and improve knowledge of critical epidemiological factors that drive virus emergence, respectively. Two tospoviruses cause significant losses for lettuce production in California and Arizona where nearly 80% of U.S. lettuce is grown. In particular, Impatiens necrotic spot virus (INSV) has emerged over the past decade to cause significant economic losses for most coastal lettuce production areas in California. Through Subobjective 3B, a highly efficient greenhouse method for evaluation of tospovirus resistance in lettuce was developed. This method was used in collaboration with ARS Vegetable Breeding Program in Salinas to evaluate wide ranging cultivated and wild lettuce for resistance to Impatiens necrotic spot virus. Although studies demonstrated no complete resistance in cultivated lettuce (Lactuca sativa) and related Lactuca species that can be crossed with lettuce, results demonstrated differences in severity and virus accumulation, which has led to selection and advancement of lettuce with improved resistance to INSV. Lettuce dieback disease, caused by either of two tombusviruses (viruses of the genus, Tombusvirus), Moroccan pepper virus (MPV) and Tomato bushy stunt virus (TBSV), results in severe stunting and necrosis of lettuce plants and can cause complete loss of crop in severely affected fields. The disease occurs in all western U.S. lettuce production areas, but disease development in infested fields varies from year-to-year. Previous studies sequenced the genomes of several MPV isolates and demonstrated a role for poor movement of solutes (leaching) through soil as a factor contributing to disease symptom development in lettuce. New research through this project in support of Subobjective 3A added to previous information on how growing conditions influence development of tombusvirus symptoms on lettuce. Light and temperature were found to contribute to symptom development with lettuce inoculated with MPV and TBSV but were not sufficient alone to confer reliable infection or development of typical disease symptoms. Additional studies identified MPV and TBSV in the eastern U.S. for the first time on escarole and endive, and genetic comparisons found the New Jersey MPV isolate to be distinct from previously characterized isolates. This shows that these viruses are not restricted to fields in California and Arizona, and can also infect other leafy green vegetables. Continuing research involving virus transmission and bioinformatics analysis is examining the likelihood that a previously unknown virus may also contribute to disease development. Forthcoming results should provide greater clarity on factors that contribute to development of lettuce dieback symptoms, the viruses that cause dieback disease in lettuce and escarole, and improved methods to minimize losses through management and resistance. Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV) and transmitted by the soil-borne organism, Polymyxa betae, severely impacts sugarbeet production throughout the world. Disease management is based on a small number of individual resistance genes, and BNYVV is now evolving to overcome the most widely used gene. In an effort to understand the physiology of rhizomania disease susceptibility and the ability for the virus to overcome resistance genes, ARS scientists and collaborators at ARS Ft. Collins, Colorado and Colorado State University, conducted studies on proteomic and metabolic changes associated with BNYVV infection of resistant and susceptible sugarbeet. Proteome analysis identified over 200 proteins with significant changes in expression among the different treatments. Comparable studies in progress are examining metabolic changes. Together, results enhance our knowledge of how BNYVV infects sugarbeet and overcomes resistance. This will contribute to development of new methods for control of this disease by either prolonging the effectiveness of resistance and/or development of novel resistance strategies for disease control. This work relates to Objective 1. Furthermore, in support of Subobjective 2A, ARS scientists collected P. betae isolates from throughout U.S. sugarbeet production areas. Pure culture isolates from each location were used to compare virus transmission, and demonstrated differences exist in transmission efficiency. This suggests variation in P. betae isolates in sugarbeet fields may influence disease development, but continuing studies are necessary.

4. Accomplishments
1. RNA interference (RNAi) methods for control of whiteflies on tomato, melon, and cassava. Whiteflies and whitefly-transmitted viruses result in significantly decreased agricultural productivity throughout the world through reduced yields and plant longevity, yet little resistance exists to whitefly and only limited natural resistance is available to many virus diseases. ARS researchers at Salinas, California, Charleston, South Carolina and Ft. Pierce, Florida, and scientists at the Boyce Thompson Institute, Ithaca, New York sequenced the genome of the whitefly, Bemisia tabaci, and used this information to make 70 RNAi constructs for use in development of genetically-modified whitefly-resistant cassava, an important food staple for sub-Saharan Africa. Ten of these constructs, including two produced at Salinas, were used to demonstrate efficacy for reducing whitefly populations when delivered as a spray to tomato plants. Further tests of these constructs are being done in tomato, melon, and cassava. Eventual field application should improve whitefly control across many affected U.S. crops, as well as for food supplies in the developing world.

2. Biological characterization of a Tomato necrotic dwarf virus (ToNDV) isolate from an agricultural weed and evaluation of potential threat to tomato. Tomato necrotic dwarf virus (ToNDV) was identified in California in the 1980s, when it caused severe losses to tomato seed production in Imperial County. After tomato seed production ceased in the region, the virus was not found again in California until 2015 when it was discovered in Imperial County in the weed, Datura (D.) discolor, and in Kern County in tomato. ARS researchers in Salinas, California sequenced the genomes of the new isolates and found the two genomic RNAs of the D. discolor isolate shared relatively low similarity to comparable RNAs from the 1980s California tomato isolates, with only 79 and 88% identity for RNA1 and RNA2, respectively. Whitefly transmission studies with Bemisia tabaci MEAM1, the most common whitefly vector in the region, demonstrated poor transmission efficiency to tomato and reduced symptom severity compared to traditional isolates. Seed transmission results from D. discolor suggest the virus is most likely not seed transmitted in this common weed and may survive in deep taproots from year-to-year. Results demonstrate a need for further monitoring of not only tomato, which can be severely impacted by ToNDV, but also weeds that may serve as long-term reservoirs for ToNDV variants that could lead to new infections of tomato, which is important for sustainability of tomato production in California.

3. New viruses found in association with lettuce dieback disease. 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 crop loss. Two related and highly stable soil-borne viruses, Tomato bushy stunt virus (TBSV) and Moroccan pepper virus (MPV) 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 as well. An ARS researcher in Salinas, California with assistance from University of California Cooperative Extension, collected isolates throughout the Salinas Valley region in 2016 and 2017, and confirmed the absence of MPV and TBSV in the collected plants. However, infection by a virus was demonstrated based on infection of test plants with sap from diseased lettuce and the ability to pass this agent to additional plants. RNA sequencing and small RNA analysis from the lettuce plants confirmed the presence of at least three viruses previously not associated with lettuce dieback. Characterization of the relationships between these newly discovered viruses and disease development will be necessary to determine if they are able to cause lettuce dieback disease symptoms.

4. Curly top strain characterization and competitiveness. The predominant forms of Beet curly top virus causing losses to tomato and sugarbeet production in the western United States have been changing over the past five years, in some cases leading to increased disease severity. An ARS researcher at Salinas, California examined accumulation of these emergent variants in important crop and weed hosts, and further evaluated transmission from important regional crops and weeds as a component of a multi-year epidemiological study focused on improved management of curly top in the West. Laboratory studies demonstrated differences between new and traditional virus strains in accumulation among tested crops and weeds, and that the host plant influences accumulation and virus transmission efficiency. Results clarified host-plant related factors driving emergence and dominance of new forms of Beet curly top virus and related curtoviruses, and are being used to develop new control strategies for curly top in sugarbeet and tomato.