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

Location: Crop Improvement and Protection Research

2018 Annual Report


Objectives
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 inhouse 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.


Approach
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.


Progress Report
This is the final report for bridging project 2038-22000-017-00D. Research will continue under 2038-22000-018-00D, "Epidemiology, Vector-Host Plant Interactions, and Biology of Vegetable and Cucurbit Viruses." 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” (Agreement #59-2038-006-F) 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, California 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 and identified over 1,000 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 to develop strategies to prevent transmission of not only individual viruses, but entire groups of related viruses. Development of RNA interference (RNAi) targeting the whitefly vector of these viruses (Bemisia tabaci) will lead to specific elimination of the whitefly vector (and not other insects) in vegetable crops and cassava, an important food staple in the developing world. During the course of this project more than 35 constructs were tested for whitefly control. Six have been delivered to collaborators in Africa to evaluate control of whitefly on cassava, and four will be used for plant transformation. An invention disclosure has been filed on the six most effective constructs (to facilitate commercial licensing). Related to Sub-objective 2B, cucurbit yellow stunting disorder virus (CYSDV) impacts production of melon and other cucurbit crops in the southwestern and southeastern U.S., and Texas. Scientists in Salinas, California, first identified this virus as it emerged in 2006, thoroughly characterized the epidemiology of the virus in the southwestern U.S., and identified new and independent sources of resistance in melon to both CYSDV and its whitefly vector, B. tabaci. Steady advancement of resistant material improves virus and vector control globally, through selection for reduced CYSDV titer in a host, and improved epidemiologically based management. This has implications for management of, not only CYSDV, but also other insect transmitted viruses throughout the world. For example, new studies focused on epidemiology-based management of tomato chlorosis virus in collaboration with the University of Sao Paulo Brazil are continuing and parallel research on CYSDV. 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. Scientists in Salinas, California, have an ongoing collaboration with the sugar beet and tomato industries to examine competitiveness of these new variants to determine their potential for long-term persistence in the field. Studies related to Sub-objective 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. Scientists in Salinas, California, partnered with ARS scientists 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. 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. In support of Objective 3B, scientists developed a highly efficient greenhouse method for evaluation of tospovirus resistance in lettuce. This method was used to evaluate wide ranging cultivated and wild lettuce for resistance to INSV. Although studies demonstrated no complete resistance in cultivated lettuce (Lactuca sativa), new breeding is focused on higher levels of 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. In previous studies, scientists in Salinas, California, 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 is related to Sub-objective 3A, adding 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, demonstrating a wider distribution of these viruses. New 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 provide greater clarity on factors that contribute to development of lettuce dieback symptoms and enhanced management strategies. Rhizomania severely impacts sugarbeet production throughout the world. It is caused by beet necrotic yellow vein virus (BNYVV) and is transmitted by the soil-borne organism, Polymyxa betae. 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, scientists and collaborators at ARS Ft. Collins, Colorado and Colorado State University, identified metabolic changes associated with BNYVV infection of resistant and susceptible sugarbeet in support of Objective 1. Results enhance our knowledge of how BNYVV infects sugarbeet and overcomes resistance. Furthermore, scientists collected P. betae isolates from throughout U.S. sugarbeet production areas. In support of Sub-objective 2A, pure culture isolates from each location were used to compare virus transmission, confirming that demonstrated differences exist in transmission efficiency. This suggests variation in P. betae isolates in sugarbeet fields influence disease development. Studies contribute to development of new methods for pathogen control and disease management by either prolonging the effectiveness of resistance and/or developing novel resistance strategies for disease control.


Accomplishments
1. Identification of common differentially expressed genes in whitefly as a result of feeding on host plants infected with distinct whitefly-transmitted viruses. It has been established that insect vectors of plant viruses are attracted to plants infected by the viruses they transmit due to yellow-coloration of leaves or to volatile compounds exuded by the plant. However, limited information exists with regard to the unique physiological changes that occur in the whitefly after feeding on such plants, nor is it known how this relates to virus transmission. Scientists in Salinas, California, and Charleston, South Carolina, previously characterized differences in gene expression between whiteflies fed on healthy tomato and tomato infected with either the semipersistently transmitted, mouthpart-borne crinivirus, tomato chlorosis virus, or the persistently transmitted, circulative begomovirus, tomato yellow leaf curl virus. They then compared genes differentially expressed between whiteflies fed on healthy melon plants and melon plants infected with the crinivirus, cucurbit yellow stunting disorder virus, and identified 59 genes differentially expressed in common between both criniviruses, as well as several in common with the begomovirus. This information will lead to development of targeted methods to interfere with transmission of a broad spectrum of whitefly-transmitted viruses affecting vegetables and other crops.

2. 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 via reduced yields and decreased plant longevity; yet, little host plant resistance exists to whitefly and only limited natural resistance is available to many virus diseases. Previously, researchers in Salinas, California, in collaboration with ARS in Charleston, South Carolina, and Fort Pierce, Florida, evaluated over 70 RNAi constructs for inducing whitefly mortality. Two constructs were selected and cloned for development of genetically modified cassava and tomato, while two additional high-performing constructs have been developed for further transformations, and methods to enhance induced resistance delivered as a foliar application were evaluated. The results have led to further tests for application of induced resistance in tomato and melon, and stable transformation of cassava, an important food staple for sub-Saharan Africa. Eventual field application should improve whitefly control across many affected U.S. crops, as well as for food supplies in the developing world.

3. Development of an infectious clone of a Tomato necrotic dwarf virus (ToNDV) isolate from an agricultural weed and evaluation of potential threat to tomato. ToNDV was first identified to cause severe losses to tomato seed production in Imperial County, California, in the 1980s. When tomato seed production ceased in the region, the virus was not identified in California until 2015, when it was found in Imperial County in the weed, Datura discolor, and in Kern County in tomato. To characterize and further understand this virus, scientists in Salinas, California, cloned the datura isolate into an agrobacterium-DNA vector using the most divergent isolate of those characterized to date, which shares only 79 and 88% sequence identity with comparable RNAs from the 1980s California tomato isolates. Like the original isolate, the cloned virus can be transmitted from infected plants by the whitefly, B. tabaci, and was infectious on both tomato and Datura. However, transmission by inoculation of cloned DNA was more efficient than whitefly transmission. The clone is being used to determine the range of host plants infected by this isolate, virus titer in these hosts, and virus transmission; all of which contributes knowledge toward disease management.

4. Identification of reservoir hosts important to epidemiology of Tomato chlorosis virus (ToCV). Tomato chlorosis virus is an important whitefly-transmitted virus with an expanding geographical range that causes losses to tomato production throughout the world. Scientists in Salinas, California, in collaboration with the University of Sao Paulo, Piracicaba, Brazil, systematically evaluated ToCV accumulation in common weed and crop hosts of the virus, as well as virus accumulation and transmission efficiency over time after feeding on the same host plants. There was highly efficient accumulation from two types of nightshade (Solanum nigrum and S. americanum), two important hosts in Brazil and common plants in U.S. tomato production regions, as well as efficient virus accumulation and transmission from several other host plants. These results enhance knowledge of ToCV epidemiology, and will improve management practices to reduce virus influx into fields not only in the U.S., but also in many areas of the world where ToCV impacts vegetable production.

5. Differential expression of metabolites associated with resistance and susceptibility of sugar beet to beet necrotic yellow vein virus. Rhizomania, caused by beet necrotic yellow vein virus (BNYVV), is one of the most important diseases of sugar beet. The virus is distributed in most growing areas of the world, and is controlled exclusively by four, possibly five, resistance genes, two of which are now deployed commercially. However, BNYVV “resistance-breaking” strains with the ability to overcome the widely used Rz1 gene have been emerging in many production areas, and it is anticipated that other resistance genes may be prone to breakdown as well. To understand how susceptible and resistant sugar beet respond differentially to BNYVV, scientists at Salinas, California, and Fort Collins, Colorado, used gas chromatography–mass spectrometry (GC-MS) and ultra performance liquid chromatography–mass spectrometry (UPLC-MS) to compare differences among traditional and resistance-breaking BNYVV in susceptible and resistant sugar beet plants. Results showed significant differences among treatments, with primary metabolomic differences associated with a strain of BNYVV as compared to healthy (non-infected) sugar beet. These studies build on the knowledge generated through previous proteomic research, and are expected to lead to identification of specific targets for genetic modification to prevent BNYVV infection of sugar beet.


Review Publications
Simko, I., Richardson, C.E., Wintermantel, W.M. 2018. Variation within lactuca spp. for resistance to impatiens necrotic spot virus. Plant Disease. 102(2):341-348. https://doi.org/10.1094/PDIS-06-17-0790-RE.
Wintermantel, W.M. 2017. Lettuce dieback. In: Subbarao, K.V., Davis, R.M., Gibertson, R.L., Raid, R.N., editors. Compendium of Lettuce Diseases and Pests. 2nd edition. St. Paul, MN: APS Press. p. 80-81.
Wintermantel, W.M. 2017. Beet western yellows. In: Subbarao, K.V., Davis, R.M., Gibertson, R.L., Raid, R.N., editors. Compendium of Lettuce Diseases and Pests. 2nd edition. St. Paul, MN: APS Press. p. 68-70.
Wintermantel, W.M. 2017. Beet yellow stunt. In: Subbarao, K.V., Davis, R.M., Gibertson, R.L., Raid, R.N., editors. Compendium of Lettuce Diseases and Pests. 2nd edition. St. Paul, MN: APS Press. p. 70-71.
Wintermantel, W.M. 2017. Lettuce chlorosis. In: Subbarao, K.V., Davis, R.M., Gibertson, R.L., Raid, R.N., editors. Compendium of Lettuce Diseases and Pests. 2nd edition. St. Paul, MN: APS Press. p. 78-79.
Wintermantel, W.M. 2017. Lettuce big vein. In: Subbarao, K.V., Davis, R.M., Gibertson, R.L., Raid, R.N., editors. Compendium of Lettuce Diseases and Pests. 2nd edition. St. Paul, MN: APS Press. p. 76-78.
Gundersen, D.E., Adrianos, S.L., Allen, M.L., Becnel, J.J., Chen, Y., Choi, M.Y., Estep, A., Evans, J.D., Garczynski, S.F., Geib, S.M., Ghosh, S.B., Handler, A.M., Hasegawa, D.K., Heerman, M.C., Hull, J.J., Hunter, W.B., Kaur, N., Li, J., Li, W., Ling, K., Nayduch, D., Oppert, B.S., Perera, O.P., Perkin, L.C., Sanscrainte, N.D., Sim, S.B., Sparks, M., Temeyer, K.B., Vander Meer, R.K., Wintermantel, W.M., James, R.R., Hackett, K.J., Coates, B.S. 2017. Arthropod genomics research in the United States Department of Agriculture-Agricultural Research Service: Applications of RNA interference and CRISPR gene editing technologies in pest control. Trends in Entomology. 13:109-137.
Hasegawa, D.K., Chen, W., Zheng, Y., Kaur, N., Wintermantel, W.M., Simmons, A.M., Fei, Z., Ling, K. 2018. Comparative transcriptome analysis reveals networks of genes activated in the whitefly, Bemisia tabaci when fed on tomato plants infected with Tomato yellow leaf curl virus. Virology. 513:52-64. https://doi.org/10.1016/j.virol.2017.10.008.