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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

2016 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:
The Salinas lab is recognized internationally as a leader in the study of insect-transmitted and soil-borne viruses affecting sugar beet and vegetable production. The lab has characterized most of viruses in the genus Crinivirus as well as other viruses transmitted by insects and through soil. The lab has developed detection methods, and is actively working toward understanding the interacting epidemiological factors that drive emergence and establishment of these viruses. The lab works with breeding programs to improve and enhance the availability and performance of resistant varieties, and uses a wide range of applied, molecular, genomic, and technological approaches to address emergence, epidemiology, and control of sugar beet and vegetable viruses. A complex collaborative project is focused on control of whiteflies, a damaging insect pest of agricultural and horticultural crops throughout the world, and a vector of numerous plant viruses. Recently completed studies involving collaboration between the ARS Virology Lab in Salinas, California, as well as the ARS Virology Lab in Charleston, South Carolina, sequenced the genome of the whitefly, Bemisia tabaci. The Salinas Lab sequenced the transcriptome (RNA used for gene expression) of whitefly in response to transmission of two important and related whitefly-transmitted viruses, Tomato chlorosis virus (2015) and Cucurbit yellow stunting disorder virus (2016) 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 is being used to understand how these viruses interact with the whitefly vector and for development of RNA interference (RNAi) to specifically eliminate 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 past year more than 30 RNAi constructs were tested for whitefly control by the ARS Virology Lab in Salinas, and 10 have been delivered to collaborators in Africa for evaluation for control of whitefly on cassava. Rhizomania disease of sugarbeet is caused by Beet necrotic yellow vein virus (BNYVV), and in the absence of viable resistance genes the virus can cause severe yield reductions. Studies conducted through this project have compared and are continuing to study efficiency of BNYVV transmission by different isolates of Polymyxa betae, the soil-borne organism that transmits the virus; and new studies are comparing transmissibility of different BNYVV isolates by specific isolates of the P. betae vector. This work is focused on determining differences in transmission of BNYVV by this soil-borne organism, and should eventually lead to development of control strategies. Previous studies by the ARS Virology Lab in Salinas identified changes in the proteome of sugarbeet in response to BNYVV infection of susceptible and resistant plants. Although this information was valuable, additional information is needed to understand how BNYVV causes rhizomania disease on sugarbeet in order to use new technologies to interfere with the infection process and develop resistance. To this end, new research, begun during 2016, is evaluating the metabolome of resistant and susceptible sugarbeet in response to BNYVV infection. Tomato necrotic dwarf virus (ToNDV), a virus that causes severe stunting and necrosis on tomato that had not been identified in the field in over 30 years. It was identified by the ARS virology lab in Salinas in weeds from Imperial County, California and in tomato from Kern County, California by University of California (UC), Davis collaborators. Previously only one isolate of ToNDV had been characterized (by the Salinas Lab). New studies demonstrated a close genetic relationship between the Kern isolate and isolates from Imperial County, collected in the 1980's, but much greater sequence divergence was found in the weed isolate from Imperial County. Surveys for the virus will continue in fall 2016 to determine if the virus is re-emerging and poses a significant risk to tomato production in California. New variants of Beet curly top virus (BCTV) have been emerging and replacing traditional strains of the virus in several locations and crops in California’s San Joaquin Valley since 2013, and similar but distinct variants have been identified in Idaho and other areas of the Mountain West. The ARS Virology Lab in Salinas has an ongoing collaboration with the sugarbeet and tomato industries to examine competitiveness of these new variants in sugar beet and tomato to determine their potential for long-term persistence in the field. Furthermore, studies are focused on improving field management of these viruses through improved knowledge of critical epidemiological factors that drive virus emergence. Lettuce dieback disease, caused by either of two tombusviruses, Moroccan pepper virus (MPV) and Tomato bushy stunt virus, results in severe stunting and necrosis of lettuce plants and can cause complete loss of crop in severely affected fields. However, disease development in fields varies from year-to-year. Previous studies by the ARS virology lab in Salinas sequenced the genomes of several MPV isolates and demonstrated a role for poor movement of solutes (leaching) through soil as a likely factor leading to symptom development of these viruses in lettuce. New research is examining the role of a previously unknown factor that appears to contribute to disease development. Results should provide greater clarity as to the factors contributing to development of lettuce dieback symptoms and should lead to improved methods to minimize losses through management and resistance. The ARS Virology Lab in Salinas works closely with the ARS Vegetable Breeding Program in Salinas. Recent collaborative studies by the virology lab have developed a highly efficient greenhouse evaluation method for tospovirus resistance in lettuce. The method was used to evaluate wide ranging cultivated and wild lettuce for resistance to Impatiens necrotic spot virus in 2016. In addition, ongoing cooperative studies with the melon breeding program in Salinas have led to the identification of several sources of resistance to Cucurbit yellow stunting disorder virus and reliable methods to monitor virus accumulation in host plants.

4. Accomplishments
1. RNA interference (RNAi) methods for control of whiteflies on tomato 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. Researchers in Salinas, California in collaboration with ARS in Charleston, South Carolina, and the Boyce Thompson Institute in Ithaca, New York sequenced the genome of the whitefly, Bemisia tabaci. Researchers in Salinas, California evaluated more than 30 RNAi constructs for control of whitefly by targeting genes identified through the genome sequencing project. Several of the constructs provided high levels of whitefly mortality in laboratory studies. Results are leading to further tests for application of these constructs to tomato, as well as cassava, an important food staple for sub-Saharan Africa, and eventual field application should improve whitefly control across many affected U.S. crops, as well as for food supplies in the developing world.

2. Sequenced the transcriptome of the whitefly, Bemisia tabaci in response to feeding on plants infected by Cucurbit yellow stunting disorder virus (CYSDV). 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. Researchers in Salinas, California, and the Boyce Thompson Institute in Ithaca, New York compared differences in gene expression between whiteflies fed on healthy melon plants and melon plants infected with the semi-persistent Cucurbit yellow stunting disorder virus (CYSDV). The data were compared to a previous study on tomato that examined differential whitefly gene expression in response to the related virus, Tomato chlorosis virus (ToCV). There were fewer differentially expressed proteins and delayed appearance of gene expression differences in response to CYSDV infection than was found with ToCV infection of source plants in the previous study. Results will lead to knowledge of critical biochemical pathways involved in virus transmission, and development of targeted approaches for control of semi-persistent plant viruses in melon, tomato, and other crops.

3. Identification and sequence analysis of new isolates of the torradovirus, Tomato necrotic dwarf virus (ToNDV) from Kern and Imperial Counties in California. ToNDV was first identified causing severe losses to tomato production in Imperial County, California in the 1980's, but has not been identified in the field since that time. A late season tomato field in Kern County during the fall of 2015 developed severe mosaic and stunting symptoms, and scientists in Salinas, California, in collaboration with scientists from the University of California, Davis identified ToNDV and Tomato mosaic virus in these plants. Subsequent surveys by researchers in Salinas, California, in collaboration with University of California, Division of Agriculture and Natural Resources, Cooperative Extension personnel also identified ToNDV in weeds from Imperial County, California. Salinas scientists sequenced the genomes of both isolates and found the Kern isolate to be closely related to the 1980s isolates, but the Imperial County isolate was significantly diverged, demonstrating greater variability in the sequence of ToNDV than was previously known. Results demonstrate a need for further monitoring of tomato and adjacent weeds in both the San Joaquin and Imperial Valleys for ToNDV, and will be valuable in understanding epidemiological factors that allowed the virus to persist undetected for three decades.

4. Curly top strain characterization and competitiveness. The predominant forms of Beet curly top virus causing losses for tomato and sugarbeet production have been changing over the past few years, in some cases leading to increased severity. ARS scientists in Salinas, California compared competitiveness of these emergent variants in mixed infections, and efficiency of their transmission from important regional crops and weeds. Laboratory studies demonstrated differences in competitive accumulation of the new variants among tested crops and weeds, and that for these new variants, the host plant influences accumulation and this in turn influences transmission efficiency. Results clarified factors driving emergence and dominance of new curtovirus species and are being used in ongoing research focused on development of new control strategies for curly top in sugarbeet and tomato.

5. Screening for tospovirus resistance. Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) have become serious economic threats to production of lettuce (Lactuca sativa) in California; however, no reliable resistance has been identified to date. A system was previously developed by researchers in Salinas to test lettuce for resistance to tospoviruses by ongoing propagation of a thrips population on lettuce infected with either INSV or TSWV in a greenhouse, since field-testing is inconsistent and unreliable. The method has been proven highly reliable over the past year with 90-100% infection of susceptible lines, and is being used to evaluate germplasm for sources of resistance to INSV through collaboration between this project and another ARS project in Salinas. Results will benefit the lettuce industry in the U.S. and throughout the world by identification of new sources of genetic resistance to INSV and TSWV that can be incorporated into commercial varieties.

5. Significant Activities that Support Special Target Populations:
An ARS researcher in Salinas, California provided internships and training to one Hartnell College student and one California State University, Monterey Bay student (minority student) in laboratory and greenhouse research including specific research projects and hands-on experience with basic laboratory skills. An ARS researcher in Salinas, California presented to Salinas Grower-Shipper Association’s Ag-Knowledge Class on the many and varied research projects conducted in Salinas, as well as how this research benefits local agriculture and agriculture throughout the world.

Review Publications
Sabanadazovic, S., Wintermantel, W.M., Valverde, R.A., McCreight, J.D., Aboughanem-Sabanadaz, N. 2016. Cucumis melo endornavirus: Genome organization, host range and codivergence with the host. Virus Research. 214:49-58.
Coates, B.S., Poelchau, M.F., Childers, C., Evans, J.D., Handler, A.M., Guerrero, F., Skoda, S.R., Hopper, K.R., Wintermantel, W.M., Ling, K., Hunter, W.B., Oppert, B.S., Perez De Leon, A.A., Hackett, K.J., Shoemaker, D.D. 2015. Arthropod genomics research in the United States Department of Agriculture-Agricultural Research Service: Current impacts and future prospects. Trends in Entomology. 11(1):1-27.
Chen, W., Hasegawa, D., Arumuganathan, K., Simmons, A.M., Wintermantel, W.M., Fei, Z., Ling, K. 2015. Estimation of the whitefly Bemisia tabaci genome size based on k-mer and flow cytometry analyses. Insects. 6:704-715.
Luan, J., Chen, W., Hasegawa, D.K., Simmons, A.M., Wintermantel, W.M., Ling, K., Fei, Z., Liu, S., Douglas, A.E. 2015. Metabolic coevolution in the bacterial symbiosis of whiteflies and related plant sap-feeding insects. Genome Biology and Evolution. 7(9):2635-2647. doi: 10.1093/gbe/evv170.
Wintermantel, W.M. 2016. Semi-persistent whitefly-transmitted viruses: Crinivirus. In: Brown, J.K., editor. Vector-Mediated Transmission of Plant Pathogens. St. Paul, MN: APS Press. p. 111-119.
Kaur, N., Hasegawa, D.K., Ling, K., Wintermantel, W.M. 2016. Application of genomics for understanding plant virus-insect vector interactions and insect vector control. Phytopathology. doi: 10.1094/PHYTO-02-16-0111-FI.