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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

2014 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. 2.D. Elucidate biological and environmental factors influencing mealybug transmission of grapevine leafroll-associated viruses in order to develop and test options for vector monitoring and management. Research will involve in-house funds with completion within 5 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.

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 in their ability to transmit BNYVV to sugarbeet and this is associated with increased presence of resistance-breaking forms of BNYVV in sugarbeet production. Single spore isolates of P. betae will be tested for ability to transmit BNYVV to examine transmission efficiency and effect of vector isolate on virus competitiveness with virus titer determined by ELISA or qRT-PCR. 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 using either qRT-PCR or ELISA. 2.C. Individual curtoviruses accumulate to higher or lower titers during single and mixed infections, and this varies by host plant, and this influences virus dominance in the field. Curtoviruses will be transmitted by beet leafhoppers from single and mixed infections and qPCR 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. 2.D. The proposed studies will elucidate grapevine leafroll transmission by two mealybug speciesand explore options for monitoring and management of mealybugs. Greenhouse studies involving single-virus isolates and each mealybug species will be conducted to determine efficiency for transmission of grapevine leafroll associated viruses isolated from California vineyards using viruliferous mealybugs. RT-PCR or ELISA will be used to detect virus. 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.

3. Progress Report:
The Salinas, California, lab is recognized internationally as a leader in the study of insect-transmitted and soil-borne viruses affecting sugarbeet and vegetable production. The lab has characterized most viruses in the genus Crinivirus, developed detection methods, and is actively working toward understanding the interacting epidemiological factors that drive emergence and establishment of these viruses in agricultural regions. The lab works with breeding programs to improve and enhance the availability and performance of virus-resistant varieties, and uses a wide range of applied, molecular and technological approaches to address emergence and epidemiology of viruses affecting sugar beet and vegetables, as well as genetic and biological characterization of relevant viruses. New studies are evaluating whitefly gene expression differences in relation to virus infection in an effort to identify new strategies for whitefly control. Progress on objectives 1 and 2: We identified protein changes in sugar beet associated with Beet necrotic yellow vein virus (BNYVV) infection, indicating specific biological pathways altered by virus infection that differ during infection by the virus. A follow-up study is nearing completion that is comparing differences in protein expression in sugar beet resulting from virus interactions with susceptible as well as virus-resistant sugar beet, and complementing two previous studies by the ARS virology lab in Salinas, California, and ARS collaborators in Fort Collins, Colorado. Comparisons of findings in sugarbeet with other pathogen systems may lead to improved knowledge of how pathogens infect plants. Additional studies are examining variation in efficiency of virus transmission by diverse collections of Polymyxa betae isolates. Progress on objective 3: Genetic relationships among tombusviruses that cause the disease lettuce dieback were clarified. One of these, Moroccan pepper virus (MPV), was found in the eastern U.S. for the first time on a new crop, escarole, demonstrating the virus is not restricted to fields in California and Arizona. Furthermore, genetic comparisons found the New Jersey isolate to be distinct from previously characterized isolates. We continue to examine environmental influences affecting lettuce dieback disease development, as studies on light and temperature were found to contribute to symptom development but modification of these factors alone was not sufficient to confer reliable infection. Additional studies in collaboration with the lettuce breeding program at ARS in Salinas are focused on identifying sources of resistance to not only MPV, but also Tomato spotted wilt virus and Impatiens necrotic spot virus. New variants of Beet curly top virus (BCTV) were identified in several locations and crops in California’s San Joaquin Valley in 2013, with lower incidence of traditional strains/species. The ARS Virology Lab in Salinas recently completed evaluation of competitiveness among traditional strains of BCTV and is now examining competitiveness of these new variants in crops to determine their potential for persistence in the field and potential to impact efforts toward development of new sources of resistance to the traditional forms of BCTV.

4. Accomplishments
1. Moroccan pepper virus infected escarole found in New Jersey. Moroccan pepper virus (MPV) was identified for the first time in the eastern U.S. (New Jersey) affecting escarole, a crop not previously known to be infected by MPV. ARS researchers in Salinas, California, determined that MPV was associated with a disease known as yellows, for which no pathogen had been identified previously, and diseased plants were found in sandy loam soils following periods of high soil moisture. Cores of escarole heads were necrotic and rotted, while outer leaves were yellow with more pronounced yellowing of veins and occasional veinal necrosis, and resulted in unmarketable heads. The coat protein gene sequence of the New Jersey isolate varied from other U.S. isolates by 3% and from a Japanese isolate (most distant of sequenced MPV isolates) by 6%. Further studies will be necessary to determine if MPV is the sole cause of escarole yellows or if it is part of a disease complex. The identification of a genetically distinct isolate of MPV from a new host in the eastern United States demonstrates MPV is more widely distributed than previously believed.

2. A new tombus-like virus associated with lettuce dieback disease in California. Lettuce dieback disease, caused by the soil-borne viruses, Tomato bushy stunt virus (TBSV) and Moroccan pepper virus (MPV; formerly known as Lettuce necrotic stunt virus), results in necrosis, stunting and death of lettuce plants in the western U.S., often with complete loss of a crop. ARS researchers in Salinas, California, isolated a previously unknown virus in spring 2014, from romaine lettuce exhibiting symptoms of lettuce dieback near King City, California. The virus could not be detected using standard methods to confirm the presence of TBSV and LNSV, which suggested the possibility of a new virus. The isolate was passed to test plants for host range analysis, with host range and symptom development on test plants typical of those for a virus in the Tombusviridae, but clearly distinct from those of TBSV and MPV. Results contribute to understanding the group of viruses associated with lettuce dieback disease and environmental factors that influence disease development.

3. Developed strategies for evaluation of resistance to tospoviruses in lettuce. Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) are emerging as serious economic threats to production of lettuce in California and throughout the world; however, only limited sources of resistance to the virus have been identified, and field studies for evaluation of resistance is challenging due to inconsistent rates of infection. ARS researchers in Salinas, California, evaluated methods for consistent greenhouse infection of lettuce using both mechanical inoculation (rubbing plants with sap of infected plants) and transmission by the thrips vector. Multiple experiments were conducted to optimize mechanical transmission of these viruses; however, results were variable and supported previous studies that without exposure to thrips these viruses lose transmissibility to most plant species, including lettuce. Additional studies evaluated transmission by thrips, and preliminary results indicate thrips transmission will be necessary for reliable evaluation of resistance. Development of methods for reliably high rates of greenhouse infection will benefit the lettuce industry in the U.S. and throughout the world through development of evaluation methods and identification of new sources of genetic resistance to these viruses.

4. Proteome differences between resistant and susceptible sugarbeet. Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV), severely impacts sugarbeet (Beta vulgaris) production throughout the world. In an effort to understand disease susceptibility and the ability for the virus to overcome resistance genes, we sought new methods to identify proteins associated with infection of sugarbeet by BNYVV. Studies conducted by ARS researchers in Salinas, California, and Fort Collins, Colorado, in collaboration with Colorado State University, compared protein expression during infection of susceptible sugarbeet by BNYVV with healthy sugarbeet using mass spectrometry, while additional studies evaluated differences in protein expression between BNYVV-resistant and susceptible sugarbeet lines during virus infection. Statistically significant differences were identified between healthy and BNYVV-infected sugarbeet, and additional significant differences were identified between resistant and susceptible sugarbeet. These studies should lead to the identification of biological targets that can be exploited to develop novel and environmentally-friendly disease control strategies for management of this very serious threat to sugarbeet production.

5. Determined competitiveness of emergent curtovirus relative to “traditional” forms of the virus. Three recently identified variants of Beet curly top virus (BCTV) emerged in 2013 at substantially high levels compared to traditional forms in some areas of California, and caused losses to tomato production and other vegetables. ARS scientists in the virology lab at Salinas, California, in collaboration with the University of California, Davis, compared competitiveness of these emergent BCTV isolates with “traditional” or typical forms of the virus to determine risk of long-term establishment and impact of the new variants on important crops. Preliminary results indicated a higher rate of infection by one emergent isolate in cucurbits, and an increased prevalence by another emergent isolate in sugarbeet compared to traditional forms of the virus. Laboratory studies demonstrated that the new species can co-infect plants along with traditional forms of the virus. Ongoing studies are evaluating competitiveness of traditional and emergent isolates in mixed infections. These findings add to the knowledge of factors driving emergence and dominance among curtoviruses, contribute to overall knowledge of curtovirus epidemiology, and raise awareness that changes in abundance of different isolates can alter disease severity on crops.

6. Developed a system for evaluating variation among isolates of Polymyxa betae for acquisition and transmission of Beet necrotic yellow vein virus (BNYVV). Rhizomania disease of sugarbeet, caused by BNYVV, is transmitted by the soil-borne organism, P. betae. Methods were developed and tested by the ARS Virology Lab in Salinas, California, to evaluate the amount of BNYVV in sugarbeet roots, for use in collaborative studies with the ARS soil-borne fungus research lab in Salinas on variation in the ability of different P. betae isolates to transmit BNYVV. These methods are being used to determine variation in efficiency of BNYVV transmission by P. betae isolates, as well as understand virus-vector interactions that may influence performance of resistant lines. This will lead to more effective methods for evaluation of resistance to both virus and vector, as well as broader understanding of variability among P. betae isolates.

7. Developed a method for evaluation of resistance in melon to Cucurbit yellow stunting disorder virus (CYSDV) under controlled environment conditions. CYSDV impacts production of melon and other cucurbit crops in the southwestern U.S., Florida and Texas. ARS scientists from the virology and vegetable breeding programs in Salinas, California, characterized epidemiology of the virus in the Southwest and have identified new sources of resistance in melon. A method for greenhouse/growth chamber evaluation of resistance was developed using whitefly-transmission and insecticide-based control of whiteflies in cages to quantify and clarify resistance responses to CYSDV. This method will be used in combination with molecular tools to assess the impact of host plant resistance in melon on primary infection and subsequent spread of CYSDV by comparing virus titer in resistant and susceptible source and target melons. Results will lead to development of improved virus resistance and enhance management practices through more effective pest management incorporating enhanced resistance to CYSDV.

8. Methods for comparative transcriptome analysis of whitefly species in response to virus infection. Whiteflies and whitefly-transmitted viruses result in significantly decreased agricultural productivity throughout the world through reduced yields and plant longevity. There are limited sources of natural resistance to the whitefly or to many virus diseases. Standardized methods were developed by ARS scientists in Salinas, California, for evaluation of whitefly transcriptome differences. Methods for whitefly propagation, host plants, controlled environmental conditions, and whitefly nucleic acid extraction were optimized for standardization of collaborative research at the ARS in Salinas, California, with ARS researchers in Charleston, South Carolina, with transcriptome analysis to be performed at the Boyce Thompson Institute in Ithaca, New York. These methods are being used in current studies to determine differences in whitefly gene expression during infection by persistent and semi-persistent viruses. These and additional studies to compare whitefly transcriptome differences will lead to identification of specific genes and genetic pathways activated in common during feeding of whiteflies on plants infected with different types of plant viruses, and development of targeted approaches for control of both whitefly vector and plant viruses in general through plant biotechnology strategies.

Review Publications
Poudel, B., Wintermantel, W.M., Cortez, A.A., Ho, T., Khadgi, A., Tzanetakis, I.E. 2013. Epidemiology of Blackberry yellow vein associated virus. Plant Disease. 97:1352-1357.
Wintermantel, W.M., Bachinsky, D. 2014. First report of Moroccan pepper virus in association with yellows on escarole in the US and world. Plant Disease. DOI: 10.1094/PDIS-04-14-0394-PDN.
Webb, K.M., Broccardo, C.J., Prenni, J.E., Wintermantel, W.M. 2014. Proteomic profiling of Beta vulgaris leaves during rhizomania compatible interactions. Proteomes. 2(2):208-223.