2013 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.
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):
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.
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.
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.
The Salinas lab is recognized internationally as a leader in the study of whitefly-transmitted viruses as well as other insect-transmitted and soil-borne viruses affecting sugarbeet and vegetable production. The lab has characterized most of viruses in the genus Criniviruses known to infect sugarbeet and vegetable crops, 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 resistant varieties, and uses a wide range of applied, molecular and technological approaches to address emergence, study, and epidemiology of viruses affecting sugarbeet and vegetable agriculture.
Beet necrotic yellow vein virus (BNYVV) which causes rhizomania, and its vector, the soil-borne fungus, Polymyxa betae, were detected for the first time in the Western Hemisphere by the Salinas Virology Lab. Research on BNYVV and related viruses has been critical to understanding of the disease and facilitating development by the lab of highly specific and sensitive diagnostic assays. The laboratory is currently characterizing a recently identified soil-borne virus of sugarbeet, and working toward understanding the dynamics of interactions between these soil-borne viruses, the soil-borne fungus that transmits them, Polymyxa betae, and sugarbeet. Complementary studies in conjunction with the sugarbeet breeding program at Salinas are seeking sources of resistance to P. betae as part of an ongoing effort to reduce losses. Studies conducted through this project have identified protein changes in sugarbeet associated with BNYVV infection, indicating specific biological pathways altered by virus infection that differ during resistance and susceptibility. Comparison of findings in sugarbeet with other pathogen systems may lead to improved knowledge of how pathogens infect plants.
Studies over the past two years with virus isolates from Salinas and international locations have reclassified a soil-borne virus affecting lettuce, Lettuce necrotic stunt virus, as Moroccan pepper virus (MPV), a previously poorly characterized virus from Africa and Europe. Research identified temperature and day length as well as other factors influencing the development of MPV symptoms on both lettuce and other host plants, and will contribute toward efforts at breeding for resistance to MPV and related viruses in lettuce. Additional studies in collaboration with the lettuce breeding program in Salinas are focused on identifying sources of resistance to three viruses affecting lettuce.
Curly top, transmitted by the beet leafhopper (Circulifer tenellus) and caused by Beet curly top virus (BCTV) and related curtovirus members of the genus, Curtovirus, has impacted yields of sugar beet and vegetables since the late 1800s. The Salinas lab has identified crop and weed reservoirs, is examining factors that drive changes in curtovirus population structure in the western US, and has applied for a patent on novel methods for control across several crops.
Sequencing and taxonomic classification of Tomato necrotic dwarf virus (ToNDV). ToNDV is a whitefly-transmitted virus that caused significant losses for tomato production in southern California during the 1980s, but was never fully characterized. ARS researchers in Salinas, California, sequenced an isolate of ToNDV, which was originally collected from tomato in Imperial County, California, to determine its relationship to other viruses. The ToNDV genome is composed of two RNA molecules, and its closest relative is the torradovirus, Tomato marchitez virus. Results of sequence analysis and comparison of genomic and biological features confirm ToNDV should be recognized as a distinct member of the genus Torradovirus. Results will facilitate improved detection and monitoring, as well as clarification of relationships among members of this emerging genus of whitefly-transmitted viruses.
Expanded knowledge on environmental factors influencing tombusvirus infection of lettuce. Lettuce dieback disease, caused by the soil-borne viruses, Tomato bushy stunt virus (TBSV) and the closely related Moroccan pepper virus (MPV), results in necrosis, stunting and death of lettuce plants in western US production regions, often with complete loss of crop. ARS researchers in Salinas, California, evaluated factors that lead to infection of lettuce and disease development. Temperature and day length as well as virus type (TBSV or MPV) influenced development of symptoms on both lettuce and other host plants. This work builds on previous studies in Salinas, California, that identified soil moisture and salinity as factors influencing disease development. Results contribute to continuing studies toward understanding what causes disease to occur sporadically in fields infested with these soil-borne viruses, and enhancing the ability of the lettuce industry to screen varieties for resistance.
Methods for evaluation of lettuce germplasm for resistance to Tospoviruses. Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) are emerging as a serious economic threat to production of lettuce (Lactuca sativa) in California and throughout the world; however, only limited sources of resistance have been identified. ARS researchers in Salinas, California, developed methods for routine transmission of INSV and TSWV to lettuce. These methods are being used to evaluate diverse Lactuca species to identify novel sources of resistance through collaboration between this project and the lettuce breeding program at ARS in Salinas. Results will benefit the lettuce industry in the US and throughout the world through development of evaluation methods and identification of new sources of genetic resistance to these viruses.
Methods for identification of resistance to Polymyxa betae in sugarbeet. Rhizomania disease of sugarbeet, caused by Beet necrotic yellow vein virus (BNYVV) is transmitted by the soil-borne organism, Polymyxa betae. ARS researchers in Salinas, California, discovered that P. betae has a significant, adverse impact on sugarbeet protein expression. Experiments were conducted to optimize experimental conditions, determine presence or absence of virus, and facilitate evaluation of sugarbeet cultivars for resistance to P. betae. These methods are being used to screen sugarbeet breeding lines and wild beet varieties for sources of resistance to P. betae, as well as understand virus-vector interactions that may influence performance of resistant lines. Results should lead to identification of sugarbeet breeding lines with resistance to P. betae, that may enhance performance of BNYVV resistance genes, improving sugar production.
Host plant, virus competition and concentration transmission of curtoviruses. Beet curly top virus and related species regularly cause losses to vegetable and sugarbeet production in the western United States. ARS scientists in Salinas, California, sampled weed and crop hosts from the San Joaquin and Salinas Valleys, California during the severe 2013 outbreak, and identified major virus species and specific variants associated with infection of different host plants. Companion laboratory studies demonstrated that some host plants accumulated specific strains preferentially in single and mixed infections (more than one virus strain or species), and this was correlated with efficiency of transmission by the beet leafhopper. These results demonstrate the influence of host plant selection on emergence and prevalence of curtovirus species and strains. Results add to the knowledge of factors driving emergence and dominance among curtoviruses and contribute to overall knowledge of curtovirus epidemiology.
Effect of resistance to Cucurbit yellow stunting disorder virus (CYSDV) in melon on virus transmission. CYSDV impacts production of melon and other cucurbit crops in the southwestern US, 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. New studies are examining the impact of resistant melon on the ability of CYSDV to move to additional crop plants by examining both virus titer in resistant and susceptible melon and how this relates to transmission. Results will contribute to larger epidemiological studies focused on diminishing virus incidence and improved management practices to reduce impact and pesticide use in affected areas.
Detection of virus RNA segments from Beet oak-leaf virus (BOLV). BOLV is a poorly characterized virus that produces mild symptoms, but is often found in association with the economically damaging Beet necrotic yellow vein virus (BNYVV) that causes rhizomania disease on sugarbeet. Previous studies have indicated the presence of BOLV can alter severity of BNYVV in sugarbeet. ARS researchers in Salinas, California, isolated portions of the RNA of BOLV, and research in progress is seeking to characterize this virus genetically. Knowledge of its genetics will lead to research that should determine the virus interactions that lead to changes in rhizomania severity when BOLV is present, and should determine whether development of resistance to BOLV is warranted.
The impact of Beet necrotic yellow vein virus (BNYVV) infection on sugarbeet protein expression. Rhizomania, caused by 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 to identify proteins associated with infection of sugarbeet by BNYVV. Studies conducted by USDA-ARS in Salinas, California, and Ft. Collins, Colorado, with collaboration from Colorado State University, identified proteins produced in susceptible sugarbeet during infection with BNYVV using mass spectrometry to clarify the types of proteins prevalent during virus infection. These results expand on limited proteomic data available for sugarbeet and provide the groundwork for continuing studies comparing protein expression differences between infected and uninfected sugarbeet as well as resistant and susceptible sugarbeet. These studies should lead to the identification of biological targets that can be exploited to develop novel and biologically friendly disease control strategies for management of this very serious threat to sugarbeet production.
Robertson, N.L. 2013. Molecular detection of Tobacco rattle virus in Bleeding Heart [Dicentra spectabilis (L.) Lem.] growing in Alaska. Plant Health Progress. doi: 10.1094/PHP-2013-0227-01-BR.
Wintermantel, W.M., Hladky, L.L. 2013. Complete genome sequence and biological characterization of Moroccan pepper virus (MPV) and reclassification of Lettuce necrotic stunt virus as MPV. Phytopathology. 105:501-508.