Location: Crop Improvement and Protection Research2007 Annual Report
1a. Objectives (from AD-416)
Study vector transmission specificity, biology, epidemiology, detection and management of whitefly-transmitted criniviruses. Address pathogenicity and infection physiology of BNYVV and other soil-borne viruses of sugarbeet. Develop virus-induced gene silencing for control of curtoviruses in tomato and sugarbeet. Identify and address problems associated with emerging and re-emerging viruses affecting sugarbeet and vegetable production in the United States, develop detection technologies for these viruses, and work toward effective management.
1b. Approach (from AD-416)
Evaluate factors contributing to the specificity of crinivirus transmission by whitefly vectors. Insect proteins will be separated and tested for interaction with whole virus and individual and combinations of virus proteins expressed in vitro. We will also conduct genetic and biological characterization of the criniviruses Lettuce chlorosis virus and Cucurbit yellow stunting disorder virus, and develop improved methods for detection and differentiation of criniviruses. Examine virus-host interactions, including differential protein expression and pathway activation in healthy sugarbeet and in sugarbeet infected with BNYVV, the causal agent of rhizomania. Studies will involve fractionation and separation of proteins and protein-protein binding studies. Attempt to develop infectious clones of BNYVV and BSBMV, and use these for generation of recombinant and pseudo-recombinant viruses that elucidate the viral genetic components responsible for BNYVV pathogenicity in sugarbeet, and increased disease severity during co-infection. Monitor for the emergence of BNYVV variants capable of overcoming known sources of resistance throughout the US beet industry using standard methods developed previously by our laboratory, and develop new methods for differentiation of resistance breaking isolates. Gene silencing constructs will be designed for control of curtoviruses in tomato and sugarbeet. Silencing constructs will be delivered in testing using either a virus-based vector carrying silencing constructs, or by delivery using Agrobacterium tumefaciens, and will be expressed as small interfering RNAs (siRNA). Identify and address problems associated with emerging and re-emerging viruses affecting sugarbeet and vegetable production in the United States, develop detection technologies for these viruses, and work toward effective management. This will involve biological, molecular and serological analyses including development of rapid detection tools, genetic characterization, vector identification and identification of factors contributing to virus emergence. (IBC info pending). Replaces 5305-22000-010-00D (3/07).
3. Progress Report
This project began in April 2007, information from the first 3 months will be presented in the 2008 Project Report when accomplishments are more complete.
Identification and Management of Cucurbit Yellow Stunting Disorder Virus (CSYDV) in the American Desert Southwest: In the fall of 2006 a new crinivirus, Cucurbit yellow stunting disorder virus (CYSDV) was identified for the first time in the southwestern desert melon production areas of California and Arizona, by scientists from the USDA-ARS Virology Lab, as well as scientists from the University of California-Davis and the University Arizona. The USDA Virology lab has developed a broad array of detection methodologies to accurately identify and confirm infection, both advanced and early stage infections, that are being used by an increasing number of labs throughout the U.S. We are monitoring spring and fall crops for CYSDV incidence and associated incidence of the whitefly vector, B. tabaci. Our lab is working closely with state and county personnel to determine incidence (nearly 100% in fall 2006, but lower in the spring 2007 crop, as expected) and distribution. Our lab is conducting host range studies on regional crops and weeds, working closely with the melon breeding program at the USDA-ARS in Salinas, CA to evaluate a putative new source of resistance in melon, and is actively involved in educating growers and developing management tactics to minimize losses. All research described above is being conducted by the USDA-ARS Virology Lab in Salinas. This project supports NP303 Component 2, Biology, Ecology, epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2C: Population Dynamics, Spread, and Epidemiology of Pathogens. Distribution and Molecular Analysis of Resistance-breaking Isolates of Beet Necrotic Yellow Vein Virus (BNYVV) in the United States: The economically devastating rhizomania disease of sugarbeet is caused by the soil-borne, Polymyxa betae-transmitted BNYVV, and can only be controlled effectively by the use of resistant cultivars. We determined that new resistance-breaking BNYVV isolates, first identified in 2003-2004 in the Imperial Valley of California, likely evolved from the existing A-pathotype based on single-strand conformation polymorphism and sequence analyses, and have identified amino acid changes unique to the resistance breaking isolates. Soil surveys recently completed indicated that the resistance-breaking isolates not only exist in the Imperial Valley, but also throughout many of the major U.S. sugarbeet production regions. The identification of resistance-breaking isolates from throughout the U.S. demonstrates that selection pressure resulting from widespread planting of Rz1 resistance is driving emergence of resistance-breaking BNYVV strains, and illustrates the urgency for developing new sources or methods of resistance. All work performed by the USDA-ARS Virology Lab in Salinas, CA. This project supports NP303 Component 2, Biology, Ecology, epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2A: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen. Characterization of Lettuce Chlorosis Virus and Development of Efficient Molecular Detection: Lettuce chlorosis virus (LCV), family Closteroviridae, genus Crinivirus, emerged as a threat to lettuce and sugar beet production in the desert regions of the southwestern U.S. in the early 1990’s, producing interveinal yellowing, stunting, and brittleness of affected leaves. We (the USDA-ARS Virology Lab in Salinas, CA) developed molecular probes and LCV-specific RT-PCR primer pairs and demonstrated their efficacy for rapid identification of LCV infected lettuce and sugarbeet plants, and are continuing to characterize LCV on a molecular level. Results of our studies are providing industry with the diagnostic tools necessary for early identification, facilitating more effective disease control and decreasing losses. This project supports NP303 Component 2, Biology, Ecology, epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2A: Pathogen biology, Virulence Determinants, and genetics of the Pathogen. First Report of Pelargonium Zonate Spot Virus from Tomato in the United States: Pelargonium zonate spot virus (PZSV) was first isolated from tomato in southern Italy in 1982 and has since been identified in France and Spain. In June of 2006, more than 100 tomato (Lycopersicon esculentum Mill.) plants exhibiting symptoms of stunting, malformation, yellow rings and line patterns on the leaves, and concentric chlorotic ringspots on the stems, similar to those of PZSVwere observed in 7 acres of tomato in Yolo County, California. The causal agent was mechanically transmitted to several indicator species. Two field infected tomato plants and one each of the mechanically inoculated host plant were positive with the double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using a commercial PZSV IdentiKit (Neogen Europe Ltd., Scotland, UK). Partially purified virions stained with 2% uranyl acetate contained spherical to ovate particles ranging between 25 to 35 nm. Published sequences of PZSV were used to design 3 sets of primer pairs specific for PZSV RNAs 1 through 3. Total nucleic acids were extracted from field infected tomato plants. Amplicons had 92%, 94%, and 96% nucleotide sequence identity to PZSV RNA1, RNA2 and RNA3, respectively. The symptomatology, serology, particle morphology, and nucleotide sequences confirm the presence of PZSV in a tomato field in California, and we have developed tools for the rapid detection of this virus in the United States. All work performed by the USDA-ARS Virology Lab in Salinas, CA. This project supports NP303 Component 2, Biology, Ecology, epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2A: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen. Development of Novel Sources of Resistance to Beet Severe Curly Top Virus (BSCTV) in Sugarbeet and Tomato: BSCTV and related curtoviruses are responsible for severe losses in numerous crops each year, including tomato and sugarbeet. Current studies are focused on development of virus induced gene silencing (VIGS) to obtain complete resistance against BSCTV and other curtoviruses in tomato and sugarbeet, and have identified two types of constructs capable of significantly reducing virus concentrations in greenhouse experiments using two independent delivery systems. Additional studies are continuing in efforts to obtain complete resistance in both crops. The ability to elicit VIGS for control of curtoviruses will provide the vegetable and sugarbeet industries with alternative and potentially more effective control methods, reducing the need for excessive pesticide application. All work performed by the USDA-ARS Virology Lab in Salinas, CA. This project supports NP303 Component 2, Biology, Ecology, epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2A: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen. Subtractive Proteomics to Characterize Resistance and Susceptibility of Sugarbeet to Beet Necrotic Yellow Vein Virus (BNYVV): New studies are using subtractive proteomics to identify proteins induced in resistant and susceptible sugarbeet (Beta vulgaris) as a result of infection with BNYVV. Studies conducted at the Sugarbeet Research Unit at the USDA-ARS in Ft. Collins, CO in collaboration with the Virology Lab at the USDA-ARS in Salinas, CA have identified differences in protein expression in susceptible sugarbeet among healthy sugarbeet, sugarbeet exposed to the virus-free vector of BNYVV, Polymyxa betae, and BNYVV infected sugarbeet. In vitro expression of BNYVV proteins is in progress for use in protein interaction arrays to identify functional interactions between virus and host plant. Results should elucidate physiological and biochemical changes that differ between healthy and BNYVV infected sugarbeet, leading to targeted methods to prevent BNYVV from eliciting symptoms of rhizomania disease. This project supports NP303 Component 2, Biology, Ecology, epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2B: Plant-Microbe-Vector Interactions. Characterization of a U.S. Isolate of Beet Black Scorch Virus: The first reported U.S. isolate of Beet black scorch necrovirus (BBSV) was obtained and characterized by the USDA-ARS Virology Lab in Salinas, CA in collaboration with the USDA-ARS in Fargo, ND. The complete nucleotide sequence of the genomic RNA of the virus, designated BBSV-Co, exhibits 93% similarity to the genome of the ‘Ningxia’ isolate of BBSV from China, with higher similarity for individual proteins (up to 97%), and it was determined that the U.S. isolate of BBSV may contain and additional gene absent from the Chinese isolate. Analysis of the coat protein by isoelectric focusing and by mass spectroscopy indicates the presence of phosphorylated resides, and it was determined that the American isolate lacked the two terminal adenosine nucleotides in the published sequences of BBSV from China. Rabbit anti-BBSV antiserum with high sensitivity was produced from a purified preparation of the virus. Results will benefit detection and monitoring for this virus in U.S. beet producing regions, and knowledge of its relationship to international BBSV isolates. This project supports NP303 Component 2, Biology, Ecology, epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2A: Pathogen biology, Virulence Determinants, and genetics of the Pathogen.
5. Significant Activities that Support Special Target Populations