Location: Foreign Disease-weed Science Research2012 Annual Report
1a. Objectives (from AD-416):
These objectives are designed to address the risks of foreign plant viral and bacterial diseases, via the collection and characterization of foreign viruses and bacteria, the development of broad range and pathogen specific diagnostics, and the assessment of biological factors associated with disease epidemiology, including evolutionary capacity, alternative hosts and transmission efficiency. 1: Collect germplasm, characterize accessions, and generate reagents for the development of diagnostic assays for foreign and emerging bacterial plant pathogens. 1A. Collect and characterize foreign and emerging bacterial plant pathogens. 1B. Characterization of toxin production among Rathyibacter species. 1C. Develop immunodiagnostic reagents for specific and sensitive detection and diagnosis of emerging bacterial pathogens, such as Rathayibacter toxicus. 2: Develop broad range diagnostics for plant pathogens using massively parallel sequencing and high-throughput screening. 2A. Develop massively parallel sequencing based diagnostics for the detection of viral and bacterial plant pathogens. 2B. Develop massively parallel sequencing based diagnostics for the detection of pathogens in vectors. 3: Assess the effects of host shifting and constant insect presence on viral evolution and pathogenesis. 3A. Develop a mechanism for assessing the effect of constant vector presence on a persistently transmitted virus (Soybean dwarf virus). 3B. Develop a mechanism for assessing the effect of constant vector presence on a semi-persistently transmitted virus. (Citrus tristeza virus) 3C. Develop a mechanism for assessing the effect of constant vector presence on a non-persistently transmitted virus (Plum pox virus). 4: Conduct vector transmission and vector interaction studies for emerging insect-transmitted plant pathogens. 4A. Determine potential host range (commercial and wild reservoir) for Cotton leaf roll dwarf virus (CLRDV). 4B. Determine potential vectors for CLRDV.
1b. Approach (from AD-416):
Obtain cultures of target bacteria from major international collections, foreign collaborators, and by traveling abroad. Accessions will be cloned, checked for authenticity using biochemical tests and added to the FDWSRU International Collection of Phytopathogenic Bacteria. Generate a complete phage genome and a draft Rathayibacter toxicus genome, compare them to genomes of other characterized corynetoxin producing bacteria/phage systems to identify candidate genes that may be associated with toxin. Identify soluble, high abundance, extracellular and/or secreted pathogen proteins as potential diagnostic targets. Potential immunogen proteins will be used to generate polyclonal and monoclonal antibodies for diagnostics development. Develop massively parallel sequencing (MPS) based diagnostics for the detection of viral and bacterial plant pathogens, nucleic acids are extracted from infected plants or vectors will be sequenced as a metagenome. The MPS sample database will serve as a target for a series of pathogen specific queries to indicate the presence of the pathogen. Assess the effect of constant vector presence on A) persistently transmitted virus (Soybean dwarf virus); B) semi-persistently transmitted virus (Citrus tristeza virus); and C) non-persistently transmitted virus (Plum pox virus), in each case the subject virus will be transmitted into multiple new hosts. The fitness of strains will be assessed by the resulting titer (measured by real-time PCR), symptom development, transmission efficiency and the rate of adaptive mutation fixation. Determine potential host range for Cotton leaf roll dwarf virus, isolates of CLRDV will be used to inoculate cotton cultivars and related host species using cotton aphids. Plants will be observed and symptom data recorded up to 30 days or longer, with virus presence confirmed by real-time PCR. Positive related hosts will be back-inoculated to cotton to check the reservoir capacity of wild relatives in field environments. Determine potential vectors for CLRDV, we will test acquisition efficiency by US biotypes of cotton aphids and other potential vectors to determine if CLRDV vectors already exist in the U.S.
3. Progress Report:
This project is a new In-House Project, approved in April of 2012. Work has just begun on the first year objectives, as work is wrapping-up on the previous In-House Project. There are no milestones or sufficient progress to report at this time.