2013 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
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
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.
To initiate work on developing diagnostics and understanding the mechanisms of toxin production for the bacteria Rathayibacter toxicus, multiple Rathayibacter species, including several R. toxicus strains were collected and characterized. Genomic sequencing was initiated for R. toxicus, and the initial data was used in conjunction with protein extraction and 2-D gel analysis to generate a number of potential antigen targets for antibody production.
With regards to developing next-generation sequencing (NGS) as a plant pathogen diagnostic tool, Soybean dwarf virus (SbDV), Citrus tristeza virus (CTV) and Plum pox virus (PPV) infected plants were raised and tissue harvested for anaylsis by NGS. In addition, several unknown viral infections of plants were harvested for NGS analysis. Known virus infections were validated by quantitative RT-PCR.
To initiate work on the relationship between aphid presence and viral evolution, parallel experimental lines with constant or periodic aphid presence were established for SbDV and CTV. Passaging experiments were completed and nucleic acids were extracted for future sequence analysis.
Next-generation sequencing (NGS) as a plant pathogen diagnostic tool. Regulatory agencies, inspections stations, diagnostic laboratories and stakeholders all need rapid diagnostic assays capable of handling a broad array of viral, bacterial, stramenopile and fungal pathogens. NGS is an extremely powerful technology that allows for the in depth sequencing of any and all organisms in a given sample, such as an infected crop plant. ARS researchers at Fort Detrick, Maryland have demonstrated that with the development of better bioinformatic tools, this powerful technology can be effectively used to simultaneously identify multiple known and unknown plant pathogens in an infected plant. This is a significant breakthrough for the field of plant pathogen diagnostics, and will have huge impact in situations where there is a need to screen for numerous plant pathogens (i.e. importation of breeding stocks) or unknown plant pathogens.
Damsteegt, V.D., Stone, A.L., Smith, O.P., Mcdaniel, L., Sherman, D.J., Dardick, C.D., Hammond, J., Jordan, R.L., Schneider, W.L. 2013. A previously undescribed potyvirus isolated and characterized from arborescent Brugmansia. Archives of Virology. 158:1235-1244.