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ARS Home » Northeast Area » Frederick, Maryland » Foreign Disease-Weed Science Research » Research » Research Project #422773

Research Project: Identification, Characterization, and Biology of Foreign and Emerging Viral and Bacterial Plant Pathogens

Location: Foreign Disease-Weed Science Research

2014 Annual Report

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.

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.

Progress Report
To initiate work on developing diagnostics and understanding the mechanisms of toxin production for the bacteria Rathayibacter toxicus (Objective 1) completed for R. toxicus, as well as, the associated phage. The genes that are likely responsible for toxin production were identified and assay development has begun for a PCR assay that can detect any Rathayibacter species capable of toxin production. The genome 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 (Objective 2), Soybean dwarf virus (SbDV) infected plant, SbDV infected aphid and Citrus tristeza virus (CTV) infected plant metagenomes were generated using 454 sequencing. Virus titers were validated by quantitative RT-PCR. The EDNA analysis of these samples was completed. To initiate work on the relationship between aphid presence and viral evolution (Objective 3), 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 sequence analysis. SbDV populations were analyzed by sequencing, and results are under examination.


Review Publications
Roy, A., Shao, J.Y., Hartung, J.S., Schneider, W.L., Brlansky, R. 2013. A case study on discovery of novel Citrus leprosis virus cytoplasmic type 2 utilizing small RNA libraries by next generation sequencing and bioinformatic analyses. Journal of Data Mining in Genomics & Proteomics. 4:129. doi: 10.4172/2153-0602.1000129.
Roy, A., Shao, J.Y., Schneider, W.L., Hartung, J.S., Brlansky, R.H. 2013. A population of endogenous pararetrovirus genomes in carrizo citrange. Genome Announcements. 2(1):e01063-13.
Ananthakrishnan, G., Choudhary, N., Roy, A., Sengoda, V.G., Postnikova, E.N., Hartung, J.S., Stone, A.L., Damsteegt, V.D., Schneider, W.L., Munyaneza, J.E., Brlansky, R.H. 2013. Development of primers and probes for genus and species specific detection of 'Candidatus Liberibacter species' by real-time PCR. Plant Disease. 97:1235-1243.
Stobbe, A., Daniels, J., Espindola, A., Verma, R., Melcher, U., Ochoa-Corona, F., Garzon, C., Fletcher, J., Schneider, W.L. 2013. E-probe Diagnostic Nucleic acid Analysis (EDNA): A theoretical approach for handling of next generation sequencing data for diagnostics. Journal of Microbiological Methods. 94:356-366.
Roy, A., Choudary, N., Colina, G., Stone, A.L., Shao, J.Y., Levy, L., Nakhla, M., Hollingsworth, C., Hartung, J.S., Schneider, W.L., Brlansky, R. 2013. Genome assembly of citurs leprosis virus nuclear type reveals a close association with orchid fleck virus. Genome Announcements. doi:10.1128/genomeA.00519-13.