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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

2015 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), genome sequencing and annotation was completed for R. toxicus as well as the associated phage. The genes that are likely responsible for toxin production were identified, and 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 two dimensional gel analysis to generate a number of potential antigen targets for antibody production. Initially polyclonal antibodies were developed and are currently being screened for specificity and efficacy prior to monoclonal production. Next-generation sequencing (NGS) (Objective 2) technology as a plant pathogen diagnostic tool was transitioned to simultaneous detection of insect vectors and pathogens. E-probe Diagnostic Nucleic acid Assay (EDNA), the bioinformatic tool was capable of detecting Ca. Liberibacter asiaticus in Diaphorina citria (the causal agent and vector of citrus greening) and Soybean dwarf virus (SbDV) in the pea aphid (Acythrosiphon pisum). To continue 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 on soybeans. Passaging experiments were completed and nucleic acids were extracted for sequence analysis. SbDV populations were analyzed by sequencing, and results are under examination. Initial sequence analysis of SbDV adapting to peas with and without aphid presence does not suggest that vector presence plays a significant role.

1. Assays for toxin production in bacterial pathogens. Rathayibacter toxicus is a USDA-APHIS select agent because infections produce a toxin in forage grasses that is lethal to livestock, resulting in 40 million dollars of damage yearly to Australia. Despite the potential threat to U.S. agriculture and food supplies, the mechanisms of toxin production for R. toxicus had never been elucidated. Scientists at the USDA ARS Foreign Disease-Weed Science Research Unit in Ft. Detrick, Maryland, and the Emerging Pests and Pathogens Research Unit in Ithaca, New York, completely sequenced the genome of three R. toxicus strains plus an associated bacteriophage, and determined that the genes responsible for toxin production are part of a transposable element housed in the bacterial DNA. From the genome information, assays capable of finding any toxin producing Rathayibacter species were developed. The assays will be useful for protecting against the introduction of the select agent plant pathogen to the United States, as well as for study of mechanisms by which toxin production is initiated.

Review Publications
Roy, A., Stone, A.L., Shao, J.Y., Colina, G., Wei, G., Choudary, N., Achor, D., Nakhla, M., Levy, L., Hartung, J.S., Schneider, W.L., Brlansky, R. 2015. Identification and molecular characterization of nuclear Citrus leprosis virus, an unassigned Dichorhavirus genus member associated with citrus leprosis disease in Mexico. Virus Research. 105:564-575.
Roy, A., Hartung, J.S., Schneider, W.L., Shao, J.Y., Leon, G.M., Melzer, M.J., Beard, J.J., Otero-Colina, G., Bauchan, G.R., Ochoa, R., Brlansky, R.H. 2015. Role bending: complex relationships between viruses, hosts and vectors related to citrus leprosis, an emerging disease. Phytopathology. DOI: 10.1094/PHYTO-12-14-0375-FI.
Stobbe, A.H., Schneider, W.L., Hoyt, P., Melcher, U. 2014. Screening metagenomic data for viruses using the E-Probe Diagnostic Nucleic Acid Assay (EDNA). Phytopathology. 104:1125-1129.
Melcher, U.K., Verma, R., Schneider, W.L. 2014. Metagenomic search strategies for interactions among plants and multiple microbes. Frontiers in Plant Science. DOI: 10.3389/fpls.2014.00268.