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ARS Home » Northeast Area » Frederick, Maryland » Foreign Disease-Weed Science Research » Research » Research Project #431767

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

Location: Foreign Disease-Weed Science Research

2021 Annual Report

Objective 1: Develop broad range nucleic acid, antibody and metabolomics based diagnostics for vectored plant pathogens. [NP303, C1, PS1] 1-A. Develop E-probe Diagnostic Nucleic acid Assay (EDNA) diagnostics for the detection of plant pathogen vectors. 1-B. Develop massively parallel sequencing based diagnostic for the detection of bacterial pathogens in vectors. 1-C. Develop immunodiagnostic reagents for specific and sensitive detection of Rathayibacter toxicus and tunicamycin toxin in plant products. 1-D. Collect and characterize foreign and emerging bacterial plant pathogens. Objective 2: Assess the effects of host metabolism and environmental factors on transmission, biology and evolution of threatening and emerging insect-transmitted plant pathogens. [NP303, C2, PS2C] 2-A. Assess the effects of vernalization on Plum pox virus adaptation to new hosts. 2-B. Determine the effects of Plum pox virus infection on host plant metabolomics. 2-C. Xylella fastidiosa subsp. pauca comparative genomes and proteomes. 2-D. Transmission of Xylella fastidiosa subsp. pauca CoDiRO by glassy-winged sharpshooter. Objective 3: Identify genes and proteins required for infection, toxin production and pathogenicity of foreign bacterial plant pathogens. [NP303, C2, PS2A] 3-A. Control of toxin production in Rathayibacter toxicus. 3-B. Rathayibacter toxicus gall transcriptome and proteome. Objective 4: Identify factors in the retention and transmission of insect-transmitted viruses and develop strategies to disrupt this transmission, with an initial focus on the cotton leafroll dwarf virus. [NP303, C2, PS2A, PS2B; C3, PS3B]

Metagenomics based detection of pathogens and vectors will utilize E-probe Diagnostic Nucleic acid Assay (EDNA) diagnostics. E-probes will be developed for vectors and vectored bacterial pathogens, and tested on metagenomes from controlled simulated insect traps, then extended to test assay success on real world samples. Immunoassays for Rathayibacter toxicus will be developed by the identification of soluble, high abundance, extracellular and/or secreted pathogen proteins as potential diagnostic targets followed by production of monoclonal antibodies. 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. Effects of vernalization on Plum pox virus (PPV) biology will be assessed using parallel lines of PPV in peaches, one undergoing artificial vernalization and the other without undergoing vernalization. PPV effects on the metabolome of peaches will be assessed using standard methods, testing PPV positive symptomatic and non-symptomatic trees and comparing the results to metabolomic profiles from healthy and Prunus necrotic ringspot infected trees. The genomes of multiple Xylella fastidosa subsp. pauca isolates will be sequenced, and comparative genomics will be used to assess potential host range and pathogenicity factors. The transmission of the olive strain will be tested using glassy-winged sharpshooter biotypes from the U.S. The genes responsible for toxin production in Rathyaibacter toxicus will be confirmed by gene knockouts, and the regulation and control of these genes will be studied using transcriptomics and proteomics.

Progress Report
The goals of Objective 1 are to develop novel diagnostic techniques and reagents for vectored plant pathogens. The departure of the unit plant virologist and E-probe Diagnostic Nucleic acid Assay (EDNA) specialist has left a critical vacancy in our ability to proceed with the planned EDNA goals (Subobj 1A and 1B). Under subobjective 1C, we have generated monoclonal antibodies that specifically detect the USDA-Animal and Plant Health Inspection Service (APHIS) Plant Pathogen Select Agent Rathayibacter toxicus, which allow for the rapid, sensitive detection of the pathogen in immunoassays on in seed and environmental samples from surveys of the U.S. Pacific northwest. Formal diagnostic assay validation is continuing under a FY 2018 Material Transfer Agreement (MTA) with the USDA APHIS Plant Protection Quarantine (PPQ) Science and Technology Plant Pathogen Confirmatory Diagnostic Laboratory in Beltsville, Maryland. We plan to continue to conduct diagnostic immunoassays on R. toxicus -infested seed samples collected in FY 2019 from infested regions of Australia. Objective 2 focuses on characterizing two vectored pathogens: plum pox virus (PPV) and the bacterium Xylella fastidiosa. Metabolites were quantitated by our collaborator and we analyzed the data; a manuscript is in preparation reporting these results. The USDA-APHIS listed select agent Rathayibacter toxicus is the focus of Objective 3. Specifically, we aim to better understand the regulation and mechanism of toxin production. Immunoreagents and other analysis methods are in place to analyze toxin production and transcription and translation of Tunicamycin (or toxin) gene cluster (TGC) under various growth conditions and several experiments have been planned. Construction of knock-out mutants in the R. toxicus TGC was also planned for this year. In related work, a comparative secretome analysis of toxigenic and atoxigenic Rathayibacter species has been completed and published. This study identified bacterial surface-associated and secreted proteins which may influence nematode attachment, in planta disease development, and microbial competition/defense. Our project has a new Objective 4 focused on cotton blue disease caused by the cotton leafroll dwarf virus. A new scientist was hired this year who will be leading this research.

1. Proteins secreted by Rathayibacter toxicus and other Rathayibacter species. Rathayibacter toxicus is a USDA-APHIS plant pathogen select agent that makes a toxin in forage grasses lethal to livestock, resulting in 40 million dollars of damage annual to the Australian economy. Due to its complex lifecycle, we do not clearly understand how Rathayibacters infect the plant, produce toxin, and cause disease. Because of the potential threat to U.S. agriculture and food supplies, such knowledge is very valuable. ARS researchers at Ft. Detrick, Maryland, have analyzed proteins secreted by Rathayibacter toxicus and two other Rathayibacter species. A number of proteins identified are similar to proteins secreted by bacterial pathogens of animals and other proteins provide clues to aspects of the unique lifecycle of these bacteria. Highly abundant and unique secreted proteins identified in this study also provide targets for the development of protein-based diagnostic assays. The information resulting from this study will lead to diagnostic assays critical in preventing entry of this damaging plant pathogen into U.S. seed and forage producing regions.

Review Publications
Tancos, M.A., Mcmahon, M.B., Garrett, W.M., Luster, D.G., Rogers, E.E. 2021. Comparative secretome analyses of toxigenic and atoxigenic Rathayibacter species. Phytopathology. 111:1530-1540.
Tamukong, Y.B., Collum, T.D., Stone, A.L., Kappagantu, M., Sherman, D.J., Rogers, E.E., Dardick, C.D., Culver, J.N. 2020. Dynamic changes impact the plum pox virus population structure during leaf and bud development. Virology. 548:192-199.