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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

2018 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.

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. While the departure of the unit plant virologist and EDNA (E-probe Diagnostic Nucleic acid Assay) specialist slowed progress on some aspects of this objective, we did select e-probes for three classes of insect vectors: multiple aphid species, glassy winged sharpshooter and Asian citrus psyllid. DNA extraction protocols have been developed; however, sequencing has not yet started (Subobjective 1A). Under Subobjective 1C, we applied a proteomic approach to identify over 700 proteins secreted by the USDA-APHIS select agent plant pathogen Rathayibacter toxicus. From these, we are selecting protein candidates to serve as antigens for generation of Rathayibacter genus-level antibodies for use in immunoassays to detect all Rathayibacter species in seed and environmental samples from surveys of the U.S. Pacific northwest. Genus-level antibodies could also be used to identify and characterize endemic Rathayibacter species with potential to cause damage to seed and forage industries. We have isolated several new strains of R. toxicus from Australian annual ryegrass seed samples and novel strains of Rathayibacter from grass seed from the U.S. Pacific Northwest and from Maryland (Subobjective 1D). Objective 2 focuses on characterizing two vectored pathogens: plum pox virus (PPV) and the bacterium Xylella fastidiosa. The PPV vernalization projects are on schedule, with inoculated and control peach and American plums established (Subobj 2A). Metabolomic experiments with PPV infected peaches are also progressing (Subobj 2B). However, infections with a control virus have not be done. The project plan calls for using Prunus necrotic ringspot virus (PNRSV) as the control. We were not able to find a source of a severe isolate of PNRSV and many peach trees are already infected with a mild isolate of PNRSV making re-infection difficult. We have selected tomato ringspot virus (ToRSV) as a more appropriate and easily available control; once the applied-for APHIS permit is approved, ToRSV will be shipped from Univ. of California at Davis and control inoculations will begin. Under Subobjective 2C, Multilocus sequence typing analyses of available X. fastidiosa strains has been completed. DNA from three strains will be submitted for sequencing very soon. 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. Polyclonal antibodies have been made to 4 separate proteins in the putative tunicamycin gene cluster (TGC) likely to be responsible for toxin biosynthesis (Subobjective 3A). At least two of these antibodies are sensitive and specific to their targets (TunB and TunC). However, antibodies to TunA and TunF lack specificity. Work on developing a transformation system for R. toxicus is on-going; however, a number of technical challenges have arisen and progress has been slow

1. Diagnostic assays for the plant pathogen select agent Rathayibacter toxicus. 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 annually to the Australian economy. Because of the potential threat to U.S. agriculture and food supplies, accurate and rapid diagnostics for R. toxicus are critical to agricultural security. ARS researchers at Ft. Detrick, Maryland, have generated highly specific and sensitive antibodies that detect R. toxicus, which will allow for the rapid, sensitive detection of the pathogen in multiple diagnostic assay formats. Antibodies have been transitioned to USDA-APHIS for diagnostic assay validation and subsequent deployment at ports of entry for detection of R. toxicus in incoming germplasm and seed commodities, thus preventing entry of this damaging plant pathogen into U.S. seed and forage producing regions.

2. Identification of the toxin biosynthetic cluster (TGC) in the plant pathogen select agent Rathayibacter toxicus. Rathayibacter toxicus is a USDA-APHIS plant pathogen select agent that makes a toxin in forage grasses lethal to livestock, resulting in up to 40 million dollars of damage annually to the Australian economy. Previous work has found a bacteriophage associated with toxin production in most, but not all, cases. ARS researchers at Ft. Detrick, Maryland, have sequenced the DNA of both R. toxicus and its bacteriophage. A tunicamycin biosynthetic cluster has been identified in R. toxicus that is similar to tunicamycin biosynthetic genes from other bacteria. The bacteriophage genome does not contain any genes with predicted toxin biosynthetic functions. Identification of the toxin biosynthetic cluster is the first step in understanding and potentially preventing toxin production by this damaging plant pathogen. Researchers at ARS and universities will use this knowledge to characterize novel Rathayibacter strains and to aid in risk assessment.

Review Publications
Fennessey, C.M., McMahon, M.B., Sechler, A.J., Kaiser, J., Garrett, W.M., Tancos, M.A., Luster, D.G., Rogers, E.E., Schneider, W.L. 2018. Partial proteome of the corynetoxin-producing Gram-positive bacterium, Rathayibacter toxicus. Proteomics. 18:1700350.
Schneider, W.L., Sechler, A.J., Rogers, E.E. 2017. Complete genome sequence of the Rathayibacter toxicus phage NCPPB3778. Genome Announcements. 5(42):e00671-17.
Sechler, A.J., Tancos, M.A., Schneider, D.J., King, J.G., Fennesey, C.M., Schroeder, B.K., Luster, D.G., Schneider, W.L., Rogers, E.E. 2017. Whole genome sequence of two Rathayibacter toxicus strains reveals a tunicamycin biosynthetic cluster similar to Streptomyces chartreusis. PLoS One. 12(8):e0183005.
Schroeder, B.K., Schneider, W.L., Luster, D.G., Sechler, A.J. 2018. Rathayibacter agropyri (non O’Gara 1916) comb. nov., nom. rev., isolated from western wheatgrass (Pascopyrum smithii). International Journal of Systematic and Evolutionary Microbiology. 68:1519–1525.