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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

2019 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. The departure of the unit plant virologist and EDNA specialist has left a critical vacancy in our ability to proceed with the planned EDNA (E-probe Diagnostic Nucleic acid Assay) goals (Subojective 1A). Under subobjective 1C, we generated highly specific antibodies that specifically detect the USDA-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. This will allow us to identify and characterize endemic Rathayibacter species with potential to cause damage to seed and forage industries. We have isolated additional 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 (Subojective 1D). Objective 2 focuses on characterizing two vectored pathogens: plum pox virus (PPV) and the bacterium Xylella fastidiosa. The PPV vernalization projects have been delayed by a powdery mildew epidemic in our containment greenhouse. The lack of treatment options during the FY2019 shutdown allowed this epidemic to get out of control and has set back progress especially on Subojective 2B. Infection lines for peach have been selected and selection is in process with American plum (Subojective 2A). Metabolomic experiments were severely impacted by the shutdown and powdery mildew epidemic and are behind schedule. Last year we decided to change the control virus used to tomato ringspot virus (ToRSV); we are still waiting on shipment of ToRSV infected budwood to begin control inoculations. Under Subojective 2C, sequencing of Xylella fastidiosa genomes has been completed and will be submitted for publication this fiscal year. 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 and protocols are available to two proteins from the putative tunicamycin gene cluster (TGC): TunB and TunC. Quantitative reverse-transcription PCR (qRT-PCR) primers and protocols have been developed for 8 TGC genes. 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. Lethal plant pathogens of livestock. Rathayibacter agropyri is a plant pathogen closely related to Rathayibacter toxicus, an APHIS-listed select agent found in Australia that makes a toxin in forage grasses lethal to livestock. R. agropyri is endemic to the Pacific Northwest U.S. ARS researchers at Ft. Detrick, Maryland have sequenced the entire DNA genome of several strains of R. agropyri, and at least three strains encode complete toxins of two bacteria that are known to make toxins. This discovery of some R. agropyri strains makes it possible that toxin production could occur.

Review Publications
Ribiero-Bronzato, A., Sherman, D.J., Stone, A.L., Wilson, V., Schneider, W.L., King, J. 2018. Transcriptome amplification coupled with nanopore sequencing as a surveillance tool for plant pathogens in plant and insect tissues. Plant Disease. 102:1648-1652.
Roy, A., Stone, A.L., Leon, M.G., Hartung, J.S., Wei, G., Mavrodieva, V., Nakhla, M.K., Schneider, W.L., Brlansky, R.H. 2018. Sweet orange showing leprosis symptoms in Colombia are naturally infected with Hibiscus infecting cilevirus and citrus leprosis virus C2. Plant Disease.
Roy, A., Stone, A.L., Melzer, M.J., Hartung, J.S., Shao, J.Y., Mavrodieva, V., Nakhla, M.K., Brlansky, R.H., Schneider, W.L. 2018. Complete nucleotide sequence of a novel Hibiscus-infecting Cilevirus from Florida and its relationship with closely associated Cileviruses. Genome Announcements. 6(4):e01521-17.
Roy, A., Stone, A.L., Melzer, M.J., Hartung, J.S., Mavrodieva, V., Nakhla, M.K., Brlansky, R.H., Schneider, W.L. 2017. First report of Cilevirus associated with green ringspot on senescent hibiscus leaves in Tampa, Florida. Plant Disease.
Davis, E.W., Tabima, J.F., Weisberg, A.J., Lopes, L.D., Wiseman, M.S., Wiseman, M.S., Pupko, T., Belcher, M.S., Sechler, A.J., Tancos, M.A., Schroeder, B.K., Murray, T.D., Luster, D.G., Schneider, W.L., Rogers, E.E., Andreote, F., Grunwald, N.J., Putman, M.L., Chang, J.H. 2018. Bacteriophage NCPPB3778 and a type I-E CRISPR drive the evolution of the U.S. biological select agent Rathayibacter toxicus. mBio. 9:e01280-18.