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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

2022 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
Under Objective 1, a critical deficiency in expertise prevented sequencing bacterial pathogens in vectors and downstream EDNA analysis. COVID19 maximized telework and 25% occupancy limits prevented collection of new foreign bacterial plant pathogens. Antibodies against Rathayibacter toxicus had already been transferred to APHIS; no additional assay development or validation was possible. Similarly for Objective 2, the combination of lack of expertise and COVID19 maximized telework and 25% occupancy limits prevented completion of both the analysis of plum pox sequence data and Xylella fastidiosa transmission testing. Again, COVID19 maximized telework and 25% occupancy limits prevented the creation of R. toxicus knock-out mutants, so we were not able to test any knock-outs for TGC transcription and translation. APHIS permits and biological materials were obtained for work on cotton leafroll dwarf virus (CLRDV) under Objective 4. Over the course of the project (2017-2022), the overall goals of Objective 1 were to develop novel diagnostic techniques and reagents for vectored plant pathogens. We generated both polyclonal and monoclonal antibodies that specifically and sensitively detect the USDA-APHIS plant pathogen select agent Rathayibacter toxicus. This technology was transferred to the USDA APHIS Plant Protection Quarantine (PPQ), Science and Technology Plant Pathogen Confirmatory Diagnostic Laboratory where formal diagnostic assay development and validation will take place. A rapid, specific, and sensitive antibody-based detection assay for R. toxicus will allow the identification and characterization of Rathayibacter-like organisms isolated from grass seed and environmental samples from the U.S. Pacific Northwest and elsewhere. The project received more than 100 cultures of various plant pathogenic bacteria from Monsanto. In addition, project personnel isolated new strains of R. toxicus from Australian annual ryegrass seed samples and novel strains of other Rathayibacter species from the U.S. Pacific Northwest and from Maryland. All viable cultures have been added to the ICPB (International Collection of Phytopathogen Bacteria) which is housed at the Foreign Disease-Weed Science Research Unit (FDWSRU). Objective 2 focused on characterizing two vectored pathogens: plum pox virus (PPV) and the bacterium Xylella fastidiosa. The PPV projects were negatively impacted by a three-year vacancy in the virologist position. However, final experiments and data analysis were completed for this objective to describe the effect of vernalization on PPV titers and transmissibility in three wild Prunus species prevalent in peach and plum cultivation areas of the eastern U.S. Metabolomic samples were collected and extracted from infected, mock infected, and control peach. Extracts were sent to our collaborator for quantitation and that data has been analyzed. However, only very preliminary aphid feeding experiments were possible and DNA sequencing of viral samples could not be completed due to COVID19 maximized telework and 25% occupancy limits in place during the final two years of the project. Three X. fastidiosa subsp. pauca genomes have been sequenced. However, the proteomic work could not be completed due to COVID19 maximized telework and 25% occupancy limits in place during the final two years of the project. Similarly, project collaborators were not able to collect glassy-winged sharpshooters for transmission experiments due to COVID19 pandemic-related restrictions at their institutions. The USDA-APHIS listed select agent Rathayibacter toxicus was the focus of Objective 3. Specifically, the project set out to better understand the regulation and mechanism of toxin production by R. toxicus. We developed polyclonal antibodies and detection protocols for two proteins from the putative tunicamycin gene cluster (TGC): TunB and TunC. Quantitative reverse-transcription PCR (qRT-PCR) primers and protocols were developed for eight TGC genes. We purchased a compact mass spectrometer to detect and quantify tunicamycin-like toxins from R. toxicus cultures. All of these new reagents and protocols were needed to examine toxin production and transcription and translation of the TGC under various growth conditions. However, COVID19 maximized telework and 25% occupancy limits in place during the final two years of the project delayed the laboratory-based time course experiments to examine toxin production. 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. A transformation system is needed to knock-out key TGC genes and definitively prove the TGC is in fact responsible for toxin production. Between pandemic restrictions and a lack of available R. toxicus galls, it was not possible to characterize the gall transcriptome and proteome. In related work, tunicamycin gene clusters were identified in three additional species: R. agropyri, R. iranicus, and R. woodii. A comparative secretome analysis of toxigenic and atoxigenic Rathayibacter species was 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. Objective 4 was added in FY20 and focuses on cotton leafroll dwarf virus. As with other components of the project, Objective 4 progress for laboratory experiments was hindered due to COVID19 maximized telework and 25% occupancy limits in place during the final two years of the project. However, APHIS permits were obtained, along with critical biological components for the experiments, including cotton aphids and infected cotton plants.

1. First transcriptome of corn leafhopper (Dalbulus maidis). The corn leafhopper (Dalbulus maidis) is an insect pest and vector of several economically important pathogens to maize, including corn stunt spiroplasma and maize rayado fino virus. Vector capability for these plant pathogens is highly specific, but little is known about the leafhopper components that enable pathogen transmission to occur. ARS scientists at Frederick, Maryland, and Wooster, Ohio, developed the first transcriptome for the corn leafhopper and identified genes regulated by interaction with maize rayado fino virus using deep sequencing and expression comparisons. Leafhopper genomes are large and complex; therefore, this data provides a basis for understanding leafhopper biology and genes underlying the virus vector interaction.

Review Publications
Mlotshwa, S., Khatri, N., Willie, K.J., Xu, J., Todd, J.C., Hong, H., Stewart, L.R. 2022. Coat protein expression strategy of maize rayado fino virus and evidence for requirement of CP1 for leafhopper transmission. Virology. 507:96-106.
Krugner, R., Rogers, E.E., Burbank, L.P., Wallis, C.M., Ledbetter, C.A. 2022. Insights regarding resistance of ‘Nemaguard’ rootstock to the bacterium Xylella fastidiosa. Plant Disease. 106(8):2074-2081.
Collum, T.D., Stone, A.L., Sherman, D.J., Damsteegt, V.D., Schneider, W.L., Rogers, E.E. 2022. Viral reservoir capacity of wild Prunus alternative hosts of plum pox virus through multiple cycles of transmission and dormancy. Plant Disease. 106:101-106. 10.1094/PDIS-04-21-0802-RE.
Stewart, L.R., Willman, M.R., Marty, D., Cole, A., Willie, K.J. 2022. Critical residues for proteolysis activity of maize chlorotic dwarf virus (MCDV) 3C-like protease and comparison of activity of orthologous waikavirus proteases. Virology. 567:57-64.
Todd, J.C., Stewart, L.R., Redinbaugh, M.G., Wilson, J.R. 2022. Soybean aphid (Hemiptera: Aphididae) feeding behavior is largely unchanged by soybean mosaic virus but significantly altered by the beetle-transmitted bean pod mottle virus. Journal of Economic Entomology.
Fletcher, J., Davis, J., Luster, D.G., Murch, R. 2022. Tactical applications of microbial forensics in agricultural biosecurity. IGI Global. 323-361.
Xu, J., Willman, M., Todd, J., Kim, K., Redinbaugh, M.G., Stewart, L.R. 2021. Transcriptome of the maize leafhopper (Dalbulus maidis) and its transcriptional response to maize rayado fino virus (MRFV), which it transmits in a persistent, propagative manner. Microbiology Spectrum. 9:e0061221.