Location: Foreign Disease-Weed Science Research2017 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.
Rathayibacter toxicus is a select agent bacteria due to the fact that it generates a lethal toxin (tunicamycin) in seed heads of forage grasses. At the initiation of this project, it was not clear whether the production of tunicamycin was a function of the bacteria itself, or if tunicamycin production was reliant on the presence of a phage. To determine the genetics behind toxin production (Objective 1) both the bacterial and phage genomes were completely sequenced and annotated. A cluster of genes on the bacterial chromosome with similarity to an operon responsible for tunicamycin production in other bacteria. The presence of this cluster of genes, termed the Tunicamycin Gene Cluster (TGC), on the bacterial chromosome indicated that the bacteria did not necessarily need the presence of the phage for toxin production. Interestingly, the TGC was identified as a likely mobile element, and probably arose via horizontal transfer. Assays for genes in the TGC were developed for both diagnostic purposes and to assess TGC expression in toxin producing and non-producing cultures. Surprisingly, these TGC genes are transcribed in all growth formats, regardless of toxin production, indicating that toxin production may be controlled post-transcriptionally. 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. These antibodies were very thoroughly screened and the results, plus the antibodies, made available to USDA-APHIS. 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 pathogens in plants in simulated exercises, the work of this project was to transition the tool to real world samples and expand the scope to include vectors and insect traps. E-probes were developed for three important viral pathogens plus HLB, as well as, two aphid vectors and the Asian Citrus Psyllid. Generating e-probes for insect vectors required the development of a new e-probe selection method, utilizing mitochondrial genomes. DNA and RNA extraction procedures were optimized for metagenomics samples from insects and insect traps, and three difference platforms of NGS (Illumina, 454 and Minion) were used to generate the sample databases to be analyzed by EDNA. EDNA was fully capable of detecting all pathogens in both plant and insect backgrounds using both the Illumina and Minion platforms. The results from the 454 platform varied depending on pathogen titer, due to the limited number of reads. Overall, EDNA succeeded at levels needed for diagnostic purposes, and the tool was also adapted for the detection of all virus families from any metagenome as well as the detection of human pathogens on food. The technology was presented at workshops at USDA-APHIS Center for Plant Health Science and Technology (CPHST) and Oklahoma State University, and both partners are currently beta testing the web-based version of the software successfully. To examine the relationship between aphid presence and viral evolution (Objective 3), parallel experimental lines with constant or periodic aphid presence were established for Soybean dwarf virus (SbDV) on soybeans. The goal of this work was to assess the hypothesis that constant aphid presence would enable virus adaptation to new hosts by picking up and spreading rare adaptive mutants due to a bottleneck/founder effect both on the original plant and during passage to naïve plants. The passages were carried out on two hosts, soybean and pea, that differed from the original host, clover. Passaging experiments were completed two times on each host and nucleic acids were extracted for sequence analysis. SbDV populations were analyzed by sequencing, and the final analysis of SbDV populations adapting to peas and soybeans did not show any significant differences between the constant aphid presence passage line and the typical periodic aphid presence passage line. These data do not suggest that vector presence plays a significant role in the rate of viral adaptation to new hosts. Summary of important accomplishments during the life of the project (2012-2017): Rathayibacter toxicus is a USDA-APHIS select agent plant pathogen due to the bacteria’s ability to make 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 were useful for USDA-APHIS 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. Rathayibacter toxicus affects seed heads of forage grasses, where toxin accumulates and leads to the death of foraging livestock. Infected forage grasses show little or no symptoms, and because of the widespread and diverse nature of forage grass growth and seed production diagnostics for R. toxicus in field settings requires cheap and easily usable diagnostic tool. Utilizing genome resources, we identified numerous potential antigenic targets for R. toxicus, expressed peptides, and produced and screened antibodies to generate a highly specific immunoassay. These antibodies have been transferred to USDA-APHIS, and are currently being prepped for more advanced approaches to detection of this important pathogen. Regulators and scientists face difficult challenges in identifying pathogens in diverse backgrounds such as plants and insect vectors. Advances in DNA sequencing have made it possible to identify any and all living things in a biological sample (a technique called metagenomics), but traditional methods used to analyze the sequence data were time consuming and inaccurate. We developed a bioinformatics tool called E-probe Diagnostic Nucleic acid Assay (EDNA) that makes analysis of sequence data much faster and more accurate, and transitioned the tool to an easily usable web based interface. The resulting product is currently being used by USDA-APHIS, and has helped to facilitate quarantine procedures, even demonstrating that US exports were disease free in key trade situations. Emerging plant viruses are frequently existing plant viruses that have adapted to expand their host range to important agricultural hosts. Despite this, there is very little knowledge about the selection pressures and evolutionary forces behind host switching in plant viruses. We examined the relationship between vector presence and viral evolution by establishing parallel Soybean dwarf virus (SbDV) experimental lines with constant or periodic aphid presence on soybeans peas, where we observed that SbDV populations adapting to peas and soybeans did not show any significant differences between the constant aphid presence passage line and the typical periodic aphid presence passage line. These data do not suggest that vector presence plays a significant role in the rate of viral adaptation to new hosts, and this allows scientists and regulators to focus efforts at identifying potential emerging plant virus threats.
1. Development of diagnostic assay for pathogens. Regulators and scientists face difficult challenges in identifying pathogens in diverse backgrounds, such as plants and insect vectors. Advances in DNA sequencing have made it possible to identify any and all living things in a biological sample (a technique called metagenomics), but traditional methods used to analyze the sequence data were time consuming and inaccurate. ARS researchers at Fort Detrick, Maryland developed a bioinformatics tool called E-probe Diagnostic Nucleic acid Assay (EDNA) that makes analysis of sequence data much faster and more accurate, and transitioned the tool to an easily usable web based interface. The resulting product is currently being used by USDA-APHIS, and has helped to facilitate quarantine procedures, even demonstrating that U.S. exports were disease free in key trade situations.
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