Location: Foreign Disease-Weed Science Research2016 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.
The goals of Objective 1 are to develop diagnostics and understand the mechanisms of toxin production for the bacteria Rathayibacter toxicus, utilizing genome sequences for R. toxicus and the associated phage. The genes that are likely responsible for toxin production were identified, and a PCR assay was developed that can detect any Rathayibacter species capable of toxin production as well as polyclonal and monoclonal antibodies for the specific detection of R. toxicus. The expression of genes responsible for toxin production was assessed by quantitative RT-PCR and next generation sequencing, and it was determined that these genes are expressed in all culturing conditions. The goals of Objective 2 are to develop tools for the rapid diagnostic analysis of next generation sequence data. E-probe diagnostic nucleic acid analysis (EDNA), the bioinformatics tool originally developed for the detection of plant viruses was transitioned to simultaneous detection of insect vectors and pathogens. EDNA was capable of detecting Ca. Liberibacter asiaticus (Huanglongbing or citrus greening disease) in Diaphorina citri (Asian citrus psyllid) and Soybean dwarf virus in Acythrosiphon pisum (pea aphid), and current work suggests that EDNA is capable of detecting vectors and pathogens out of complex insect trap backgrounds. The goal of Objective 3 is to understand the relationship between aphid presence and viral evolution. Parallel experimental lines with constant or periodic aphid presence were established for Soybean dwarf virus (SbDV) on soybeans. Passaging experiments were completed and nucleic acids were extracted for sequence analysis. SbDV populations were analyzed by sequencing, and results indicated that there were differences in SBDV populations adapting to peas with and without aphid presence, suggesting that vector presence plays a significant role. A parallel set of experiments was initiated with Plum pox virus.
1. Antibodies developed to select agent. 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. Because of the potential threat to U.S. agriculture and food supplies, accurate and rapid diagnostics that can distinguish R. toxicus from other Rathayibacter species that already exist in the United States are critical to food security and trade. ARS researchers in Frederick, Maryland have generated a highly specific polyclonal antibody that specifically detects R. toxicus, which will allow for the detection of the pathogen by ELISA. The peptide expressed to generate this antibody is now being used to generate monoclonal antibodies that will be transitioned to USDA-APHIS for rapid, on-location biosensor applications.
Blagden, T., Schneider, W.L., Melcher, U., Daniels, J., Fletcher, J. 2016. Adaptation and validation of E-probe diagnostic nucleic acid analysis for detection of Escherichia coli O157:H7 in metagenomic data of complex food matrices. Journal of Food Protection. 79(4) 574-581. DOI: 10.4315/0362-028X.JFP-15-440.