Characterizing and Detecting Pathogens to Ensure Safe Exchange of Plant Germplasm

Objective 1: Characterize unknown and poorly described pathogens and diseases which are priorities of the USDA-APHIS Plant Germplasm Quarantine Program. The emphasis is on viruses and viroids because they comprise most of the pathogens of quarantine significance and are the most difficult to detect and eliminate. ? Sub-objective 1A. Identify unknown and poorly characterized plant viruses using Next Generation Sequencing (NGS) technology. ? Sub-objective 1B. Validate Next Generation Sequencing (NGS) discovery of viruses using biological and/or molecular techniques. ? Sub-objective 1C. Characterize viral diseases of prohibited genus germplasm and production crops using biological and/or molecular techniques. The sub-objectives reflect the growing interest in NGS as a tool for routine use in service and diagnostic programs. NGS may, in some cases, eventually replace other techniques for etiology and characterization research. However, considerable efforts are required to optimize, compare, and validate such tools before they can be used with confidence. Regulatory and clean stock programs require, to the maximum extent possible, definitive conclusions about plant health based on the best scientific data available. Biological and molecular assays are still required to augment or confirm NGS results, perhaps more so than ever, because it is likely that NGS will reveal previously undetected viruses in clonally propagated crops. Objective 2: Develop sensitive, reliable and time efficient methods to detect viruses and virus-like pathogens of quarantine significance. ? Sub-objective 2A. Develop Next Generation Sequencing (NGS) methods to detect virus and virus?like pathogens of quarantine significance. ? Sub-objective 2B. Develop molecular (non?NGS) methods to detect virus and virus-like pathogens of quarantine significance. Sub-objective 2A parallels sub-objective 1A in advancing the use of NGS as a detection technique for known viruses by quarantine programs, in addition to its use for investigating disease etiology. However, many virus detection problems still require other (non-NGS) solutions, and confirmatory test methods for NGS results are advisable. Assays such as polymerase chain reaction (PCR) and enzyme linked immunosorbent assays (ELISA) still have widespread utility as routine detection techniques (sub-objective 2b).