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Research Project: Characterizing and Detecting Pathogens to Ensure Safe Exchange of Plant Germplasm

Location: National Germplasm Resources Laboratory

2018 Annual Report

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).

Conduct laboratory and greenhouse research to develop and transfer new or improved methods to detect viruses in plant germplasm undergoing quarantine testing. The emphasis is on higly sensitive techniques to detect virus-specific nucleic acids, including high throughput sequencing. Conduct biological and molecular studies to characterize poorly described virus and virus-like pathogens of quarantine significane, or diseases of unknown etiology that may be associated with such causal agents. Use sequencing based appraoches to investigate the genetic diversity of quarantine viruses, therby allowing the continual refinement, improvement, and validation of nucleic acid detection protocols.

Progress Report
Characterizing and developing detection methods for a virus of sugarcane (sugarcane streak mosaic virus) is ongoing. This is a collaborative project between National Germplasm Resource Laboratory (NGRL), USDA Sugarcane Research Unit in Houma, Louisiana and USDA-Animal Plant and Health Inspection Service (APHIS). Another new virus of sugarcane is being investigated with collaborators from the University of Florida and the French Agricultural Research Centre for International Development (CIRAD). Research on a previously undescribed virus infecting a weed (Sorghum almum) that occurs frequently near sugarcane production fields is ongoing in collaboration with the University of Florida. The concern is that the weed may be a reservoir from which the virus spreads to sugarcane. A collaborative project with the University of Idaho on detection techniques for two viruses that infect potato (tobacco rattle virus, potato mop top virus) is ongoing. A previously undescribed virus of potato is being investigated in collaboration with the International Potato Center (CIP) in Peru.

1. Molecular characterization of blackcurrant-associated rhabdovirus (BaRV). Several viruses cause diseases in the genus Ribes. Symptoms suggestive of rhabdovirus (a bullet-shaped RNA virus) infections in blackcurrants were reported more than 20 years ago, but the virus was never characterized. The virus was not included in the USDA plant germplasm quarantine testing protocols because a positive control was not available. A rhabdovirus was identified from a quarantined blackcurrant by a high-throughput sequencing (HTS) technology. Its complete genomic sequence was determined and a molecular detection test was developed. Additional testing of quarantined samples also detected the new rhabdovirus in material from France. This technology will help expand testing for this virus and exclude it from germplasm entering the U.S.

2. Molecular characterization of blackcurrant closterovirus 1 (BCCV-1). A new closterovirus (a filamentous-shaped RNA virus) infecting blackcurrants was identified from a plant that was used as a positive control (for a different virus) in the USDA quarantine program. Its complete genomic sequence was determined and a molecular detection test was developed. Additional testing of quarantined samples also detected the new virus in nine additional plants. This technology will be useful to expand testing for this virus and exclude it from germplasm entering the U.S.

3. Molecular and serological characterization of a new tymovirus infecting Solanum quitoense. Naranjilla (Solanum quitoense) is a subtropical perennial plant whose fruit is used primarily for juices and jams in South America. A new virus, Naranjilla chlorotic mosaic virus (NarCMV), was identified by high throughput sequencing from a stunted plant with mosic symptoms. The complete genomic sequence was determined and both RNA and protein based molecular tests were developed for its detection. We also determined that the virus will infect more commercially significant solanaceous (nightshade family) crops such as eggplant and peppers, but not potato. This information will benefit quarantine and clean stock programs for solanaceous crops.

Review Publications
Wu, L., Liu, H., Postman, J.D., Li, R. 2018. Molecular characterization of a novel Nucleorhabdovirus from black currant identified by high-throughput sequencing. Archives of Virology.
Eicholtz, M.I., Grinstead, S.C., Wu, L., Kinard, G.R., Li, R. 2017. First report of Beet western yellows virus infecting Epiphyllum spp. Plant Disease.
Lan, P., Zhao, J., Zhou, Y., Li, Y., Shen, D., Liao, Q., Li, R., Li, F. 2017. Complete genome sequence of Paris mosaic necrosis virus, a distinct member of the genus Potyvirus. Archives of Virology.
Filloux, D., Fernandez, E., Comstock, J.C., Mollov, D.S., Roumagnac, P., Rott, P. 2018. Viral metagenomic-based screening of sugarcane from Florida reveals occurrence of six sugarcane-infecting viruses and high prevalence of Sugarcane yellow leaf virus. Plant Disease.
Green, K., Mollov, D.S., Tran, L., Alvarez-Quinto, R., Ochoa, J., Quito-Avila, D., Karasev, A. 2018. Characterization of a new tymovirus causing stunting and chlorotic mosaic in naranjilla (Solanum quitoense). Plant Disease. 102:911-918.
Yueyue, L., Tan, G., Lan, P., Zhang, A., Liu, Y., Li, R., Li, F. 2018. Detection of tobamoviruses by RT-PCR using a novel pair of degenerate primers. Journal of Virological Methods. 259:122-128.