Project : USDA ARS

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

Location: National Germplasm Resources Laboratory

2019 Annual Report

Objectives
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).

Approach
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
The project continues to characterize and develop detection methods for viruses of sugarcane (e.g. sugarcane streak mosaic virus) including in weeds (e.g. maize stripe virus) commonly found near sugarcane production. This is a collaborative project between National Germplasm Resource Laboratory (NGRL), University of Florida and Sugar Research Australia. Another continuing sugarcane project is the production of detection antibodies for sugarcane yellow leaf virus. A potentially new virus infecting blackberries has been identified and research to characterize its genome and develop detection techniques is underway. Research also continues on apple luteovirus 1, which may be contributing to the rapid apple decline syndrome that is affecting apple orchards in the northeastern U.S.

Accomplishments
1. New virus discovered in potato. Potato tubers imported at the Port of Miami entry were sent to the Animal and Plant Health Inspection Service (APHIS) for testing. The standard protocol laboratory tests revealed no pathogens but bioassay plants inoculated in the greenhouse showed symptoms. ARS scientists in the National Germplasm Resource Laboratory (NGRL) were asked to assist with the identification using high throughput sequencing the previously uncharacterized torradovirus was discovered. The virus was concurrently identified by scientists at the International Potato Center (CIP) in Peru. ARS and CIP collaborated to complete the genomic sequence and design a diagnostic assay which is now implemented in the USDA-APHIS quarantine program. This research helped prevent the introduction of an exotic potato virus to the U.S. and devised a method to intercept the virus in future importations.

2. New viruses discovered in Prunus. Prunus is grown worldwide and is an important crop with many fruit types (e.g. peach, plum, cherry, nectarine, almond). Viruses frequently cause yield losses and quality reduction in these crops, and they are often spread inadvertently in nursery stock and/or rootstocks. We identified two new viruses in quarantined sweet cherry material from Europe, both belonging to the family Betaflexiviridae using high throughput sequencing. The complete genomic sequences of both viruses was determined. Molecular detection tests were also developed to facilitate exclusion of the viruses from the U.S. This research will be useful to stone fruit quarantine, clean stock, and certification programs.

3. New virus discovered in papaya. Papaya in an important tropical crop that is susceptible to many detrimental viral diseases. We were assisting a collaborator in Ecuador to study papaya ringspot virus epidemiology and performed high throughput sequencing on samples sent to NGRL. Analysis of the sequencing results revealed a new rhabdovirus. The genome was characterized and diagnostic assays were developed for this virus. This will help study the epidemiology of the disease and develop management strategies for it. This research will benefit scientists who work directly with papaya growers.

4. New virus discovered in naranjilla. Naranjilla is a subtropical perennial small tree with round edible fruit that resembles an orange. It is in the same plant family (Solanaceae) as tomato, potato, eggplant, and bell & chili peppers. ARS scientists in Beltsville, Maryland, used high throughput sequencing to identify a new tyomvirus from diseased naranjilla in Ecuador. The viral genome was fully sequenced. Although naranjilla is a minor regional crop in South and Central America, the concern is whether any new virus of solanaceous crops can infect the more economically important members of the family. We demonstrated that the virus can experimentally infect tamarillo, peppers and eggplants, but not potatoes. This research will be useful to design diagnostic assays and investigate the epidemiology of this new virus, it will also reduce concern for the potato industry.

5. Detection method developed for virus of blackcurrant. Blackcurrant (Ribes species) is a woody shrub grown for its berry fruit in temperate regions. It has traditionally been a minor crop, but interests in growing currants have recently increased based on reports that the berries are rich in nutrients and phytochemicals. Germplasm of the genus must be imported to the U.S. through quarantine programs. The plants are susceptible to several viruses, including one called blackcurrant closterovirus 1. We developed and validated a sensitive nucleic acid amplification (RT-PCR) detection technique for this virus. We used the assay to test 245 Ribes accessions from the USDA National Clonal Germplasm Repository in Oregon. The virus was detected in 45 accessions (18.4%) including blackcurrant, red currant and gooseberry. This research suggests that efforts to initiate therapy and maintain clean stock for Ribes collections are needed. The research will also help quarantine, clean stock and certification programs better test for the virus so that additional isolates can be excluded from material entering the U.S.

U.S. DEPARTMENT OF AGRICULTURE

National Germplasm Resources Laboratory: Beltsville, MD