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

2020 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
Developing detection methods, determining the genetic diversity, and characterizing isolates of sugarcane mosaic virus from Florida and sorghum mosaic virus from Louisiana is ongoing. This is a collaborative project between the National Germplasm Resource Laboratory (NGRL), Centre de Cooperation International en Recherche Agronomique pour le Development (CIRAD) France, and the ARS location in Houma, Louisiana. Another ongoing sugarcane project is the development of sugarcane yellow leaf antibodies for serological detection. Work continues to establish and curate a rose virus collection with funding from the National Clean Plant Network. About 60 rose accessions have been received and tested for viruses to date. We have been working to characterize and determine the genetic diversity of apple luteovirus 1 and other viruses in apple orchards affected by rapid apple decline (RAD) in Pennsylvania. Healthy apple seedlings and rootstock plants were grafted with scions from RAD symptomatic trees, and plants are currently being maintained in a screenhouse and a field nursery for observation. This is a collaborative project with Pennsylvania State University and the Pennsylvania Department of Agriculture. Apple samples have been collected from the ARS apple repository in Geneva, New York, and they will be subjected to high-throughput sequencing for virome analysis. Camellia samples with disease symptoms have been received from a germplasm collection belonging to Barlett Tree Company and will tested for viruses by high-throughput sequencing at the request of this stakeholder.

Accomplishments
1. Two new viruses identified in roses. ARS scientists discovered new viruses, provisionally named Rose virus A (RVA) and Rose virus B (RVB), in rose plants and characterized their genomes. RVA was detected in roses from Maryland and RVB was found in material from California. The genome sequences of these viruses facilitated developing reliable detection methods that can be used to screen rose genetic material. The entire rose germplasm collection at the National Clean Plant Network-Roses in California was tested for RVA and RVB. Seven rose accessions were RVB positive. This information helps develop better viral control measures for the rose trade and production industries.

2. New virus infecting papaya was identified and fully characterized. The complete genomic sequence of a new virus from papaya was determined and used to develop primers for molecular detection methods. The new virus was found in five papaya growing areas in Ecuador as a mixed infection with a different virus, papaya ring spot virus. These findings will help understand the complex nature of viral diseases affecting papaya and facilitate improved management strategies. This project was a collaboration with an Ecuadorian scientist who obtained his PhD in the U.S. working in an ARS laboratory.

3. Genome sequences of Ramu stunt virus (RmSV) were completed. Ramu stunt disease, caused by the RmSV, is a devastating disease of sugarcane and a pathogen of a quarantine concern globally. Sequences of the full-length genomes of five RmSV isolates were determined. This information is useful to validate and improve existing diagnostic RT-PCR detection assays, as well as to better understand the diversity of this virus. This research will be beneficial to sugarcane quarantine, certification, and breeding programs.

4. Molecular characterization of four novel betaflexiviruses infecting camellias was completed. Virus-like symptoms have been reported from camellias for decades, a plant with diverse uses including ornamental, tea production, and as an ethnobotanical medicine. Many species of the virus family Betaflexiviridae infect woody plants and cause diseases. Four novel viruses belonging to three different genera of the family Betaflexiviridae were identified by high-throughput sequencing technology in camellias with ringspot symptoms. The viral complete genomic sequences were determined. Molecular tests were developed to detect these viruses in more camellia samples, and the results showed that three of these viruses were frequently detected in camellias. The three common viruses were only seed transmissible, which was revealed by testing seedlings grown from infected plants. These results increased the knowledge of viral camellia diseases. It will help devise disease management and clean stock propagation approaches for camellias, most notably for the ornamental and landscape industry.

5. Molecular characterization of a novel badnavirus infecting camellias. Badnavirus is a group of DNA containing viruses that have recently emerged as important plant pathogens in woody plants. A novel virus, camellia lemon glow virus, was identified by high-throughput sequencing from an ornamental camellia tree with disease symptoms. Its complete genomic sequence was determined. A molecular test was developed to detect the virus and 47 additional camellia trees were tested. The new virus was detected in 13 of them, indicating it may be commonly found in camellias. The genomic sequence information and detection protocol will facilitate the exclusion of the virus from germplasm collections and propagation materials, which benefits the ornamental plants industry.

6. Detection methods for sugarcane white streak virus (SWSV) and sugarcane striate virus (SStrV) were developed. The ARS sugarcane germplasm collection in Miami was screened for these two viruses, SWSV and SStrV. A total of 571 sugarcane plants were tested. All samples tested negative for SWSV, but SStrV was detected in 19 accessions. This research confirmed that SStrV is present in Florida and highlights the need to test for the virus in collections, research material, and commercial breeding lines. Distributing only tested material will minimize the spread of SStrV in curated collections and in U.S. commercial sugarcane production.

7. Identification and RT-PCR detection of the newly described citrus concave-gum associated virus (CCGaV) in RAD-affected orchards. Rapid apple decline (RAD) is a recently emerging problem of apple production in the northeastern U.S. An RNA virus that was first described from citrus was identified from RAD affected apple trees by high-throughput sequencing. A sensitive RT-PCR detection protocol for CCGaV in apples was developed. A total of 55 apple samples were tested by RT-PCR and CCGaV was detected in 34 of them (62%). The possible role of this virus in causing RAD is currently unclear; however, the finding that apple is a natural host for CCGaV is important. A technology transfer document describing this assay was transferred to the USDA Animal and Plant Health Inspection Service (APHIS) and the Pennsylvania Department of Agriculture for use in quarantine and certification programs.

U.S. DEPARTMENT OF AGRICULTURE

National Germplasm Resources Laboratory: Beltsville, MD