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ARS Home Northeast Area Beltsville, Maryland (BARC) Beltsville Agricultural Research Center National Germplasm Resources Laboratory Research





- Li, Ruhui
- Abrahamian, Peter
- Kinard, Gary
- 2023
- Publications
- Plant Genetic Resources, Genomics and Genetic Improvement (301)
- Plant Diseases (303)

Research Project: Characterization and Detection of Viruses that Impact the Exchange and Curation of Plant Germplasm **Location:
                            National Germplasm Resources Laboratory** 2023 Annual Report **Objectives**
                                

Objective 1: Identify and characterize unknown and poorly described viruses and viral diseases of select clonally propagated crops

Sub-objective 1A: Identify unknown and poorly characterized plant viruses and viral diseases using  High-Throughput Sequencing (HTS) technology.
Sub-objective 1B: Validate High-Throughput Sequencing (HTS) discovery of plant viruses using  biological and molecular techniques.

Objective 2: Develop sensitive, reliable, and resource-efficient methods to detect new and emerging viruses of quarantine significance.

Sub-objective 2A: Develop and/or optimize High-Throughput Sequencing (HTS) methods to detect viruses of clonally propagated plant germplasm.
Sub-objective 2B: Develop non-HTS methods to detect viruses of clonally propagated plant germplasm. **Approach**
                                

Conduct laboratory and greenhouse research to characterize and detect viruses of clonally propagated plant germplasm.  The emphasis is on crops whose germplasm is subject to federal quarantine regulations and/or are curated by the U.S. National Plant Germplasm System. Conduct biological and molecular studies to characterize poorly described virus and virus-like pathogens of quarantine significance, or diseases of unknown etiology that may be associated with such causal agents. Develop or improve and transfer plant virus detection methods, especially high-throughput sequencing and PCR protocols that detect virus-specific nucleic acids. Use sequencing-based approaches to investigate the genetic diversity of plant viruses, thereby allowing continual improvement and validation of nucleic acid detection protocols. **Progress Report**
                                

Forty garlic accessions with or without symptoms from the National Plant Germplasm System collection in Pullman, Washington, were sequenced by high-throughput sequencing (HTS) in 10 pooled samples. Sequence analysis revealed the presence of nine known viruses [Garlic virus A, Garlic virus B, Garlic virus C, Garlic virus D (GarV-D), Garlic virus E (GarV-E), Garlic virus X, leek yellow stripe virus (LYSV), onion yellow dwarf virus (OYDV), and Shallot virus X (ShVX)]. These viruses, especially GarV-D, GarV-E, LYSV and OYDV, were present as quasi species in all plants. The preliminary result suggests that most of the tested germplasm accessions were infected with multiple viruses. Forty pear accessions without obvious symptoms from the National Plant Germplasm System germplasm collection in Corvallis, Oregon, were sequenced by HTS. Sequence analysis only revealed the presence of apple stem pitting virus and a novel partitivirus in one of eight pooled samples, respectively. The preliminary result suggests that most of the tested germplasm accessions were not infected with any viruses. We also collaborated with the ARS U.S. Horticulture Laboratory (USHL) in Ft. Pierce, Florida, to detect by high-throughput sequencing nectarine virus M (NeVM), a stone fruit virus, in a grapefruit cv. Red Ruby. This was the first report of NeVM in citrus. Follow-up studies detected the virus in other citrus trees at the USHL. It was also noticed that NeVM infected trees seemed to have resistance or tolerance to Huanglongbing (HLB) infections. This offers the intriguing possibility, which requires further experimentation, that NeVM may offer cross-protection benefits against HLB infection and could be a disease management tool for devastating HLB disease. This early work has been documented with Invention Disclosure Docket 3723. The project’s progress has been severely limited by staffing vacancies for the past year; 4 of its 5.5 FTE are currently vacant. However, this situation should improve beginning in July 2023 when a new Research Plant Pathologist and a new technician are currently scheduled to join NGRL. **Accomplishments**
                                

1. A new virus was discovered infecting pear germplasm. Several viruses infect pear and may cause virus diseases that are difficult to control. Virus infections in curated germplasm accessions, especially those imported before quarantine regulations were applicable, are not well studied. We tested samples from eight pear accessions from the NPGS genebank in Corvallis, Oregon, with symptoms suggestive of virus infection. Bioinformatic analyses from high-throughput sequencing revealed the presence of a novel partitvirus. The nearly full length sequence of the virus genome was determined and a PCR-based detection protocol was developed. Determining the infection status of clonal germplasm accessions will allow better curation practices, including the possibility that therapy can be performed on them to minimize the risks associated with distributing infected material. 2. A new virus was discovered infecting Ribes germplasm. Approximately 20 viruses have been reported to date in Ribes (currants, gooseberries), some of which cause diseases that are difficult to control. Virus infections in curated germplasm accessions, especially those imported before quarantine regulations were applicable, are not well studied. We tested samples from ten pooled Ribes accessions from the NPGS genebank in Corvallis, Oregon, with symptoms suggestive of virus infection. Bioinformatic analyses from high-throughput sequencing revealed the presence of a novel cytorhabdovirus. The fully length sequence of the virus genome was determined and a PCR-based detection protocol was developed. A follow-up random sampling of additional Ribes accessions detected the new virus in 15 of 80 (19%) Ribes accessions. Determining the infection status of clonal germplasm accessions will allow better curation practices, including the possibility that therapy can be performed on them to minimize the risks associated with distributing infected material. **Review Publications**
                            *Lan, P., He, P., Yang, J., Cam, M., Zhou, G., Chen, X., Wei, T., Li, R., Li, F. 2022. High-throughput sequencing reveals the presence of novel and known viruses in diseased Paris yunnanensis. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2022.1045750.*


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