Location: National Germplasm Resources Laboratory
2022 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
Twenty-eight apple accessions with or without virus-like symptoms from the ARS apple germplasm repository in Geneva, New York, were sequenced by high-throughput sequencing (HTS). Sequence analysis revealed the presence of three latent viruses [apple chlorotic leaf spot virus, apple stem grooving virus, and/or apple stem pitting virus (APSV)] in nine samples. These viruses, especially APSV, were present as quasi species in all plants. The preliminary result suggests that most of the tested germplasm accessions were free of viruses. Sixteen tart cherry accessions with virus-like symptoms from the ARS tart cherry germplasm repository in Geneva, New York, were sequenced by HTS. Sequence analysis revealed the presence of eight viruses [cherry green ring mottle virus, cherry necrotic rusty mottle virus, cherry virus A (CVA), cherry virus F, little cherry virus 1, prune dwarf virus, prunus necrotic ringspot virus (PNRSV) and/or prunus virus F] in nine samples. Among them, CVA was the most prevalent (16 positive samples) followed by PNRSV. Five rose plants with virus-like symptoms were sequenced by HTS. Sequence analysis revealed the presence of nine viruses [apple mosaic virus, blackberry chlorotic ringspot virus, PNRSV, rose cryptic virus 1 (RCV-1), rose spring dwarf-associated virus, rose rosette virus (RRV), rose virus A (RVA) and tobacco ringspot virus (TRSV)]. Among these viruses, RCV-1 and PNRSV were the most prevalent. Three of the viruses, RRV, RVA and TRSV, are new additions to our rose virus collection. Apple seedlings grafted with dormant budwood from three ARWV-infected apple accessions showed typical symptoms of drooping twigs (apple rubbery wood, ARW) and swollen trunks (apple flat limb, AFL). One of the accessions used as graft inoculum was also infected with ACLSV and ASPV, one with ASGV, and one was a single infection of ARWV. RT-PCR tests confirmed the presence of the original virus/viruses in the inoculated seedlings. This study fulfilled Koch’s postulate for ARWV, confirming that viruses are the causal agent of the ARW/AFL. ARWV alone appears to cause symptoms, although it cannot be excluded that the latent viruses may amplify them. This information is useful for the pome nursery, trade, and production industries. This annual report is also the final one for project 8042-22000-302-00D. This project delivered information, products, and solutions related to viruses of clonally propagated germplasm over the past five years. The crops on which new viruses were discovered and reported included blackcurrant, camellia, naranjilla (tropical fruit), potato, rose, spiderwort (herbaceous ornamental), and sweet cherry. Numerous viruses were thoroughly characterized with the most notable including blackcurrant-associated rhabdovirus, ramu stunt virus of sugarcane, citrus concave gum-associated virus from symptomatic Rapid Apple Decline (RAD) trees, maize stripe virus infecting sorghum, three cherry viruses from the ARS apple germplasm repository collection, and sugarcane umbra-like virus. Detection methods were developed and transferred for many of the viruses that were newly discovered or more thoroughly characterized by this project. One noteworthy example is sweet potato vein clearing virus, which has not been reported in the U.S. and thus is highly targeted for exclusion. The project verified that apple rubbery wood virus (ARWV) is the primary causal agent for the diseases called apple rubbery wood and apple flat limb. This project also contributed to investigations into the etiology of RAD that has affected orchards in the northeastern U.S. We discovered a new virus (apple luteovirus 1) in RAD symptomatic trees. Four other viruses were also detected in symptomatic orchards, including one (citrus concave gum-associated virus) that had not previously been reported in the northeastern U.S. RAD etiology research is ongoing among several collaborators and the disease may represent a complex problem caused by a combination of biotic and abiotic factors. In summary, this project has made significant contributions in identifying and detecting viruses of clonally propagated germplasm, which helps quarantine, clean stock, and curation programs reduce virus incidence in collections and subsequent inadvertent spread through germplasm distributions and exchanges. The ultimate beneficiaries are clonal crop growers and producers who have access to virus-tested material for planting. Future progress and accomplishments will be reported as project 8042-22000-324-000D.
Accomplishments
1. Two novel viruses of the genus Potyvirus were identified from Tradescantia spp. plants with mottle and mosaic symptoms. The diseased plants of this ornamental (commonly called spiderwort) were collected from a nursery in Pennsylvania experiencing a disease outbreak. The samples were analyzed by high throughput sequencing (HTS) as requested by the Pennsylvania Department of Agriculture (PDA). Sequence analysis revealed the presence of six viruses- three novel viruses and three known viruses. Genomic sequences of the two novel [Tradescantia mottle virus 1 (TMoV-1) and Tradescantia mottle virus 2 (TMoV-2)] and one known [Tradescantia mild mottle virus (TMMV)] potyviruses were determined. This information is useful to the ornamental industry and specifically helps develop better virus detection and control measures in production greenhouses.
