Location: National Germplasm Resources Laboratory
2021 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
Five pear accessions with stony pit symptoms and one asymptomatic accession were sequenced by high-throughput sequencing (HTS). Sequence analysis revealed apple stem pitting virus (ASPV) in all symptomatic trees, and citrus virus A in two samples. APSV was present as quasi-species in all plants. None of these viruses were found in the asymptomatic trees. The preliminary result suggests that stony pit disease might be associated with ASPV. More samples need to be analyzed to make a more definitive correlation between these viruses and diseases. A new rhabdovirus was identified from a blackcurrant accession with leaves displaying chlorotic and necrotic margins, along with early senescence. This virus is different from blackcurrant rhabdovirus 1 and analyses indicate the new virus revealed is only distantly related to lettuce yellow mottle virus, another plant rhabdovirus. We are currently sequencing the viral genome. These two fruit virus projects collaborate with the ARS National Clonal Germplasm Repository in Corvallis, OR. Five camellias with leaf ringspot or flower breaking symptoms were subject to HTS analysis, which detected three new betaflexiviruses. Camellia ringspot-associated virus 1 (CRSaV-1), CRSaV-2, and/CRSaV-3 were found in all samples. All three viruses were present as quasi-species, and genetic diversity among different variants of each of the viruses was high. These viruses may be ubiquitous, but only cause disease in some genotypes. Our analysis suggests resistant genes in some camellia varieties that suppress the viruses and result in asymptomatic infections.
Accomplishments
1. Viruses detected in ARS cherry collection. Three recently described viruses were detected in sour cherry accessions from the ARS sour cherry germplasm repository in Geneva, New York: cherry virus F (CVF), prunus virus F (PVF) and cherry virus T (CVT). This is the first report of any of these viruses in the germplasm collection, and the first report of CVT in the U.S. The complete genome sequence of this CVT isolate was determined. The genome sequences of the three viruses were used to develop reliable detection methods that can be used to screen cherry genetic material. An additional 100 sour cherry accessions from the genebank were tested for the three viruses. Both CVF and PVF were frequently detected, but CVT infections were rare (1% incidence). This information helps develop better viral control measures for the germplasm repository, along with others who breed, propagate, and use cherry germplasm.
2. PCR detection method developed for sweet potato vein clearing virus (SPVCV). SPVCV is an exotic virus that has been reported from Africa, South America, and the Pacific Islands. A sensitive PCR detection protocol was developed to prevent the introduction of the virus in sweet potato germplasm imported to the U.S. A technology transfer document describing this assay was transferred to the USDA Animal and Plant Health Inspection Service (APHIS) for use in their germplasm quarantine program.
3. A new rose virus was discovered. Rose virus r (RVR) was detected from a private rose collection (cultivar Hugh Dickson) in Maryland. Its genome was sequenced, which allowed us to determine RVR is a new plant rhabdovirus. The genome sequence of this virus facilitates developing reliable detection methods that can be used to screen rose genetic material. This information is useful for the rose trade and production industries.
4. The genome sequences of eleven maize stripe virus (MStV) isolates were determined. MStV causes severe disease of maize and sorghum in tropical and subtropical production areas worldwide. Our research provided the first complete reference genome of MStV, which is comprised of 4-5 separate segments of RNA. This study yielded a complete and updated genetic description of MStV and provided insight into potential mechanisms underpinning its diversity.
Review Publications
Diaz-Lara, A., Mollov, D.S., Golino, D., Al Rwahnih, M. 2020. Detection and characterization of a second carlavirus in Rosa sp. Archives of Virology. https://doi.org/10.1007/s00705-020-04864-4.
Al Rwahnih, M., Alabi, O., Hwang, M., Tian, T., Mollov, D.S., Golino, D. 2021. Discovery and genome characterization of a new Nepovirus infecting grapevine. Plant Disease. https://doi.org/10.1094/pdis-08-20-1831-re.
Tahir, M.N., Bolus, S.J., Grinstead, S.C., Mcfarlane, S., Mollov, D.S. 2021. New virus from the family Tombusviridae infecting sugarcane. Archives of Virology. https://doi.org/10.1007/s00705-020-04908-9.
Bolus, S.J., Al Rwanhnih, M., Grinstead, S.C., Mollov, D.S. 2021. Rose virus R, a cytorhabdovirus infecting rose. Archives of Virology. https://doi.org/10.1007/s00705-020-04927-6.
Gavrili, V., Lotos, L., Mollov, D.S., Grinstead, S.C., Tsialtas, I., Katis, N., Maliogka, V. 2021. First report of barley virus G infecting corn in Greece. Plant Disease. https://doi.org/10.1007/s42161-021-00903-4.
Medina-Salguero, A., Comejo-Franco, J., Grinstead, S.C., Mowery, J.D., Mollov, D.S., Quito-Avila, D. 2021. Characterization of a mild isolate of papaya ringspot virus type-P (PRSV-P) and assessment of its cross-protection potential. Plant Disease. https://doi.org/10.1371/journal.pone.0241652.
