Location: Foreign Disease-Weed Science Research
2023 Annual Report
Objectives
Objective 1: Develop genomic sequence resources and broad range nucleic acid and antibody-based diagnostics for novel emerging and invasive vectored plant pathogens. (NP303, C1, PS1A, PS1B)
Sub-objective 1.A: Development of rapid diagnostics for bacterial blight of grapevine caused by Xylophilus ampelinus.
Sub-objective 1.B: Identify and characterize emergent and invasive plant viruses and bacteria with potential for agronomic damage, and develop genome sequence resources.
Sub-objective 1.C: Develop immunodiagnostic reagents and field-deployable diagnostic assays for specific and sensitive detection of exotic, emergent, and quarantine plant viruses.
Sub-objective 1.D: Collect and characterize foreign and emerging bacterial plant pathogens.
Objective 2: Investigate the relationship of pathogen biology and ecology to disease expression of novel emerging and invasive vectored plant pathogens. (NP303, C2, PS2A, PS2B)
Sub-objective 2.A: Investigate virus determinants of resistance-breaking.
Sub-objective 2.B: Analyze virus protein expression and function in pathogenicity in single and mixed infections.
Sub-objective 2.C: Identify proteins and genes involved in virulence during X. fastidiosa subsp. pauca infection of citrus.
Sub-objective 2.D: Test susceptibility of New World Vitis species and hybrids to bacterial blight of grapevine caused by Xylophilus ampelinus.
Sub-objective 2.E: Genetic determinants of toxin production in Rathayibacter toxicus.
Objective 3: Understand the role of the vector in pathogen retention and transmission of viruses and bacteria. (NP303, C2, PS2B, PS2D)
Sub-objective 3.A: Develop sequence data resources for selected insect vectors and utilize data for refined taxonomic identification assays.
Sub-objective 3.B: Evaluate virus transmission determinants and test candidate molecules for transmission disruption.
Approach
Using specialized containment facilities, we will research key questions to assist in detecting and managing emerging, foreign, and invasive viral and bacterial diseases which threaten U.S. agriculture. Our research aims are to 1) develop sequence data, collections, and needed diagnostic tools for new pathogens; 2) elucidate basic molecular biology of virulence, toxicity, and pathogenicity of target pathogens; and 3) develop tools to characterize important vector populations and methods to block virus transmission. Target diseases include: foreign bacterial blight of grapevine, caused by Xylophilus ampelinus; citrus variegated chlorosis, caused by Xyllella fastidiosa; foreign and emergent maize lethal necrosis (MLN), caused by mixed infections of potyviruses in combination with the rapidly emerging maize chlorotic mottle virus (MCMV) driven by its corn thrips (Frankliniella williamsi) vector; U.S.-emergent cotton leafroll dwarf disease, caused by cotton leafroll dwarf virus (CLRDV); foreign disease caused by maize yellow mosaic virus (MaYMV/MYDV-RMV2); foreign rice tungro disease and other waikavirus-caused diseases; and foreign annual ryegrass toxicity (ARGT) livestock poisoning caused by the Select Agent Rathayibacter toxicus. Our research plan also includes the flexibility to respond to new pathogens that may emerge. Research will result in the development of sequence data, diagnostic tools, pathogen collections, and basic biological information about pathogenicity, toxicity and resistance-breaking, as well as tools to assess corn thrips populations and block spread of cotton leafroll dwarf disease.
Progress Report
The goals of Objective 1 are to develop sequence resources and diagnostics for novel emerging and invasive vectored plant pathogens. Under Subobjective 1A, the DNA of multiple strains of Xylophilus ampilinus was sequenced in preparation for diagnostic development. Under Subobjective 1B, pathogen sequencing and characterization, RNA extracted from groundnut samples with unusual viral symptoms was sequenced at a high depth of coverage to identify the virus causing the symptoms. Progress on Subobjective 1C, generating purified viral and protein antigens for antibody generation for the low titer poleroviruses maize yellow mosaic virus and cotton leafroll dwarf virus was attempted, but could not be completed until repairs are completed to the greenhouse building where this work will be performed. Maize yellow mosaic virus stocks were compromised by an ultralow temperature freezer failure brought on by an electrical power outage; the stocks were recovered after several months of work. Attempts to recover other viral isolates impacted by the freezer failure is ongoing with partial success. For Subobjective 1D, strains from the ICPB were not added to the U.S. Culture Collection website due to a temporary support staff vacancy.
Objective 2 focuses on the relationship between pathogen biology and disease expression. For Subobjective 2A, virus chimeras were not constructed because the greenhouse building where research will be performed requires repairs before the work can be completed. Containment SOPs, APHIS permits, and workflows were developed for cotton leaf roll dwarf virus (CLRDV), but work cannot proceed until the greenhouse building where research will be performed is repaired. For Subobjective 2B, transgenic maize was not screened for HCPro expression, as the greenhouse building where the research will be performed requires repairs before the work can be completed. Maize chlorotic dwarf virus (MCDV) ORF mutants were designed and constructed.
For subobjective 2C, X. fastidiosa has been grown on rich and minimal media and extractions of RNA and proteins are in process. Grape hybrids and native species have been obtained for Subobjective 2D. Under Subobjective 2E, significant progress has been made on the construction of homologous recombination knockout constructs for R. toxicus.
In research related to Objective 2, experiments to determine whether cacao mild mosaic virus (CaMMV) is seed transmissible have begun. Since its detection in 2021, CaMMV has resulted in direct, significant economic losses in the United States. Two hundred seeds from CaMMV-infected trees in Hawaii were planted at FDWSRU and leaves were sampled 1, 4, and 6 months after planting. DNA extractions from leaf samples are underway prior to PCR testing for CaMMV.
Objective 3 examines the role of vectors in pathogen retention and transmission of viruses and bacteria. For Subobjective 3A, thrips samples from Hawaii were obtained and testing begun. However, no results were completed within the sample degradation window due to a temporary support staff vacancy. Cotton aphid colonies and a CLRDV isolate have been acquired for work on Subobjective 3B. However, experiments are on hold because the greenhouse building where the research will be performed requires repairs before the work can be completed.
Accomplishments
1. First infectious clones of waikaviruses using maize chlorotic dwarf virus as a model system. Plant viruses in the genus Waikavirus cause severe economic losses to many staple human food crops, including corn, rice, and sorghum. Little is known about how waikaviruses cause disease in plants, or the mechanisms that allow them to be transmitted from one plant to another by insects. Therefore, ARS researchers at Frederick, Maryland, and Wooster, Ohio, together with Ohio State University collaborators, developed the first waikavirus infectious clones using the maize chlorotic dwarf virus. Infectious clones are molecular tools used to study how virus genes control infection, disease, and transmission. Using these tools, researchers produced infections of mild and severe versions of the maize chlorotic dwarf virus in plants and were able to track the infections using a green fluorescent protein. The research showed that a previously uncharacterized gene called P27 is essential for leafhoppers to transmit the virus to new plants. However, the P27 gene does not impact movement within infected plants, and it does not cause disease symptoms. These results are a groundbreaking step forward for fundamental waikavirus research, opening up new avenues of research for this virus group for which such studies were previously impossible.
Review Publications
Cardwell, K.F., Harmon, C.L., Luster, D.G., Stack, J.P., Hyten, A.M., Sharma, P., Nakhla, M.K. 2023. The need and a vision for a diagnostic assay validation network. PhytoFrontiers. 3:9-17. https://doi.org/10.1094/PHYTOFR-05-22-0056-FI.
Stewart, L.R., Willie, K.J., Xie, W., Todd, J.C., Tran, H. 2023. First waikavirus infectious clones and vascular expression of green fluorescent protein from maize chlorotic dwarf virus (MCDV). Frontiers in Virology. https://doi.org/10.3389/fviro.2023.1216285.
