Location: Floral and Nursery Plants Research
2022 Annual Report
Objectives
Objective 1: Identify and characterize new, emerging, and re-emerging viruses of major significance to ornamental and nursery crops, develop new technologies and specific reagents for their detection, and examine the virus-vector biology associated with selected key viral diseases. (NP303, C1, PS1A, PS1B; C2, PS2B, PS2D)
Sub-objective 1A: Identification, characterization, distribution, and detection of emerging or previously unknown viruses. [Non-hypothesis research].
Sub-objective 1B: Development of broad-spectrum and virus-specific reagents. [Non-hypothesis research]
Sub-objective 1C: Determine and characterize complete viral genome sequences and virus diversity using high-throughput sequencing. [Non-hypothesis research]
Objective 2: Determine and analyze the viral genome sequences and organization of selected high-impact ornamental and nursery crop plant-infecting viruses, including the utilization of new tools to evaluate the role of viral genes and gene products in transmission, pathogenicity, and disease development. (NP303, C2, PS2A)
Sub-objective 2A: Identify viral determinants of host specificity, pathogenicity, and vector transmissibility. [Hypothesis 1: Site-directed mutagenesis or exchange of genes or genome segments between infectious clones of virus isolates having different host ranges or symptom responses will allow attribution of host-specificity, symptom, or vector transmission determinants to specific gene/regions.]
Sub-objective 2B: Identify essential virus-host protein interactions as potential targets for disruption of viral infection [Non-hypothesis 2: Identification of crucial interactions between viral proteins and host proteins will allow determination of those interactions necessary for the establishment of infection, or for cell-to-cell and/or long-distance movement within the plant, and elucidate future potential targets for disrupting virus infection or systemic movement by gene-editing of appropriate host proteins.]
Sub-objective 2C: Identify the interactions between emaraviral particles and the eriophyid mite vector which confer transmission specificity. [Non-hypothesis research].
Objective 3: Characterize genomes and strains and bacteriophages of bacteria of major significance to ornamental and nursery crops for culture collection and develop accurate pathogen detection tools and potential effective control methods, including those for select agent strains of Ralstonia solanacearum (Rs).
Sub-objective 3.1a: Identify important genetic components contributing to cool-virulence of Rs strains and develop a rapid purification-free plant DNA extraction method for detection of R. solanacearum species complex (Rssc), including the select agent r3b2 IIB-1&2 strains of Rs. [Non-hypothesis]
Sub-objective 3.1b: Determine the role of bacteriophages isolated from Rssc-infested soil or directly from a tropical Rssc strain in virulence and competitive fitness of Rssc strains. [Non- hypothesis]
Sub-objective 3.2: Determine genomes, identify unique ORFs and develop highly specific diagnostic markers to detect and differentiate landscape tree strains from other Xf strains. [Non- hypothesis].
Approach
Approach 1. Develop knowledge, tools, and reagents to aid U.S. floricultural producers and diagnosticians to establish and apply effective virus testing protocols to improve clean stock production for vegetatively propagated annuals and perennials. Research will initially focus on "new" uncharacterized or emerging viruses affecting key ornamental crops recently identified as significant to the floral and nursery industry. Develop new virus-specific and broad-spectrum polyclonal and/or monoclonal antibody reagents, purification protocols, nucleic acid hybridization probes, PCR primers, isothermal amplification methods, and improved associated protocols. High-throughput sequencing (HTS) of nucleic acids from plants infected with unknown viruses will yield information about the genomes of previously uncharacterized viruses. HTS has the potential to identify any virus present and identify all components of mixed infections and is suited to application in situations where rapid results are important (in Quarantine operations and germplasm introduction). HTS will also be used to examine virus diversity of selected viruses, including rose rosette virus.
Approach 2. Determine the genome organization of selected viruses of major significance to ornamental and nursery crops. Determine the genes or gene products involved in replication, systemic movements, and pathogenicity to understand the role of viral pathogen genes in disease development and to identify new targets in the pathogen genome and tools for disease management. Develop and modify infectious clones of selected viruses by gene exchange and site-directed mutagenesis. Examine interactions between viral gene products, and between viral and host proteins, using yeast two-hybrid, bimolecular fluorescence complementation, and GST-pull down assays. Virus-Induced Gene Silencing (VIGS) and/or protein over-expression will also be utilized. Identify and examine mechanisms and specificity of the interactions between rose rosette virus particles and the eriophyid mite vector, in addition to determining the minimal complement of viral RNAs necessary for mite transmission.
Approach 3. Characterize genomes and strains and bacteriophages of bacteria of major significance to ornamental and nursery crops. Develop pathogen detection tools and effective control methods, including those for select agent strains of Ralstonia solanacearum (Rs). Identify genetic elements and bacteriophages contributing to cool-virulence and competitive fitness traits of Ralstonia solanacearum species complex (Rscc) strains for accurate detection and effective control of Rs. Determine the complete genomes of additional ornamental strains of Xylella fastidiosa (Xf) using HTS. Identify unique ORFs and develop highly specific diagnostic markers to detect and differentiate landscape tree strains from other Xf strains.
Progress Report
This report is for a new project 8020-22000-052-000D which began in March 2022, and continues research from 8020-22000-042-000D, "Detection, Identification, and Characterization of New and Emerging Viral and Bacterial Diseases of Ornamental Plants". As this new project has just begun, there is little significant progress to report in FY22. A few items are noted below. Please see the report for 8020-22000-042-000D for more information on overall FY22 progress.
Under Objective 1a: We examined samples of various ornamental species brought to our attention by plant disease clinics, nurseries, or individuals. These samples included a red buckeye (Aesculus pavia) showing a strong mosaic associated with an infestation by eriophyid mites, suggesting possible presence of an emaravirus.
Under Objective 1c: We previously reported the detection of a complex mixed infection of two distinct isolates of each of clover yellow mosaic virus (ClYMV) and white clover mosaic virus (WClMV), which were stably maintained through multiple passages in Nicotiana benthamiana and other bioassay hosts. Individual shoots of eight individual clover plants from the original site were tested by reverse transcription-polymerase chain assay (RT-PCR) with primers specific to both ClYMV and WClMV, and both viruses were detected in each of these individual plant samples.
Under Objective 2a: In collaboration with a scientist from Texas A&M University, genome segments of isolates of rose rosette virus differing in the length and/or amino acid sequence of the predicted encoded protein were identified for potential substitution into an infectious clone of rose rosette virus developed by the collaborator. The potential effects of the substituted genome segments on symptom severity, systemic movement, or viral titer will be examined.
Accomplishments
