Project : USDA ARS

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Research Project: Detection, Biology, and Genomics of New and Emerging Viral and Bacterial Diseases of Ornamental Plants

Location: Floral and Nursery Plants Research

2025 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
Sub-Objective 1A. The viral genus Emaravirus currently has 33 recognized species, and additional putative members are reported each year. Although eriophyid mite vectors (or putative vectors) have been identified for about two thirds of the species, several of the remaining viruses were identified via high throughput sequencing and lack any information on possible eriophyid mite vectors. We have previously identified three new emaraviruses and associated eriophyid mites and sequenced these viruses. We are currently examining several further putative emaraviruses, for each of which a collaborating ARS mite expert has identified associated eriophyid mites as the potential vectors. RNA extracts have been prepared from the presumed emaravirus-infected plants, and these will be subjected to high throughput sequencing. Samples of various ornamental and landscape plants with virus-like symptoms were received from collaborators, universities, or private diagnostic laboratories from over 10 different states and local collections, representing more than 25 different species. Eriophyid mites were observed to be associated with multiple symptomatic plants, and identifications to genus and species were made by a collaborating ARS mite expert at Beltsville, Maryland. RNA was extracted from symptomatic tissues for subsequent testing by RT-PCR and/or high throughput sequencing. Viruses previously known to infect the respective crop were identified in Rudbeckia, Hydrangea, and Prunus serotina; new viruses previously detected by us were identified in Lindera and Callicarpa, whereas viruses in other samples await more complete identification and characterization, with some thought to represent potentially undescribed emaravirus species. In collaboration with colleagues in South Korea, we identified both lettuce big-vein virus and Mirafiore lettuce big-vein virus in mixed infection in plants of chicory showing foliar necrotic spots and leaf dieback. These symptoms were observed in about 10% of the crop and are distinct from the typical symptoms observed in lettuce, the more common host of these viruses. Additionally, with 186 other plant pathologists, plant breeders, and germplasm collection managers, we reviewed named presumed virus or virus-like diseases associated with eight major vegetatively propagated crops (including citrus, currant, grape, strawberry, and members of the rose family) that have no known isolates or verifiable diagnostic tests. The resulting report recommends the removal of 120 of these “phantom agents” from regulatory lists to streamline germplasm exchange internationally. Sub-Objective 1C: Several ornamental Crinum plants growing at the U.S. National Arboretum in Washington, D.C., were identified as symptomatic and observed to have typical virus-like filamentous particles in leaf-dip preparations by electron microscopy. Total RNA was purified from the cultivar ‘Mo’Pon’ and custom complementary DNA (cDNA) libraries were prepared for high throughput sequencing protocols. Near-complete genome sequences were obtained for the carlavirus Nerine latent virus (NeLV) and two potyviruses, Crinum mosaic virus (CriMV) and Nerine yellow stripe virus (NYSV). The NeLV complete genome sequence (MG012804) was published and the CriMV sequence results have been submitted for publication. This is the first report of NYSV in Crinum in D.C. To confirm the presence of these three viruses in the original sample (and three additional symptomatic Crinum plants), NeLV-, CriMV-, and NeYSV-specific coat protein primers were designed and used in RT-PCR reactions. All four plants were positive with the NeYSV-specific primer set; two were NeLV positive, and three were CriMV positive. The host range of NeYSV appears to be confined to the Amaryllidaceae (amaryllis family). Since NeYSV (and NeLV and CriMV) can be transmitted by aphids and infected vegetative propagation material, commercial flower growers should continue to employ diagnostic and phytosanitary measures to ensure the health of their crops. Working with an international team of scientists in the Potyviridae Study Group (SG) of the International Committee on the Taxonomy of Viruses (ICTV), a new genus, Phragmivirus, was described and reported within the family Potyviridae. This proposal has been accepted by the ICTV. The genus Phragmivirus is represented by two virus species (Phragmivirus phragmii and Phragmivirus spartinae) for which complete genome sequences are available for both members, namely common reed chlorotic stripe virus (CRCSV), and Spartina mottle virus (SpMV), respectively. This updated taxonomy of Potyviridae will be useful to virologists and regulatory officials worldwide to ensure that viruses can be correctly identified, and their relationships to other viruses are understood. In collaboration with scientists in Italy, the genome sequences of viruses present in mixed infections of six Freesia plants showing necrotic symptoms were determined. These included three viruses previously known to be common in the crop (bean yellow mosaic virus, freesia mosaic virus, and freesia sneak virus), and two viruses identified as new members of recently described viral families (freesia konkovirus 1, family Konkoviridae; and freesia associated yue-like virus 1, family Yueviridae). In subsequent testing of 21 plants with varying degrees of necrotic symptoms and one apparently asymptomatic plant, freesia mosaic virus was detected in all the samples, including the asymptomatic plant, in which bean yellow mosaic virus and freesia associated yue-like virus 1 were also detected. Searching available datasets from multiple sources for sequences related to freesia konkovirus 1 and freesia associated yue-like virus 1 identified eight additional putative plant-infecting konkoviruses, and two putative new yue-like viruses from ferns and oomycetes. Freesia associated yue-like virus 1 is the only yue-like virus identified from an angiosperm, as the two previously recognized viruses were found in crustaceans. A co-authored manuscript has been submitted. Sub-Objective 3.1A: Using a modified high throughput seedling virulence assay that we previously developed, hundreds of Ralstonia solanacearum race 3 biovar 2 strain UW551 mutants (generated through random transposon mutagenesis) were screened at 20° C and 28° C for significantly reduced virulence. Several potential mutants showing attenuated virulence were selected for further confirmation in tomato plants at both temperatures. We then identified the transposon insertion sites in these mutants and determined the genes or genomic regions affected by the mutations. In collaboration with APHIS scientists in Laurel, Maryland, we also evaluated various liquid media for their effectiveness in increasing R. solanacearum populations. Specifically, we tested the media’s ability to enrich R. solanacearum spiked into healthy geranium tissue and to recover the pathogen from artificially inoculated asymptomatic and symptomatic geranium plants. These enrichment approaches were assessed as potential improvements over direct DNA extraction followed by molecular detection, with the goal of enhancing diagnostic sensibility. Sub-Objective 3.1B: Repeated attempts to delete a prophage from Ralstonia pseudosolanacearum strain GMI1000 were not successful. Therefore, efforts shifted toward isolating and characterizing Ralstonia-infecting bacteriophages from soil samples collected in Maryland. A jumbo phage was successfully isolated, and its genome was sequenced and published as a first step toward evaluating its potential as a biocontrol agent against R. solanacearum. In collaboration with scientists in Egypt, we also isolated a novel Pectobacterium brasiliense-infecting phage from Egypt. We sequenced and characterized its genome, determined its morphology and growth characteristics, and evaluated its thermal and pH stability. Additionally, we assessed its potential as a biocontrol agent against soft rot in vegetables under greenhouse conditions. A joint co-authored manuscript has been published. Sub-Objective 3.2: We isolated a Xylella fastidiosa strain from an oak tree in Maryland and assembled its draft genome sequence. By comparing this genome with other Xylella strains in GenBank, we identified unique regions specific to our isolate. Based on these regions, we designed strain-specific primers to be tested for their specificity. Such genome-based specific detection and identification of X. fastidiosa in landscape trees are essential for epidemiological studies, disease diagnosis, and a better understanding of bacterial leaf scorch of landscape trees.

Accomplishments
1. Excluding "phantom agents" from regulation to streamline global germplasm exchange. The diagnosis and control of plant diseases are complicated by what regulators refer to as “phantom agents”—putative pathogens identified in the literature based on symptoms, yet lacking supporting data such as molecular characterization or isolates to confirm their existence. Some of these virus or virus-like disease agents remain on regulatory lists, potentially creating false barriers in trade and plant certification. ARS scientists from Beltsville, Maryland, Riverside, California, and Parlier, California, in collaboration with APHIS and researchers from six other countries, conducted a comprehensive review of existing literature to summarize over 120 “phantom agents” reported across 10 genera of fruit crops. After soliciting feedback from hundreds of stakeholders, the authors of the resulting manuscript concluded that most of these "phantom agents" do not pose a threat to global agriculture. Consequently, they recommend the removal of such agents from regulatory lists to facilitate germplasm exchange and enhance global agricultural trade. Removal of “phantom agents” from quarantine lists will ease the burden of quarantine inspectors and benefit plant breeders and producers of fruits and ornamentals across multiple continents.

U.S. DEPARTMENT OF AGRICULTURE

Floral and Nursery Plants Research: Washington, DC