Project : USDA ARS

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Research Project: Detection, Identification, and Characterization of New and Emerging Viral and Bacterial Diseases of Ornamental Plants

Location: Floral and Nursery Plants Research

2018 Annual Report

Objectives
The three objectives of this project are: (1) Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops, and develop corresponding diagnostic testing methods. [NP303, C1, PS1]; (2) Determine the genome organization of selected viruses of major significance to ornamental and nursery crops. Analyze full-length infectious clones to 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. [NP303, C2, PS2A]; and, (3) Characterize genomes of bacteria of major significance to ornamental and nursery crops to develop diagnostic tests for accurate pathogen detection. Identify and characterize genes and/or phages affecting virulence and competitiveness of those bacteria to develop effective control methods. [NP303, C1, PS1]. The long-term objective of this project is to develop effective means for the detection and identification of new and emerging plant viral and bacterial diseases of ornamentals, thus allowing growers to select pathogen-free or pathogen-indexed plants (tested for absence of specific pathogens) for propagation. Improved detection and differentiation methods for these pathogens will enable state and federal regulatory officials to make timely and appropriate recommendations in safeguarding the movement of horticultural and agricultural products into the United States. Understanding viral and bacterial genome structures and functions, their mechanisms of pathogenicity and resistance, and conferring virus and bacterial resistance in plants will lead to the development of better disease control measures and increases in both productivity and quality of ornamental plants for industry and the consumer.

Approach
The overall approach is to 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 those "new" currently uncharacterized or emerging viruses affecting key ornamental crops recently identified as significant to the floral and nursery industry. Based on the knowledge and tools developed while identifying and characterizing new viruses and comparisons to previously-characterized viruses, 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 will be developed. Validation of the recently devloped Universal Plant Virus Microarray (UPVM) will continue in order to transfer the UPVM technology to potential users. Next generation sequencing (NGS) of nucleic acid extracts from plants infected with unknown viruses is expected to yield information about the genomes of previously uncharacterized viruses without any background information on what viruses might be infecting the plant. Both NGS and UPVM have 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). Determine the genome organization of selected viruses of major significance to ornamental and nursery crops. Analyze full-length infectious clones to 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. We will make modifications to infectious clones of selected viruses by gene exchange and site-directed mutagenesis. We will examine interactions between viral gene products, and between viral and host proteins, using yeast two-hybrid, bimolecular fluorescence complementation, and GST-pulldown assays. VIGS and/or protein over-expression will also be utilized. Characterize genomes of bacteria of major significance to ornamental and nursery crops to develop diagnostic tests for accurate pathogen detection. The genomic DNA sequences of ornamental strains of Xylella fastidiosa (Xf) will be determined. The genetic diversity and phylogenetic relatedness among woody ornamental and non-ornamental strains will be evaluated. This sequence information will be used to develop specific PCR detection tools for woody ornamental strains of Xf. The identification and characterization of genes and regulatory elements, including phages, affecting virulence and/or competitiveness of Ralstonia solanacearum (including Race 3 Biovar 2) will be studied. This information will be used to further develop accurate detection tools and effective control methods.

Progress Report
Objective 1a: We have continued to examine samples of various ornamental species brought to our attention by plant disease clinics, nurseries, or individuals. These samples included a plant of Zizia aurea received from the Missouri Department of Agriculture, showing fine chlorotic spotting on the foliage; electron microscopy of embedded leaf material revealed the presence of paracrystalline arrays of small isometric virus particles, but not yet further identified; and, a nucleic acid extract of symptomatic leaf tissue has been processed for high throughput sequencing (HTS). Total RNAs from several gardenia plants from Deleware and Mar exhibiting chlorotic ringspot and line pattern symptoms were prepared for HTS. Onion yellow dwarf virus was detected in an ornamental Allium plant for the first time. No virus was detected in samples of beech trees from Ohio affected by decline, by either electron microscopy of leaf extracts or embedded tissue, or by HTS. In collaboration with scientists from the Systematic Entomology Laboratory and the Electron and Confocal Microscopy Unit at Beltsville, Maryland, we continue to examine the interactions between eriophyid mites and different rose species and genotypes. Material from additional rose varieties has been received from collaborators at several universities as part of a USDA-NIFA-SCRI project to examine the viral, vector, and host factors influencing the spread of Rose rosette virus and its ability to cause disease in roses. In collaboration with a visiting scientist from India, we initiated development of both serological and nucleic acid-based (RT-RPA) lateral flow assays for detection of Plantago asiatica mosaic virus (PlAMV). Specific detection of PlAMV was achieved by both methods, but further optimization of materials and extraction conditions is required to increase the sensitivity of the serological lateral flow assay. Improved rapid extraction methods are necessary for effective utilization of the RT-RPA lateral flow assay. In partnership with collaborators in Costa Rica, Plantago asiatica mosaic virus was detected in imported lilies grown in Costa Rica, the first time the presence of PlAMV was confirmed in that country. Objective 1b: We have continued to utilize existing broad-spectrum PCR primers for analysis of a wide range of suspected virus-infected plants, allowing the detection of previously reported viruses in new hosts, or obtaining products for sequence analysis of newly-discovered viruses allowing their comparison to previously characterized viruses. Using these generic primers, amplified products were obtained and sequenced from a variety of ornamental plant species, including narcissus (a potyvirus, a potexvirus, and a carlavirus from a single plant), veronica (the carlavirus HelVS), and lemon balm (the potexvirus TVX). In our ongoing research on the development of broad-spectrum reacting antibodies to important plant virus species, rabbit polyclonal antibodies (PcAbs) have been generated to synthetic peptide immunogens representing highly conserved carlavirus antigenic sites on viral coat proteins. Hybridoma cell lines secreting mouse monoclonal antibodies (McAbs) reactive to key epitopes are being developed. We will screen the PcAb and McAbs against the immunogen peptides, plus virions and bacterially-expressed cloned CPs from our current collection of more than 20 diverse carlaviruses. Synthetic peptides representing highly conserved potexvirus antigenic coat protein sites have also been used as immunogen for rabbit PcAb production. These will be evaluated for their broad-spectrum reactivity to purified virions and expressed cloned CPs from our current collection of about 20 diverse potexviruses. In ongoing collaborative research (a USDA-NIFA-SCRI Project) to develop efficient serological diagnostic tools to enable the rapid, user-friendly and accurate detection of Rose rosette virus (RRV), our previously developed RRV NP-specific rabbit PcAbs and ten selected McAbs were shown to detect RRV antigen in Western-blots and several ELISA formats. Twelve synthetic peptides representing predicted antigenic sites on the NP were used to define the epitope-specificity of the PcAb and McAbs. These tests showed that using the PcAb as an ELISA-trapping antibody and an admixture of two McAbs as the detecting antibodies would predictably be the best format for the detection of RRV in plants. This is currently being tested with healthy and RRV-infected rose samples from across the US. Objective 1d: The full genome sequence of a HelVS isolate from a cultivar of Veronica was determined by Illumina MiSeq high throughput sequencing (HTS); this represents the first full genome sequence of this carlavirus, which is known to infect several species of ornamentals, including Helenium and Impatiens, as well as Veronica. The full genome sequence of an isolate of Tulip virus X was also obtained by HTS from a lemon balm plant showing mosaic symptoms. The partial genome sequence of an isolate of Apple mosaic virus was determined from a highly symptomatic sample of Magnolia tripetala. Total RNA from a Callicarpa americana beautyberry plant growing in Alabama and exhibiting mosaic symptoms was analyzed by HTS. Viral sequences were identified that had significant putative protein homologies with members of the genus Emaravirus. Complete or near-complete sequences were obtained for genomic RNAs 1-4. HTS and RT-PCR analysis of other Callicarpa samples is ongoing. To our knowledge, this is the first report of an emaravirus infecting Callicarpa. Objective 2a: In partnership with Korean collaborators, full length infectious clones of two isolates of the tobamovirus Youcai mosaic virus (YoMV) were produced and shown to induce symptoms of differing severity in Nicotiana benthamiana. Sequence comparisons and subsequent gene exchange between the mild and severe symptom variants demonstrated that the symptom differences were dependent on a single residue change in the coat protein gene. Full length infectious clones of three isolates of Zucchini yellow mosaic virus differing in symptom severity were also produced in partnership with Korean collaborators, with efficiency of infectivity, but not the symptom differences, found to depend on the length of the 3'-terminal poly(A) tail, with much greater infectivity when the length was increased from about 30 to 60 residues. Objective 2b: We demonstrated that specific sequences of the Lolium latent virus coat proteins, which are unique to this type of virus, target specific sequences of proteins associated with chloroplasts of the plant cell. It is this interaction that ultimately facilitates the transfer and spread of the virus. This work identifies a method by which the virus spreads through the host plant which virologists, pathologists and disease managers can target as the first step in developing countermeasures for mitigating the disease and thus reducing disease losses in agriculturally important plants. In partnership with Korean collaborators, interactions between the two coat proteins and the movement protein of Radish mosaic virus were examined, together with their subcellular localization. When expressed individually as fluorescent fusion proteins, and examined by confocal microscopy, the movement protein (MP) was localized at the cell periphery, whereas the large coat protein (LCP) subunit accumulated as punctate aggregates in the cytoplasm, and the small coat protein (SCP) subunit was distributed throughout the cytoplasm and at the cell periphery. Results from co-expression of free and fluorescently-labeled proteins showed that SCP can interact separately with both LCP and MP, and that MP determines the localization of combinations. In collaboration with a Japanese scientist, the localization patterns and RNA silencing suppression efficiency of variants of the TGB1 protein of a lily isolate of Plantago asiatica mosaic virus (PlAMV) were examined. As we have previously demonstrated with other potexvirus TGB1 proteins, certain amino acid substitutions affect the ability of the TGB1 protein to localize to the nucleus and nucleolus, and these variants also differ in their efficacy of RNA silencing suppression. Objective 3.1: We propagated and verified a tree strain of X. fastidiosa isolated from Virginia, and prepared genomic DNAs and sent the DNAs for sequencing. We compared these genomic sequences to other landscape tree strains of X. fastidiosa that we had sequenced before or identified in GenBank for conservation of unique genes and phylogenetic relationships. We also searched GenBank for potential functions of the 150 different R. solanacearum race 3 biovar 2-unique DNA regions identified previously and were able to cluster these into 32 regions in the genome of R. solanacearum as potential targets for mutagenesis. Objective 3.2: We isolated a novel Ralstonia phage from soil in Egypt and have designated it Ralstonia phage RsoP1EGY. This phage specifically infects only r3b2 phylotype IIB sequevar 1, and not non-r3b2 strains of R. solanacearum and has properties similar to other podovirus phages. The 41 kbp genome shares no significant sequence identity to any other reported R. solanacearum or non-Ralstonia phages and is the first sequenced and characterized R. solanacearum phage isolated in Egypt. It has potential as a biocontrol agent for r3b2 strains of R. solanacearum in all countries where potato brown rot is a problem. We determined the effect of another phage, Rs551, on its carrier R. solanacearum strain UW551 by deleting 60% of the phage’s prophage region from the UW551 genome. The deletion resulted in significantly increased virulence as compared to the wild type UW551. Our results suggest that phage Rs551 may play an important ecological role by regulating the virulence of and offering a competitive fitness advantage to its carrier bacterial strain for persistence of the bacterium in the environment.

Accomplishments
1. A datasheet on Plantago asiatica mosaic virus added to Invasive Species Compendium. Plantago asiatica mosaic virus is a rapidly emerging pathogen first discovered in the mid 1970s in the Russian Far East that is affecting multiple crops throughout the world, including wild-growing and cultivated lilies. In lilies, the virus is thought to be spread through the international bulb trade, although the original source of infection is not known. An ARS scientist in Beltsville, Maryland, published a datasheet that presents a summary of what is known of the basic biology of the virus, including both natural and experimental host range, transmission in the absence of any known biotic vector, and methods which may be useful to control infection. This information will be useful to quarantine officials, producers, and consumers to prevent the spread to new crops and countries where the virus is not yet established, and to reduce disease losses in agriculturally and horticulturally important crops.

2. Discovery of novel pelarspoviruses infecting jasmine from Hawaii, DC, Maryland, and California. A myriad of foliar symptoms, including ringspots, line patterns, mosaic, mottling, and leaf deformation were observed on species of jasmine plants growing in Hawaii and at the U.S. National Arboretum in Washington, DC. ARS scientists in Beltsville, Maryland, discovered two distinct pelarspovirus species (called JaVH and JMaV) infecting these plants, and developed an assay to detect and differentiate these virus species in jasmine plants from other locations. These assays revealed that both virus species were found in samples tested from Hawaii, DC, and Maryland, but that only one species (JMaV) was detected in a symptomatic sample from California. This information will increase our understanding of the genetic diversity of these two new viruses and will be useful to ornamental nurseries, public and private plant disease diagnostic clinics, and other scientists who study ornamental viruses.

3. Prophage from select agent Ralstonia solanacearum affects virulence of this bacteria. R. solanacearum is a bacterial species that causes millions of dollars of crop losses in a wide range of plant species worldwide. One strain in particular, the r3b2 subgroup, is such a threat to U.S. agriculture that it has been designated a select agent and is subject to strict quarantine regulations. ARS scientists in Beltsville, Maryland, characterized a filamentous lysogenic bacteriophage (named Rs551) that is stably maintained as a prophage in the genome of a r3b2 strain. Infection of the Rs551 phage into a phage-free strain of R. solanacearum significantly reduced the pathogen’s virulence, while deletion of the phage genome from a naturally-infected r3b2 strain resulted in significantly increased virulence compared to the wild type strain. The fact that this phage genome was also present in the genomes of 11 other r3b2 strains suggests an evolutionary and biological role as well as a possible target to manage and control this important pathogen.

U.S. DEPARTMENT OF AGRICULTURE

Floral and Nursery Plants Research: Washington, DC