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

2024 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
Under Sub-objective 1A: RNA was extracted from samples of various ornamental and landscape plants with symptoms typical of emaravirus or other virus infections. In several instances, eriophyid mites were associated with the symptomatic plants and were tentatively identified by a collaborating ARS mite expert, with some potentially representing previously uncharacterized species. Samples included Cephalanthus and Sambucus species from Missouri, Callicarpa from North Carolina, Celtis from North Carolina, Lindera from Maryland and Ohio, oleander from California, Magnolia from Oklahoma and Maryland, and lilac and aspen from Colorado. The emaraviruses Callicarpa mosaic-associated virus 2 and Lindera severe mosaic-associated virus were identified from North Carolina, and from both Maryland and Ohio, respectively. Mixed infections of elderberry carlavirus C, elderberry carlavirus D, and tobacco ringspot virus were detected in Sambucus samples from Missouri. Under Sub-objective 1A: In collaboration with colleagues at the RDA, National Institute of Horticultural and Herbal Science, Wanju, and at Chungnam National University, Daejeon, South Korea, tomato spotted wilt virus was identified affecting lobelia in South Korea for the first time, affecting about 80% of a commercial field. Under Sub-objective 1B: Reverse transcription polymerase chain reaction (RT-PCR) was used to analyze samples including freesia, clover, hydrangea, Crinum, and Spathiphyllum with broad spectrum PCR primers capable of detecting potyviruses, potexviruses or carlaviruses, with subsequent virus identification either by sequencing of the resulting PCR products, or selection of appropriate virus-specific primers to identify the particular viruses present in the samples analyzed. Under Sub-objective 1C: The complete genome sequence of a novel potyvirus, Crinum mosaic virus, was determined by high-throughput sequencing of total RNA from one of three symptomatic Crinum plants co-infected with another potyvirus and a carlavirus. The viral genome sequence was determined to be 9,873 nt and had a typical potyvirus genome organization. The putative 3188 amino acids large polyprotein precursor shared the highest, yet distinct, sequence identity (<58% amino acid) with other known bulb-infecting potyviruses. Phylogenetic analysis confirmed the designation of this virus as a novel Potyvirus, for which we propose the species name Potyvirus crinum for this new member of the Potyviridae. Under Sub-objective 1C: In collaboration with colleagues at the CNR Institute for Sustainable Plant Protection in Turin, Italy, high-throughput sequencing of RNA from freesia plants affected by necrotic leaf disease revealed the first full genome sequence of the ophiovirus, freesia sneak virus, including a fourth genomic RNA which had not been detected by prior methods. The protein encoded by the newly detected RNA4 has a low degree of sequence identity with the equivalent proteins of other ophioviruses known to have an RNA4, apart from a conserved region of 115 nucleotides and the encoded amino acids near the N-terminus of the 324 amino acid protein. Under Sub-objective 1C: In collaboration with ARS and APHIS colleagues at Beltsville and scientists at Agrosavia, Centro de Investigacíon La Libertad, Villavicencio, Colombia, multiple viruses were detected by high-throughput sequencing from a single plant of Hibiscus rosa-sinensis showing black spots on the foliage. The viruses identified included full genomes of four novel viruses (three carlaviruses and a potexvirus), and additional viruses of the genera Betacarmovirus, Cilevirus, Nepovirus, and Tobamovirus. A hibiscus soymovirus was also detected. Some of these viruses were also detected in additional hibiscus plants in other areas of Colombia. Under Sub-objective 2A: In collaboration with colleagues at Chungnam National University, Daejeon, South Korea, a single amino acid difference in the HC-Pro protein of two closely related isolates of turnip mosaic virus differentially affected both symptom severity and the efficiency of viral RNA silencing suppression activity in infected plants of radish and Nicotiana benthamiana. The single amino acid difference was found to occur within a six amino acid motif, which is fully conserved in all other isolates of turnip mosaic virus examined. Under Objective 3.1a: We modified the high-throughput virulence screen method we developed previously by dipping a toothpick into sterile water, then into a microcentrifuge tube containing N. glutinosa seeds to easily inoculate water agar in 1.5 microcentrifuge tubes with at least 5 seeds. Using this modified assay, we screened hundreds of R. solanacearum race 3 biovar 2 strain UW551 mutants at 20 or 28oC generated by random transposon mutagenesis for significantly reduced virulence. Mutants that displayed lost virulence at cool (20 oC) or warm (28oC) temperatures were selected for further testing at warm and cool temperatures, respectively.

Accomplishments
1. Discovery of lytic bacteriophages isolated from Egypt for biocontrol of potato soft rot caused by Pectobacterium carotovorum. Pectobacterium carotovorum is an economically important phytopathogen causing destructive bacterial soft rot disease in many ornamental plants and fruit and vegetable crops worldwide, including potatoes. Phage therapy is a promising and environmentally safe alternative to combat bacterial diseases either in the field or during storage. An ARS scientist in Beltsville, Maryland, in collaboration with scientists in Egypt, discovered and determined the genome sequences of two novel bacteriophages (designated PcaP1EGY and PcaP2EGY) isolated from a potato field in Egypt. These phages specifically target P. carotovorum strains, displaying visible lysis activity and significantly protecting both potato tubers and plants from soft rot disease. Their potential as biocontrol agents against P. carotovorum is promising, whether in the field or during post-harvest storage.

U.S. DEPARTMENT OF AGRICULTURE

Floral and Nursery Plants Research: Washington, DC