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

2022 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. Additional resources in the merged project will strengthen the research in the current Objective 1: Objective 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]

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
This is the final report for Project 8020-22000-042-000D, which ended in March 2022, and has been replaced by the new Project 8020-22000-052-000D, “Detection, Biology, and Genomics of New and Emerging Viral and Bacterial Diseases of Ornamental Plants”. Objective 1a: We examined diseased samples of various ornamental species brought to our attention by plant disease clinics, nurseries, or individuals. These samples included: a) bur oak (Quercus macrocarpa), having symptoms similar to those previously reported on common oak (Quercus robor) infected with an emaravirus in Germany, and currently under examination; b) two petunia samples showing a bright mosaic. Electron microscopy of the petunia samples revealed flexuous virions of c.560-570 nm consistent with a potexvirus, and further characterization is in progress; c) Passiflora samples infected with a potyvirus; and d) Baptisia presumed to be infected with a potyvirus. We previously reported detection and identification in Maryland of an emaravirus infecting spicebush (Lindera benzoin), and tentatively named Lindera severe mosaic virus (LSMV). Seed of spicebush collected in the fall of 2021 and germinated in a greenhouse yielded a small number of seedlings with symptoms associated with LSMV infection. No spicebush plants were known to occur in the vicinity of the greenhouse, suggesting the possibility of seed transmission of LSMV. The presumed eriophyid mite vector (Phyllocoptes linderifolius) and symptoms were readily detected on leaves of spring 2022 growth of spicebush in the area where the seeds were collected, and mites were also observed on the immature fruit by a collaborating ARS mite expert. Experiments are underway to test the ability of the mite to transmit LSMV to healthy spicebush seedlings. Objective 1d: The complete genomes (RNAs 1-7) of two distinct rose rosette virus (RRV) isolates from the District of Columbia and Delaware were determined by high-throughput sequencing of total RNAs from symptomatic tissues. The complete genomes of two other co-infecting viruses (blackberry chlorotic ringspot virus and rose spring dwarf-associated virus) in the District of Columbia source were also determined. In initial collaborative RRV virus diversity studies with Texas A&M University colleagues, these RRV sequences were compared with 95 other RRV genomes deposited in the NCBI virus database (of which only three other isolates have their complete genomes determined). FY18-FY22: Substantial results were realized over the five years of the Project in each objective, which are summarized here. Objective 1: Determined the full genome sequences of three viruses co-infecting Crinum, Nerine latent carlavirus (and first report in the U.S.), Nerine yellow stripe potyvirus, and Crinum mosaic potyvirus (first report). Detection and full genome sequencing of two novel pelarspoviruses infecting star jasmine (Jasminum multiflorum) and angelwing jasmine (J. nitidum) plants from Hawaii, District of Columbia, Maryland, and California; and a third novel pelarspovirus in ornamental Clematis. Determined and first reported complete genome sequence of carnation latent virus, the type member of the genus Carlavirus. Detected and determined the near-complete sequences of isolates of apple mosaic virus and a novel carlavirus from plants of Magnolia tripetala. Determined the full sequence of an isolate of tulip virus X from Melissa officinalis. Developed virus-specific rabbit polyclonal and mouse monoclonal antibodies that can detect rose rosette virus, the rose rosette disease pathogen, in Western-blots and several ELISA formats. Determined the complete genomes (with 4 to 7 genome segments) of several new or emerging emaraviruses infecting landscape shrubs and trees, including beautyberry (Callicarpa americana), spicebush (Lindera benzoin), and rose (Rosa spp.). Determined the first full genome sequence of Helenium virus S from Veronica found in a mixed infection with two distinct isolates of Butterbur mosaic virus, one of which has a major deletion in an essential gene. Determined the near-complete genome sequence of two distinct isolates each of clover yellow mosaic virus and of white clover mosaic virus from co-infection in plants of while clover. Determined the near complete genomes of a novel allexivirus and an isolate of tobacco streak virus from a co-infected plant of Liriope muscari. Developed a highly specific and sensitive serological assay for the detection of Plantago asiatica mosaic virus in ornamental lily, which was applied for screening of multiple Asiatic, Oriental, and Tiger lilies. Demonstrated common occurrence of two non-vector eriophyid mites on roses and developed a high-resolution 3-D model of Phyllocoptes fructiphilus, the rosebud mite vector of rose rosette virus. Objective 2: Demonstrated that the interaction of Lolium latent virus major coat protein with a host ankyrin repeat protein NbANKr redirects it to chloroplasts and modulates virus infection. Determined viral sequence variations among 17 infectious clones of Turnip mosaic virus showing differential pathogenicity and infectivity in Nicotiana benthamiana, turnip, and Chinese cabbage. Developed chimeric infectious clones that affect symptoms and pathogenicity of Turnip mosaic virus; two determinants were shown to be required to induce stem necrosis in Nicotiana benthamiana, and to break resistance in Chinese cabbage. Determined the first sequences of Plantain virus X isolates and demonstrated that it is synonymous with the later-described Actinidia virus X and has a broader host range and geographic distribution than previously known. Demonstrated interactions between the dual coat proteins and the movement protein of Radish mosaic virus and their subcellular localization as free proteins and interacting aggregates. Demonstrated differential subcellular localization and RNA silencing efficiency of variants of the TGB1 protein of lily isolates of Plantago asiatica mosaic virus. Demonstrated that a single nucleotide change in the overlapping movement and coat protein genes of Youcai mosaic virus altering one amino acid residue in the coat protein caused milder symptoms. Objective 3: Developed and validated TaqMan-based real-time PCR assays for detection and differentiation of Ralstonia solanacearum select agent strains. Developed detection methods of select agent pathogen R. solanacearum race 3 biovar 2 with portable POCKIT™ and BLItz® systems. Identified a DNA region associated with cool virulence of R. solanacearum strain UW551 and developed the first PCR assay utilizing this region for specific detection of the select agent race 3 biovar 2 strains of R. solanacearum. Determined the molecular and biological characteristics of Rs551, a new filamentous bacteriophage isolated from a select agent race 3 biovar 2 strain of Ralstonia solanacearum. Discovered that prophage Rs551 and its repressor gene orf14 reduce virulence and increase completive fitness of its lysogenic select agent carrier strain UW551 of Ralstonia solanacearum. Discovered and determined the biological and molecular characteristics of four lytic Ralstonia phages isolated from Egypt, Indonesia, and the U.S. for their potential as biocontrol agents against Ralstonia solanacearum species complex strains. Identified and characterized a global LuxR-type transcriptional regulator, AclR, in Acidovorax citrulli, a seedborne bacterial pathogen responsible for bacterial fruit blotch. Discoved and determined the suggested significant role of the type III effector RipS1 in the cool virulence trait of the select agent Ralstonia solanacearum race 3 biovar 2 strains and as a potential target for the development of cool virulence-specific diagnostic tools to differentiate the highly regulated cool-virulent strains from non-cool-virulent strains of R. solanacearum. Developed specific detection and identification of American mulberry-infecting and Italian olive-associated strains of X. fastidiosa by FTP-LMAM (Fluorescence of TaqMan Probe upon Dequenching- Loop-Mediated Isothermal Amplification).

Accomplishments
1. Discovery of a jumbo Ralstonia-infecting phage isolated from the U.S. with promising biocontrol potential. The economically important bacterial wilt disease caused by R. solanacearum is difficult to control; using Ralstonia-infecting phages is a promising re-emerging control strategy. An ARS scientist in Beltsville, Maryland, isolated and characterized a jumbo phage that is the largest Ralstonia-infecting phage sequenced and reported to date. The phage has a wide host range, infecting each of the three newly established Ralstonia species in R. solanacearum species complex: R. solanacearum, R. pseudosolanacearum, and R. syzygii, and significantly reduced the virulence of a strain of R. solanacearum in tomato plants. This suggests that the jumbo phage has the potential to be developed into an effective control against diseases caused by R. solanacearum species complex strains.

2. Discovery of the type III effector RipS1 in the cool virulence of the select agent Ralstonia solanacearum race 3 biovar 2 strains. R. solanacearum race 3 biovar 2 strains cause brown rot of potato at cool temperatures and are listed as select agents in the U.S. and highly regulated. To gain a better understanding of cool-virulence mechanisms, ARS scientists in Beltsville, Maryland, generated libraries of transposon mutants in the cool virulent R. solanacearum strain UW551 and screened 10,000 mutants, using our previously published seedling assay, for significantly reduced virulence at cool, but not warm, temperatures. One mutant with the transposon altered the expression of a gene (RipS1) that may serve as a potential target for the development of cool virulence-specific diagnostic tools to differentiate the highly regulated cool-virulent strains from non-cool-virulent strains of R. solanacearum to safeguard U.S. agriculture.

U.S. DEPARTMENT OF AGRICULTURE

Floral and Nursery Plants Research: Washington, DC