Location: Floral and Nursery Plants Research
2020 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
Under Objective 1a: We examined diseased samples of various ornamental species brought to our attention by plant disease clinics, nurseries, or individuals. These samples include: i) a plant of Phlox caroliniana previously found to be infected with a potyvirus was further shown to also be infected with cucumber mosaic virus (CMV), confirmed by RT-PCR and sequencing; ii) plants of honeysuckle (Lonicera sempervirens and two named hybrids) with poor rooting success were examined. One hybrid was found to be infected with a probable carlavirus (slightly flexuous particles of 620-640 nm), for which further characterization is in progress; iii) foliage of Southern hackberry (Celtis laevigata) exhibiting decline after development of generalized mottle and chlorosis. Electron microscopy of sections revealed presence of many fine filaments, and multiple membranous abnormalities in the cytoplasm of affected leaves; iv) multiple samples of lilyturf (Liriope muscari) with chlorotic to white streaking on the foliage were obtained from different sources. Electron microscopy revealed flexuous particles of c.800 nm, and further characterization is on-going; v) plants of garlic mustard (Alliaria petiolata) with obvious mosaic were found to be infected with a presumed potyvirus, for which further characterization is in progress; vi) plants of closed bottle gentian (Gentiana andrewsii) showing necrotic spotting and some veinal necrosis were infected with a putative potyvirus, which is undergoing further characterization.
Under Objective 1d: We determined the full-length genome of Carnation latent virus (CLV), which is the type species for the Carlavirus genus. The complete CLV-KL sequence was determined to be 8,513 nt using high-throughput sequencing and was validated by Sanger sequencing of CLV-specific RT-PCR generated amplicons and 5' and 3' RACE protocols. In pairwise analysis, the genome shares 40-46% identity with other recognized carlaviruses and the six in silico translated proteins have 15-62% amino acid identities with their respective carlavirus orthologs. In phylogenetic analysis, CLV clusters with Butterbur mosaic virus, Coleus vein necrosis virus, and Garlic common latent virus. The CLV-KL coat protein shares 98% identity with the CLV-UK NCBI Reference Sequence.
In collaboration with Korean colleagues, Pyrus pyrifolia cryptic virus was identified infecting pear trees in Korea; no specific symptoms could be attributed to this virus, as the infected trees had mixed infections with up to four viruses commonly known to infect pear.
Work continued on historic and contemporary isolates of plantain virus X (PlVX) with collaborators in the United Kingdom (UK) and the Netherlands, and expanded to include additional colleagues in the Netherlands and Belgium. This expanded collaboration has resulted in determination of essentially full genome sequences from two historic PlVX isolates from Plantago lanceolata in the UK, one from P. lanceolata in the Netherlands, one from Capsicum annuum originating from Ethiopia, and one from Browallia americana in Belgium, plus multiple additional partial sequences from the UK, the Netherlands, and Belgium. In addition to the previous determination that plantain virus X is synonymous with actinidia virus X (AVX; reported in New Zealand from Actinidia chinensis imported from China, and in Canada from Ribes nigrum imported from an undisclosed source), it is now clear that PlVX/AVX has a natural host range including plants from at least four taxonomically diverse plant families (the Plantaginaceae, Solanaceae, Actinidiaceae, and Grossulariaceae), and has an apparent geographic range including the UK, the Netherlands, Belgium, Ethiopia, and China, with documented importations into New Zealand and Canada.
Under Objective 2a: In partnership with Korean collaborators, full-length infection clones of two additional Korean isolates of turnip mosaic virus (TuMV) were generated. Isolate TuMV-KBC belongs to the World-B phylogenetic group of isolates, and infects Chinese cabbage but not radish; in contrast, isolate TuMV-KRS belongs to the Basal-BR phylogenetic group, and infects radish but not Chinese cabbage. Chimeric infectious clones were prepared by exchanging an internal (~48%) portion of the genomes of isolates KBC and KRS in order to determine which viral genes control host range; additional chimeric constructs exchanging smaller fragments of the viral genome are currently in progress to more finely map the viral determinants of host range specificity.
In collaboration with Korean colleagues, the full sequence of a Korean isolate of radish mosaic virus (RaMV), only recently reported in Korea, was determined. Subsequently, infectious clones of the two genomic segments of this RaMV isolate were developed, and modified by insertion of a unique restriction enzyme site into the 3’-terminal untranslated region of the infectious clone of RNA 2, to allow use as a Virus-Induced Gene Silencing (VIGS) vector for cruciferous crops. A fragment of the phytoene desaturase gene was inserted to demonstrate the efficacy of the modified RaMV as a VIGS vector, resulting in the expected development of chlorotic bleaching of leaf tissue. This RaMV-derived VIGS vector will be further tested to aid in determining the function of host genes of radish and other field crops and ornamental species of cruciferous crops.
In collaboration with other ARS colleagues at the Beltsville location, the complete sequence of a Maryland isolate of pepino mosaic virus (PepMV) was determined, and shown to belong to the ‘European’ clade of PepMV isolates. Infectious clones were subsequently developed, and variants prepared to express the gene for the green fluorescent protein (GFP) as a marker to track systemic infection of inoculated plants.
Under Objective 3.1: In collaboration with scientists in Italy, a gene sequence that we previously identified that is uniquely present in Italian olive and American mulberry strains of Xylella fastidiosa was used to design primers and probe for the development of real-time detection methods for these Italian olive and American mulberry strains. These methods included TaqMan qPCR and LAMP assays, and a new technology, “Fluorescence of TaqMan Probe upon Dequenching -Loop-Mediated Isothermal Amplification” (FTP-LAMP) assay.
Under Objective 3.2a: We isolated a 'jumbo' R. solanacearum-infecting phage from soil in the United States and characterized its genome. We also determined the morphology, growth characteristics, thermal stability, host range, and effect on the virulence of its host bacterial strain. Under Objective 3.2b: We performed genetic screens to identify temperature-responsive regulatory elements in cool-virulent R. solanacearum by random mutagenesis of the cool-virulent R. solanacearum strain UW551 with a transposon carrying a promoter-less bacterial luciferase (luxCDABE) operon as a reporter. We also screened the transposon mutants at cool (20oC) and tropical (28oC) temperatures and identified specific mutants in which expression of the reporter is temperature-dependent, indicating that the transposon inserted downstream of a temperature-dependent promoter. We also developed high-throughput R. solanacearum virulence screening methods using tobacco seedlings and used this method to screen random transposon mutants of the cool virulent strain UW551 of R. solanacearum to identify mutants that lost virulence at cool but not at high temperatures, in order to study mechanisms that differentiate cool-virulent strains from tropical strains of in causing disease at cool temperature.
Accomplishments
1. Identification and characterization of a global regulator, AcrR, in Acidovorax citrulli. Identification and characterization of a global regulator, AcrR, in Acidovorax citrulli. Acidovorax citrulli is a seedborne bacterial pathogen responsible for bacterial fruit blotch, which causes significant losses in melon and watermelon worldwide. Bacterial motility and virulence in this and other species are controlled by LuxR-type regulators. ARS scientists in Beltsville, Maryland, collaborated with scientists in China to identify a LuxR-type regulator, AcrR, in a strain of A. citrulli, Aac-5. They found that the acrR mutant had reduced virulence and lost twitching and swimming motilities and flagellar formation, but increased biofilm formation and growth ability. This new knowledge indicates that AcrR acts as a global regulator affecting multiple important biological functions of A. citrulli, and should lead to the development of effective control strategies to combat this economically important bacterial pathogen.
Review Publications
Choi, G., Cho, I., Ju, H., Hu, W., Kim, B., Oh, J., Seo, E., Park, J., Domier, L.L., Lim, H., Hammond, J., Song, K. 2019. Full-length infectious clones of two new isolates of tomato mosaic virus induce distinct symptoms associated with two differential amino acid residues in 128 kDa protein. Plant Pathology Journal. 35(5):538-542. https://doi.org/10.5423/PPJ.NT.12.2018.0286.
Guan, W., Wang, T., Huang, Q., Tian, E., Liu, B., Yang, Y., Zhao, T. 2020. A LuxR-type regulator, AcrR, regulates flagellar assembly and contributes to virulence, motility, biofilm formation and growth ability of acidovorax citrulli. Molecular Plant Pathology. 00:1-13. https://doi.org/10.1111/mpp.12910.
Hu, W., Kim, B., Kwak, Y., Seo, E., Kim, J., Han, J., Kim, I., Lim, Y., Cho, I., Domier, L.L., Hammond, J., Lim, H. 2019. Five newly collected turnip mosaic virus (TuMV) isolates from Jeju Island, Korea are closely related to previously reported Korean TuMV isolates but show distinctive symptom development. Plant Pathology Journal. 35(4):381-387. https://doi.org/10.5423/PPJ.NT.11.2018.0238.
Oh, J., Choi, G., Kim, J., Oh, M., Kim, K., Park, J., Domier, L.L., Hammond, J., Lim, H. 2019. Differences in isolates of tomato yellow leaf curl virus in tomato fields located in Daejeon and Chungcheongnam-do between 2017 and 2018. Korean Journal of Agricultural Science. 46(3):507-517. https://doi.org/10.7744/kjoas.20190034.
Cho, I., Yang, C., Kwon, S., Yoon, J., Kim, D., Choi, G., Hammond, J., Moon, J., Lim, H. 2019. First report of Grapevine Syrah virus 1 infecting grapevines in Korea. Plant Disease. 103(11):29-70. https://doi.org/10.1094/PDIS-05-19-1037-PDN.
Abrahamian, P., Hammond, J., Hammond, R. 2020. Complete genome sequence of an American isolate of Pepino mosaic virus. Microbiology Resource Announcements. 9/e01124-19. https://doi.org/10.1128/MRA.01124-19.
Jordan, R.L., Hammond, J. 2020. Bean common mosaic virus and bean common mosaic necrosis virus (Potyviridae). Encyclopedia of Virology. https://doi.org/10.1016/B978-0-12-809633-8.21293-4.
Kreuze, J.F., Vaira, A., Menzel, W., Candresse, T., Zariev, S.K., Hammond, J., Ryu, K., Report Consortium, I. 2020. ICTV Virus Taxonomy Profile: Alphaflexiviridae. Journal of General Virology. https://doi.org/10.1099/jgv.0.001436.
Cho, I., Yang, C., Kwon, S., Yoon, J., Kwon, T., Hammond, J., Lim, H. 2020. First report of Pyrus pyrifolia cryptic virus infecting pear in Korea. Plant Disease. https://doi.org/10.1094/PDIS-09-19-1959-PDN.
