Source: UNIV OF MINNESOTA submitted to NRP
IDENTIFICATION, CHARACTERIZATION, AND IMPORTANCE OF PLANT PARARETROVIRUSES AND CONFORMATIONAL DISEASE AGENTS AND IDENTIFICATION, DETECTION, AND MANAGEMENT OF NEW VIRAL DISEASES OF HORTICULTURAL CROPS IN MINNESOTA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1009997
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2016
Project End Date
Sep 30, 2021
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108
Performing Department
Plant Pathology
Non Technical Summary
The aims of this project are to a) provide practical information including modified breeding and strategies that can be used to reduce crop and financial losses caused by DNA viruses that can integrate into plant genomes (pararetroviruses), b) aberrant forms of normal plant proteins that are transformed into filamentous prion-like structures that accumulate to high levels in all plant cells, and c) new viruses that affect horticultural crops.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21215101101100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1510 - Corn;

Field Of Science
1101 - Virology;
Goals / Objectives
Plant PararetrovirusesIdentify, characterize and develop detection protocols for new badnaviruses occurring as episomal disease-inducing viruses. First study: three new badnaviruses of cultivated rose, turf grass (Lolium) and hot pepper (Capsicum Frutescens) in Minnesota.Determine whether plant pararetroviruses are transmitted vertically only (i.e. integrated in the host genome), or whether the alternate modes of vertical transmission exist.Determine what genetic elements (pararetroviral sequences) in host genotypes that could be used to predict the susceptibility for episomal infection and disease induction.Determine the external factors (e.g. type of stress, environmental changes) that may be correlated with disease expression resulting from different forms of pararetroviral elements transmitted vertically in plants.Conformational diseasesDetermine the identity of structural proteins of fibrils isolated from plants that are not members of Asteraceae.Determine if identified plant proteins assemble into fibrils in vitro or in heterologous systems.Identify agents that induce filaments.Determine the effect of fibrils on plants.New Viral Diseases of Horticultural CropsContinue our ongoing program of identifying new viruses causing disease in horticultural crops in Minnesota, as well as known viruses occurring in crops not previously recorded as hosts. The main objective, as always, is to provide rapid diagnostic capacity to our Plant Disease Clinic to respond to growers' need for recommendations for disease management and avoiding financial loss.This research also includes diseases caused by phytoplasmas which can be devastating in effect as evidenced by recent outbreaks of a disease of garlic caused by aster yellows (Mollov et al 2014) and of Spiraea caused by western X phytoplasma.
Project Methods
Plant PararetrovirusesOur investigation of new pararetroviruses for which episomal (virion) forms have been identified will follow the procedures that have been used in previous studies. This approach will be used, for example, for the study of new badnaviruses that we have recently identified in hot pepper (Capsicum frutescens x) and forage grass (Lolium perenne). For studies on pararetroviruses that occur primarily or exclusively as non-episomal (i.e. not present in virions) replicating forms we will the badnavirus Pelargonium vein-banding virus (PVBV) and the caulimoviruses Petunia vein-clearing virus (PVCV) and Dahlia mosaic virus (DaMV). The questions to be addressed are:do these pararetroviral elements occur as non-integrated circular dsDNA, as sequences integrated in the host plant genome, or both?Can the presence of these pararetroviral elements lead to disease induction in the host, and if so, under what conditions?Is the transition to disease induction a function of parental genotype composition?Pararetroviruses for which episomal (i.e. virion) forms have not been identified, or occur only exceptionally, Pelargonium vein banding virus (badnavirus) and Dahlia mosaic virus will be used as model systems. Rolling-circle amplification (RCA) and standard PCR will be used to identify the nature of the vertically transmitted viral genome (circular, non-integrated vs. linear, integrated). Evidence for presence of integration of viral genomic sequences in the host genome will be obtained by fluorescent in sita hybridization (FISH). Different stress factors (e.g. tissue culture, moisture stress, temperature shifts) will be used to determine the effect on development of episomal infection from non-episomal viral genomic sequences.Conformational DiseasesProteins associated with fibrils prepared from Trifolium pratense (red clover),Ficus benjamina (weeping fig), and Celtis occidentalis (common hackberry), will be identified by collision-induced dissociation tandem mass spectrometry (CIDMS/MS) to determine if fibrils from diverse plant species are composed of related protease inhibitors or different proteins. In addition, we will survey plants will high quality sequenced genomes for amyloid fibril-like structures and, if found, characterize the fibrils of those plants.Proteins identified in objective 1 will be expressed in E. coli. Assembly of the expressed proteins into fibrils in E. coli would indicate that fibril assembly does not require other plant proteins and provide further evidence that fibrils are composed of only the identified protein. We will determine if highly purified E. coli expressed proteins form fibrils and if the addition of purified fibrils promotes their formation. If E. coli produced proteins do not form fibrils, we will determine if fibrils form when the sunflower protease inhibitor is transiently overexpressed in Nicotiana benthamiana and if inoculation with purified fibrils promotes the processes. Fibril abundance will be determined by immunosorbent electron microscopy.We have observed fibril abundance is highly variable suggesting that it is affected by environmental factors that affect expression. Expression of many KTIs is induced by herbivore feeding, pathogens, mechanical wounding, and plant hormones (Major & Constabel 2008; Kang et al 2002). We will determine the effects of these treatments on sunflower KTI expression and fibril abundance.It is not known if fibrils have any role in the leaf and flower distortion pathologies associated with them. If the experiments in Aim 3 demonstrate that inoculation with purified fibrils promotes fibril accumulation, we will determine if inoculation also promotes leaf and flower distortion. In addition, we will address these questions by creating sunflower lines with increased protease inhibitor expression due to the presence of a 35S:KTI transgene. In addition, CRISP/Cas9 technology will be used to create sunflower mutants that do not express the protease inhibitor. If it does not work we will employ MicroRNA Induced Gene Silencing (MIGS) to reduce the protease inhibitor. We anticipate that reducing KTI expression will reduce fibril abundance while overexpression may increase fibril abundance. Using these plants, we will determine if inhibiting fibril accumulation prevents leaf and flower distortion and if increasing fibril production increases the severity of the leaf and flower distortion.Since a number of stresses are known to induce protease inhibitor expression and water stress induces fibril accumulation in Ficus benjamina, it is possible that fibrils help protect against stresses. We will test this hypothesis by determining if the transgenic plants with increased and reduced fibril accumulation have altered susceptibility to water stress, sunflower herbivores and sunflower pathogens.New Viruses of Horticultural CropsMethods for studying new viruses will be those that have been described previously (see list of publications). Virus will be isolated from infected host plant tissue by differential and isopycnic density gradient centrifugation. Genomic sequence, genome organization and phylogenetic relationships will be determined as previously. Virus detection protocols will be developed using PCR and antibody-based assays including ELISA and immunosorbent electron microscopy (ISEM). In the case of viruses for which antibody production by the conventional method (rabbit antiserum) is not feasible, rabbit antibodies will be generated using synthetic coat protein sequences expressed in E. coli as has been done recently for Orchid fleck virus (OFV).

Progress 10/01/16 to 09/30/21

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This final report is being submitted for the termination this project, as the PI is no longer employed by the University of Minnesota.

Publications


    Progress 10/01/19 to 09/30/20

    Outputs
    Target Audience:Plant disease clinic and diagnosticians, commercial crop and ornamental plant producers, research scientists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Department of Plant Patholgy is currently hosting a Ph. D research student, Robert Alvarez collaborating with this project Training of virus detection of viral diseases on apples to a graduate student from the Department of Horticulture How have the results been disseminated to communities of interest?Results have been disseminated to communities of interest through peer reviewed journals, publications, National Plant Diagnostic Network updates and presentations to professional horticultural groups and master gardeners. What do you plan to do during the next reporting period to accomplish the goals?Continue investigating new models of integrations plant pararetroviruses. Identification and characterization of newly identified viruses in commercial production in Minnesota.

    Impacts
    What was accomplished under these goals? Plant Pararetroviruses. New models of plant pararetrovirus integration have been identified. These new models studied suggest that integration is not required for vertical transmission, virions are associated with disease symptoms (Pelargonium x hortorum), research is being carried to clarify the disease etiology. Characterization of two complete genomes of Spireae yellow leaf spot badnavirus and development of improved detection protocols. Data mining of Ash genomes for endogenous pararetroviral sequences. Conformational diseases, amyloids fibrils have been detected on several different host inlcuding Hackberry trees, Hydrangea spp., Ficus spp. the identity of these new amyloids fibrils is being studied using peptide sequencing and mass spectrometry New viral diseases of horticultural crops. Detection and characterization previously unreported viruses on apple orchards and fruits, this was found to be sustantially more widespread than initially assumed. Collaboration with the Department of Horticulture at the University of Minnesota with the detection of these new viruses at the nursery level.

    Publications

    • Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Cindy B.S. Tong, Hsueh-Yuan Chang, James J. Luby, David Bedford, Nicholas P. Howard, Dimitre Mollov, Benham E.L. Lockhart (2020) Skin Dimpling, a Novel Apple Postharvest Storage Disorder, and its Association with Apple Stem Pitting Virus
    • Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Bratsch, Olszewski N., Lockhart, B.E.L (2020) Incidence of cymbidium mosaic, odontoglossum ringspot, and orchid fleck virus in orchids in Minnesota and production of antibodies for use in ELISA to detect orchid fleck virus. European Journal of Plant Pathology
    • Type: Journal Articles Status: Published Year Published: 2020 Citation: Pierre-Yves Teycheney, Andrew D. W. Geering, Idranil Dasgupta, Roger Hull, Jan F. Kreuze, Ben Lockhart, Emmanuelle Muller?, Neil Olszewski, Hanu Pappu, Mikhail M. Pooggin, Katja R. Richert-P�ggeler, James E. Schoelz?, Susan Seal?, Livia Stavolone, Marie Umber? (2020) ICTV Virus Taxonomy Profile: Caulimoviridae. Journal of General Virology
    • Type: Journal Articles Status: Accepted Year Published: 2020 Citation: John Hammond, Michael David Reinsel, Samuel C Grinstead, Benham E.L. Lockhart, Ramon L. Jordan, Dimitre S. Mollov (2020) A mixed infection of helenium virus S with two distinct isolates of butterbur mosaic virus, one of which has a major deletion in an essential gene. Frontiers in Microbiology, section Microbe and Virus Interactions with Plants
    • Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Alvarez-Quinto RA; Lockhart BEL; Olszewski N. Genome characterization of Spireae yellow leaf spot virus. Archives of Virology
    • Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Alvarez-Quinto RA; Lockhart BEL; Olszewski N. A new TRV-like virus infecting weigela spp. Archives of Virology
    • Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Alvarez-Quinto RA; Lockhart BEL; Olszewski N. Complete genome sequencing of cereal chlorotic mottle virus.Archives of Virology


    Progress 10/01/18 to 09/30/19

    Outputs
    Target Audience:Plant disease diagnosticians, commercial crop and ornamental plant producers, research scientists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We are currently hosting a Ph. D research student, Robert Alvarez, from Ecuador, supported by the government of Ecuador. How have the results been disseminated to communities of interest?1.Results have been disseminated to communities of interest through peer reviewed journals, publications, National Plant Diagnostic Network updates and presentations to professional horticultural groups and master gardeners. What do you plan to do during the next reporting period to accomplish the goals?Continue investigating new models of integrations plant pararetroviruses. Identification and characterization of newly identified viruses in commercial production in Minnesota.

    Impacts
    What was accomplished under these goals? New models of plant pararetrovirus integration have been identified. New models studied suggest that integration is not required for vertical transmission, virions are associated with disease symptoms (Pelargonium x hortorum). Important implications for breeding and parent choice as the case of Banana Streak Virus and the catasthropic effect in Banana. Complete genome sequences of badnaviruses has been obtained for Polyscias mosaic virus, Aglaonema bacilliform virus, Cycad leaf necrosis virus. Data is published and publicy avaliable, making it useful for detection. A new TRV-like virus is being characterized from Weigela sp. showing virus-like symtomps, it was detected in greenhouse production in Minnesota. Common detection techniques ELISA and RT-PCR for TRV result in false positives. The new TRV-like virus from Weigelea doesnt react with TRV antibodies during ELISA. Complete genome sequence was obtained and epidemiology is being studied. This new viral disease is a serious threat for ornamentals and vegetables crops in Minnesota. A realiable detection method will be developed.

    Publications

    • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Alvarez-Quinto, RA; Lockhart, BEL; Moreno-Martinez, JM, Olszewski NE. (2019) Complete genome sequence of aglaonema bacilliform virus (ABV). Arch. Vir. doi: 10.1007/s00705-019-04445-0
    • Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Alvarez-Quinto RA; Lockhart BEL; Olszewski N. (2019). Genomic characterization of Cycad leaf necrosis virus, the first badnavirus identified in a gymnosperm. Arch. Vir. In Press
    • Type: Journal Articles Status: Published Year Published: 2019 Citation: Mattie M. Baumann; Roy G. Kiambi; Benham E. Lockhart (2019). Ladys Slipper Orchid and Hydrangea: New Ornamental Hosts of Tobacco Rattle Virus (TRV) in Minnesota. Plant Health Progress. https://doi.org/10.1094/PHP-05-19-0034-BR
    • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Alvarez-Quinto RA; Lockhart BEL; Olszewski N. (2019) Complete genome sequence of a previously undescribed badnavirus occurring in Polyscias fruticosa L. (Ming aralia). Arch. Vir. 164(9):2371-2374


    Progress 10/01/17 to 09/30/18

    Outputs
    Target Audience:Plant disease diagnosticians, commercial crop and ornamental plant producers, research scientists Changes/Problems: No major changes are envisaged or necessary What opportunities for training and professional development has the project provided? We have hosted a visiting research scientist, Roy Gitonga Kiambi, from Kenya. We are currently hosting a Ph. D research student, Robert Alvarez, from Ecuador, supported by the government of Ecuador. How have the results been disseminated to communities of interest? Results have been disseminated to communities of interest through peer reviewed journals, publications, National Plant Diagnostic Network updates and presentations to professional horticultural groups and master gardeners. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? We have identified a novel single stranded negative sense RNA virus (SNRV) in Maize in Ecuador and have descovered the presence of this virus in 'golden bantam' in the United states. It appears to be seed transmitted and more work is being done on this. We have identified a couple new pararetroviruses of each Aralia and Agloenema and published work on these. Currently we are continuing work on maize viruses in Kenya

    Publications

    • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Kiambi, R.G., Baumann, M.M., and Lockhart, B.E. 2018. A new disease of Epimedium caused by Carnation ringspot virus. Plant Health Progress. https://doi.org/10.1094/PHP-09-18-0051-BR
    • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Baumann, M.M., Kiambi, R.G., and Lockhart, B.E. 2018. Lady's slipper and hydrangea: new ornamental hosts of Tobacco rattle virus in Minnesota. Plant Health Progress.
    • Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Baumann, M.M., Kiambi, R.G., Lockhart, B.E., and Gomez, T. 2019. Asclepias yellow vein virus: A proposed new potyvirus of Milkweed. Plant Health Progress.
    • Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Alvarez-Quinto, R.A., Lockhart, B.E., Olszewski, N. 2018. Complete genome sequence of a previously undescribed badnavirus occurring in Polyscias fruticosa L. (Ming aralia).


    Progress 10/01/16 to 09/30/17

    Outputs
    Target Audience:Plant disease diagnosticians, commercial crop and ornamental plant producers, research scientists Changes/Problems:No major changes are envisaged or necessary What opportunities for training and professional development has the project provided?We have hosted a Ph.D student, Zaigham Abbas Shah on techniques for producing antibodies for plant virus detection using coat-protein sequences expressed in E. coli. We are currently hosting a Ph. D research student, Robert Alvarez, from Ecuador, supported by the government of Ecuador. How have the results been disseminated to communities of interest?Results have been disseminated to communities of interest through peer reviewed journals, publications, National Plant Diagnostic Network updates and presentations to professional horticultural groups and master gardners. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? We have identified a novel mode of pararetrovirus infection associated with a widespread disease of ash (Fraxinus spp).

    Publications

    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Bratsch, S., Lockhart, B. (2017). Preliminary characterization of a virus associated with Euonymus alatus dieback. Acta horticulturae: Third International Symposium on Woody Ornamentals of the Temperate Zone.
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Bratsch, S., Lockhart, B., Mollov, D., Tisserat, N. (2017). Partial characterization of two new viruses in ash belonging to the families Partitiviridae and Caulimoviridae. Acta horticulturae: Third International Symposium on Woody Ornamentals of the Temperate Zone.
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Bratsch, S., Zlesak, D, Mollov, D., Lockhart, B. (2017) First Report of Rose rosette virus associated with Rose Rosette Disease in Rosa hybrida in Minnesota. Plant Health Progress 18:2, 102-103. http://dx.doi.org.10.1094/PHP-01-0008-BR
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Bratsch, S., Mollov, D., Lockhart, B. (2017). Characterization of a new nepovirus causing a leaf mottling disease in Petunia hybrida. Plant disease, 101(6), 1017-1021. https://doi.org/10.1094/PDIS-12-16-1785-RE