Source: UNIV OF MINNESOTA submitted to NRP
IDENTIFICATION, CHARACTERIZATION AND BIOLOGY OF ENDOGENOUS PARARETROVIRUSES (EPRVS) CAUSING VERTICALLY-TRANSMITTED PLANT DISEASES.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0079870
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2011
Project End Date
Sep 30, 2016
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
Previous and ongoing work on pararetroviral, conformational and new viral diseases have been essentially summarized in previous sections describing the importance, aims and experimental approaches used. In the case of pararetroviral and conformational diseases precedents from the plant world are either totally lacking or very marginal. The ongoing and proposed research described above is a foray into previously unknown territory, as was the case when the suggestion of activatable pararetroviral genomes integrated into the host genome was first advanced (Harper et al 2002). It is quite likely that conformational diseases associated with the accumulation of misfolded cellular proteins will in the near future be recognized to be of wide occurrence and importance.Conformational diseasesThis project arises from our observations that amyloid fibril-like structures (fibrils) are widely distributed across the plant kingdon. In Asteraceae, high fibril abundance is associated with symptoms of leaf and flower deformation (Figure 2). The fibrils are 5-7 nanometers (nm) in diameter, can exceed 3000 nm in length, and are similar in morphology to amyloid fibrils (Figure 3). The fibrils of sunflower (Helianthus annuus), gerbera (Gerbera jamesonii) and zinnia (Zinnia) lack nucleic acids and have a major protein that migrates with a molecular mass of ~22 kDa during SDS-PAGE. Proteins with other molecular masses are variably associated with the purified fibrils, suggesting the presence of dimers. N-terminal protein sequencing by Edman degradation was performed on the 22 kDa protein of fibrils purified from gerbera and zinnia. The sequences of all of the proteins were nearly-identical and a BLAST search of EST sequences revealed that the N-terminal sequences had perfect or almost perfect, identity with predicted mature Kunitz-type protease inhibitors from sunflower. To provide further conformation that the 22 kDa fibril protein is a KTI, the complete coding region of the sunflower KTI with highest identity to the gerbera and zinnia fibril proteins was cloned and sequenced. Then the proteins of sunflower fibrils were resolved by SDS PAGE and the resulting bands were cut out of the gel, digested with trypsin, and sequences of the resulting peptides were obtained by collision-induced dissociation tandem mass spectrometry (CID MS/MS) analysis. The sequence of four of the trypsin fragments had 100% identity with the predicted fragments from the cloned sunflower KTI. Amyloid fibrils have been identified and studied in bacteria, fungi, and animals but not in plants.Kunitz trypsin inhibitors (KTIs) are likely present in all plants. KTIs are vacuolar-localized and inhibit a wide range of proteases and invertase (Major & Constabel 2008). KTIs accumulate in response to plant hormones, biotic and abiotic stresses, and protect plants against herbivores and pathogens (Major & Constabel 2008; Kang et al 2002). In addition to impairing defense responses, experimentally reducing KTI expression causes defects in leaf shape, and shoot and root growth (Islam et al 2015). The fibrils of sunflower (Helianthus annuus), gerbera (Gerbera jamesonii) and zinnia (Zinnia) are composed of a protease inhibitor belonging to the Kunitz trypsin inhibitor family. Fibrils from other plant species will be sequenced to determine if this phenomenon is restricted to protease inhibitor proteins in plants or if it is widely distributed among plant proteins.The importance of this project lies in its principal objective to provide information that leads to practical disease elimination, control or mitigation in a wide range of crops of agricultural and horticultural interest both locally and globally, as has been demonstrated by the previous record of accomplishments. There are two components to the project, as designated in the revised project title. The first addresses pararetroviral and conformational plant diseases, two areas in which we have been pioneers. These diseases are to a large extent of genetic rather than infectious origin, and dealing with these diseases are related to choice of parental genotypes and hybridization strategies rather than pathogen elimination. In the case of pararetroviral diseases this notion has been clearly illustrated by Banana streak virus (BSV) infection in all improved musa hybrids produced worldwide in breeding programs between 1960-1990 (Frison and Sharrock 1998). Similar problems due to pararetrovirus transmission in one parental genotype developed in petunia (Petunia vein clearing virus) (Lockhart and Lesemann 1998) and Kalanchoë (Kalanchoë top-spotting virus) (Ferji and Lockhart 1988). In the case of conformational diseases we are at the stage that we were in 1990-1995 when our data pointed to the existence of a previously undescribed disease-inducing mechanism arising from the integrated viral segments present in one parent but expressed only in hybrids with a non-carrying parent (LaFleur et al. 1996)Research on new viruses of horticultural interest cannot be predicted or anticipated. Our objective and commitment are to immediately investigate new viral disease problems occurring in Minnesota (or likely to be of interest to), and to deploy all available resources to this end. Previous examples include rhizomania of sugarbeet (Peyer and Lockhart 2000), corky ringspot of potato (Lockhart and Mason 2011), new viruses of roses (Mollov et al. 2012, 2013, 2014), mosaic of white ash (Machado-Caballero et al. 2013), and phytoplasma diseases of garlic (Mollov et al. 2013) and Spiraea (Lockhart et al. 2012).Areas in which we have already identified a need for investigating are organic and high-tunnel vegetable production (Fig 1D), imported ornamentals (e.g. petunia) (Fig 1E) carrying viruses capable of infecting a wide range of species, and woody ornamentals.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2122129110133%
2121830110133%
2122123110134%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
2123 - Bedding/garden plants; 2129 - Florist crops, other; 1830 - Peanut;

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 10/01/11 to 09/30/16

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We are hosting a Ph.D student, Zaigham Abbas Shah on techniques for producing antibodies for plant virus detection using coat-protein sequences expressed in E. coli. 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? Plant pararetroviruses 1. A new badnavirus occurring in roses was identified and is being propagated following tissue culture. Virus will be extracted for transmission studies and genomic characterization. In the course of this work a second caulimovirus was discovered in roses, which is now under investigation. Of special interest is the possible role of aphid vectors in transmission of pararetroviruses in roses. 2. Pelagonium vein banding virus, PVBV, was identified as a suitable candidate for the study of vertical transmission of plant pararetroviruses via seed, but not via viral sequences (endopararetroviruses or EPRVs) integrated in the host genome. 3. The effect of stress factors on inducing de novo disease expression due to PVBV infection is being investigated Conformational diseases 1. In the family Asteraceae, the filamentous proteins whose accumulation is associated with growth abnormalities such as severe flower deformation were identified as insoluble forms of a native plant protease inhibitor. Amino acid sequences of the protease inhibitor identified by mass spectrometry were expressed in E. coli and rabbit antibodies prepared for studies on protein filament induction and cellular accumulation. New viral diseases of Horticultural crops 1. Two previously undescribed viruses of high tunnel-grown tomatoes were identified in Minnesota. Identification, characterization and epidemiological studies of these two viruses are in progress.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Identification of Tobacco streak virus in Cranberry and the Association of TSV with Berry Scarring L. D. Wells-Hansen, J. J. Polashock, N. Vorsa, B. E. L. Lockhart, and P. S. McManus Plant Disease Apr 2016, Volume 100, Number 4
  • Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Identification, transmission and genomic characterization of a new member of the family Caulimoviridae causing a flower distortion disease of Rudbeckia hirta.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: First Report of a 16SrI (Aster Yellows) Group Phytoplasma in Phlox in the United States. A. E. Sathoff, D. Rajendran, S. D. Wannemuehler, K. Sweeney, F. Manan, Z. C. Kosgey, L. H. Garber, and B. E. Lockhart.


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Target audiences include: researchers, local, national and international commodity groups, and disease clinic diagnosticians. 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?Journal publications, conference presentations, via national plant disease diagnostic network system. What do you plan to do during the next reporting period to accomplish the goals?Continue ongoing projects.

Impacts
What was accomplished under these goals? Additional information was obtained on the existence of non-integrated non-encapsidated plant pararetroviral elements that are transmitted vertically (via seed) in a number of new plant species.

Publications

  • Type: Journal Articles Status: Other Year Published: 2015 Citation: Hammond, J., Mollov, D., Reinsel, M., and Lockhart, B. 2014. Detection of Helenium virus S and two distinct isolates of Butterbur mosaic virus in a single plant of Veronica.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Mollov, D.S., Guaragna, M., Lockhart, B., Rezende, J., Jordan, R. 2014. First report of Catharanthus mosaic virus in Mandevilla in the United States. Plant Disease. 99:165.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Lockhart, B., Mollov, D.S., Mason, S., Bratsch, S. 2014. First report of natural occurrence of Turnip vein-clearing virus in garlic mustard (Alliaria petiolata) in the United States. Plant Health Progress. doi:10.1094/PHP-BR-14-0029.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Agneroh, T., Bratsch, S., Lockhart, B. (2015) First report of Canna yellow mottle virus in Kenya. Plant Health Progress 16:1, 34-35. Online at: https://www.plantmanagementnetwork.org/sub/php/volume16/number1/PHP-BR-14-0037.pdf
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Bratsch, S., Mollov, D., Lockhart, B., Johnson, D., Ehlenbeck, S. (2015). First report of Cucumber Mosaic Virus Infection in Pachysandra in the U.S.A.. Plant Disease 99:3, 422.1. Online at: http://apsjournals.apsnet.org/doi/abs/10.1094/PDIS-09-14-0974-PDN
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Bratsch, S., Flynn, J., Lockhart, B. (2014) Detection of Tobacco Rattle Virus (TRV) in Phryma leptostachya L.: A new native plant host in Minnesota. Plant Health Progress, 15:4, 184-185. Online at: https://www.plantmanagementnetwork.org/sub/php/volume15/number4/PHP-BR-14-0021.pdf
  • Type: Journal Articles Status: Other Year Published: 2016 Citation: Bratsch, S., Lockhart, B., Johnson, D. (In preparation) First report of Alternanthera Mosaic Virus in Penstemon in the U.S.A. Plant Health Progress.
  • Type: Journal Articles Status: Other Year Published: 2016 Citation: Bratsch, S., Lockhart, B., Ishimaru, C., Ehlke, K. (In preparation) Confirmation of first report of Orchid fleck virus in orchids in the U.S.A. Plant Health Progress.
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Wells-Hansen, L.D., Polashock, J.J., Vorsa, N., Lockhart, B., and McManus, P. 2015. Identification of Tobacco streak virus in cranberry and the association of TSV with berry scarring. Plant Disease First Look. Online.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Mahuku, G., Lockhart, B.E., Wanjala, B., Jones, M.W., Kimunye, J.N., Stewart, L.R., Cassone, B.J., Sevgan, S., Nyasani, J.O., Kusia, E., Kumar, P.L., Niblett, C.L., Kiggundu, A., Asea, G., Pappu, H.R., Wangai, A., Prasanna, B.M., and Redingbaugh, M.G. 2015.Maize Lethal Necrosis (MLN), an emerging threat to Maize-Based Food Security in Sub-Saharan Africa. Plant Disease: 99(7)
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Bratsch, S., Mollov, D., Lockhart, B., Johnson, D., and Ehlenbeck, S. 2015. First report of Cucumber mosaic virus infection in Pachysandra in the United States. Plant Disease: 99(1).
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Vo, J.N., Mahfuz, N.N., Lockhart, B.E., Geering, A.D.W. 2015. Improved methods for the purification and enrichment of banana streak virus for antibody production and protein detection. European Journal of Plant Pathology: 143(3): 619-626.


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Target audiences are growers, diagnosticians, plant disease clinics, plant breeders and researchers. 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? Peer-reviewed publications, national and international symposia and commodity group conferences. What do you plan to do during the next reporting period to accomplish the goals? Recently next generation sequence has identified a number of badnaviruses in crop plants but no virus particles have been found associated with the symptoms in the plants. One goal of this project is to determine whether badnaviruses can replicate and induce symptoms without producing particles.

Impacts
What was accomplished under these goals? Three new viruses were characterized and sequenced completely. These were Rose yellow leaf virus and Rudbeckia flower distortion virus (genus: caulimoviruses) and pelargonium vein-banding virus (genus: badnavirus).

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Mollov, D., M.A. Guaragna, B. Lockhart, J.A.M. Rezende, and R. Jordan. 2014. First report of Catharanthus mosaic virus in Mandevilla in the United States. Plant Disease. Online First: October 1.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Lockhart, B., D. Mollov, S. Mason and S. Bratsch. 2014. First report of natural occurrence of Turnip vein-clearing virus in garlic mustard (Alliaria petiolata) in the United States. Plant Health Progress. doi:10.1094/PHP-BR-14-0029.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Mollov, D., B. Lockhart, D. C. Zlesak. 2014. Complete nucleotide sequence of Rose yellow leaf virus, a new member of the family Tombusviridae. 159:2795-2798.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Mollov, D., B. Lockhart, E. Saalau-Rojas, C. Rosen. 2014. First report of a 16SrI (Aster Yellows) Group Phytoplasma on garlic (Allium sativum) in the USA. Plant Disease 98:419.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Mollov, D. B. Lockhart, A. Phibbs, T. Creswell, G. Ruhl, E. Dorman, G. Kinard, and R. Jordan. 2014. Clematis chlorotic mottle virus, a novel virus occurring in clematis in the USA. Phytopathology 104:S3.81.


Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Target audience for this reporting period: plant virologists, plant breeders, commercial rose producers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One Ph.D. student is in her third year of training. How have the results been disseminated to communities of interest? Results have been disseminated by publication in peer-reviewed journals, oral and poster presentations at professional international meetings, provision of primer sequences for virus detection and screening and a posting on the internet of photographs of disease symptoms, widely viewed. What do you plan to do during the next reporting period to accomplish the goals? Continue with ongoing research as described above.

Impacts
What was accomplished under these goals? A new plant pararetrovirus infecting cultivated roses was transmitted, characterized and sequenced. The virus was named Rose yellow vein virus (RYVV). Phylogenetic analysis revealed RYVV is the first member of a new genus in the family Caulimoviridae. There was no evidence that RYVV occurs as an endogenous pararetrovirus (EPRV) in roses, and it is probably transmitted primarily by clonal propagation and possibly to a lesser extent by root graft. The characterization, transmission, and sequencing of Rudbeckia flower-distortion virus (RuFDV) was completed. This virus is transmitted efficiently by aphids, but there is no evidence of seed transmission or of its occurrence as an EPRV in Rudbeckia spp. Phylogenetic anlyasis revealed that RuFDV, like RYVV mentioned above, appears to be the first member of a new genus in the family Caulimoviridae. Work is continuing on Pelargonium vein-banding virus, a new badnavirus that is unique in being transmitted vertically via seed, but does not occur as an integrant (EPRV) in the Pelargonium genome. A new caulimovirus was identified in phlox. Partial genomic sequences were obtained, and revealed on ly limited sequence similarity to known caulimoviruses. Further biological and genomic characterization of this virus is being done in collaboration with the USDA ARS ornamental virus unit at Beltsville, MD.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Mollov D, Lockhart B, Zlesak DC, Olszewski N. Arch Virol. 2013 Apr;158(4):877-80. doi: 10.1007/s00705-012-1547-9. Epub 2012 Nov 21. Complete nucleotide sequence of rose yellow vein virus, a member of the family Caulimoviridae having a novel genome organization. Mollov, D., B. Lockhart, E. Saalau-Rojas, C. Rosen. 2013. First report of a 16SrI (Aster Yellows) Group Phytoplasma on garlic (Allium sativum) in the USA. Plant Disease. First look. On line September 18. Mollov, D., B. Lockhart, D.C. Zlesak. 2013. Complete nucleotide sequence of Rosa rugosa leaf distortion virus, a new member of the family Tombusviridae. Archives of Virology. 158:2617-2620. Machado-Caballero, J.E., B.E. Lockhart, S.L. Mason, D. Mollov, and J.A. Smith. 2013. Identification, transmission, and partial characterization of a previously undescribed Flexivirus causing a mosaic disease of ash (Fraxinus spp.) in the USA. Plant Health Progress. doi:10.1094/PHP-2013-0509-01-RS. Mollov, D., B. Lockhart, D.C. Zlesak. 2013. Complete nucleotide sequence of Rose yellow mosaic virus, a novel member of the family Potyviridae. Archives of Virology. 158:1917-1923. Mollov, D., B. Lockhart, D.C. Zlesak, and N. Olszewski. 2013. Complete nucleotide sequence of Rose yellow vein virus, a member of the family Caulimoviridae having a novel genome organization. Archives of Virology 158:877-880. Guaragna, M.A., J. Lamborn, D. Groth-Helms, S. Juszczak, D. Mollov, B. Lockhart, T. van Schadewijk, J. Hammond, and R. Jordan. 2013. First Report of Nerine yellow stripe virus in Amaryllis in the United States. Plant Disease 97:1389. Lockhart, B., D. Mollov, and J. Voth-Hulshout. 2012. Association of Spirea stunt Phytoplasma with a disease of Spiraea spp. in Minnesota. Plant Health Progress. doi:10.1094/PHP-2012-1023-01-BR. Lockhart, B., D. Mollov, and M. Daughtrey. 2012. First report of Alfalfa Mosaic Virus occurrence in hydrangea in the USA. Plant Disease 97:1258. Lockhart, B. and D. Mollov. 2012. First Report of Alfalfa mosaic virus occurrence in Tecoma capensis in the USA. Plant Health Progress. doi:10.1094/PHP-2012-0824-02-BR. Abstracts Mollov, D., B. Lockhart, and D. Zlesak. 2013. Symptoms, transmission, and detection of four rose viruses. VI International Symposium on Rose Research and Cultivation. Hanover, Germany. Mollov, D., B. Lockhart, and D. Zlesak. 2012. Rose Yellow Mottle Virus, a novel virus that affects Rosa sp. Phytopathology 102:S4-82. Mollov, D., B. Lockhart, and D. Zlesak. 2012. Identification, transmission and genomic characterization of a novel member of the Caulimoviridae causing a yellow-vein disease of cultivated Rose.


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Plant pararetroviruses infecting Rudbeckia, Pelargonium and rose were completely sequenced and their genomes characterized. The viruses occurring in rose (Rose yellow vein virus, RYVV) and Rudbeckia (Redbeckia flower distortion virus, RuFDV) did not occur as endogenous pararetroviruses (EPRVs) integrated in the host genome. Based on phylogenetic analyses both RYVV and RuFDV appear to represent new genera in the family Caulimoviridae. Pelargonium vein-bounding virus (PVBV, genus Badnavirus) was found to a plant pararetrovirus that is transmitted vertically (i.e. via seed) but does not occur as an EPRV. This represents a novel form of vertical plant virus transmission. Preliminary data suggest that a similar mechanism of vertical non-EPRV transmission may be responsible for the widespread occurrence of Dahlia mosaic virus in dahlias. Results have been disseminated in the form of a scientific conference poster presentation and a publication in a peer-reviewed journal. PARTICIPANTS: Benham E. Lockhart, Dimitre Mollov, Neil Olszewski TARGET AUDIENCES: Virologists, Plant Pathology professionals, plant breeders, commercial plant propagators PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
A technique based on rolling-circle amplification (RCA) was developed to differentiate between integrated (EPRV) and non-integrated forms of plant pararetroviruses that are transmitted vertically. This has permitted rapid screening of plants for presence of either form of the viral pathogen genome.

Publications

  • Mollov, D., Lockhart, B., Zlesak, D., Olszewski, N. 2012. Complete nucleotide sequence of rose yellow vein virus, a member of the family Caulimoviridae having a novel genome organization. Archives of Virology. 10.1007/S00705-012-1547-9


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Transmission of disease-causing plant pararetroviruses (badnaviruses and caulimoviruses) through the germ line (vertical transmission) poses a potential threat to plant improvement by conventional breeding. In addition to our originally described model of vertical transmission via viral sequences integrated in the host's nuclear genome, we have discovered a second mode of virus transmission that involves circular viral elements not integrated in host nuclear DNA. The discovery of new badnaviruses in crops such as sweet potato, grapevine and figs, underlines the need to understand these mechanisms of virus transmission during hydridization for crop improvement. PARTICIPANTS: Ben Lockhart and Shauna Mason. Collaborators: Neil Olszewski, Department of Plant Biology, University of Minnesota. University of Minnesota Arboreatum. TARGET AUDIENCES: Results from project activities were communicated to various audiences including county and regional master gardener groups, plant societies (American Hosta Society, American Rose Society), and private horticultural companies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Pelegonium vein-banding virus (PVBV), a previously undescribed seed-transmitted badnavirus, was shown to be transmitted vertically in the form of free circular viral elements and not as an integrant in the host genome (endogenous pararetrovirus) as mentioned in the previous report. The newly described caulimovirus, Rudbeckia leaf-distortion virus (RuFDV) was found to have a novel genome organization and appears to represent a new genus in the family Caulimoviridae.

Publications

  • Badnavirus: Caulimoviridae. Olszewski, N.E. and Lockhart, B.E. 2011. In: The Springer Index of Viruses, 2nd Edition. Tidona, C. and Darai, G. (eds.) pp. 263-269.
  • The potato corky ringspot pathogen, Tobacco rattle virus, occurs in native habitats in Minnesota, B.E. Lockhart and S.L. Mason, 2011. Plant Health Progress doi:1094/PHP-2011-1028-01-BR.


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Further evidence of plant diseases arising from integrated pararetroviral sequences demonstrated the potential importance of this phenomenon to plant breeding programs as shown previously for the problem posed by Banana streak virus (BSV) in banana and plantain improvement programs. This information was communicated to communities of interest, including private horticultural companies in scientific meeting communications. PARTICIPANTS: Ben Lockhart and Shauna Mason. Collaborators: Neil Olszewski, Department of Plant Biology, University of Minnesota. University of Minnesota Arboreatum. TARGET AUDIENCES: Results from project activities were communicated to various audiences including county and regional master gardener groups, plant societies (American Hosta Society, American Rose Society), and private horticultural companies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Using project resources two new plant pararetroviruses were identified, characterized genomically and biologically, and shown to be the cause of the disease with which they were associated. These two new viruses were named respectively, Rudbeckia flower-distortion virus (RuFDV) (Genus Caulimovirus) and Pelargonium vein-banding virus (PVBV) (Genus Badnavirus). PVBV was shown to be integrated into the host genome and transmitted through seed. RuFDV was shown to be efficiently transmitted by aphids.

Publications

  • First Report of Tobacco etch virus Infection in Coleus in the United States B. E. L. Lockhart, S. L. Mason, D. A. Johnson, and D. S. Mollov Plant Disease Jul 2010, Volume 94, Number 7: 921.
  • First Report of Tobacco rattle virus in Sedum in Minnesota B. E. Lockhart and S. L. Mason Plant Disease Mar 2010, Volume 94, Number 3: 374.


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Discovered and completed genomic sequencing of two new plant pararetroviruses that are seed-transmitted in Pelargonium and Rudbeckia, respectively. Determined that the Pelargonium virus, a badnavirus, occurs as an endogenous pararetrovirus (EPRV) integrated into the host genome, whereas the Rudbeckia virus, a caulimovirus, is not integrated into the host genome and is therefore transmitted vertically by a mechanism novel to plant pararetroviruses. Identified and characterized a novel filamentous virus that is highly seed-transmitted and widely distributed in plant species in the family Asteraceae. PARTICIPANTS: D. Mollov is a graduate student working on this project. TARGET AUDIENCES: Plant pathologists, virologists. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The results obtained expand knowledge about and provide additional examples of occurrence of plant pararetroviruses as EPRVs in host genomes and the potential danger that this phenomenon poses in plant breeding programs, as evidenced by our previous research on the widespread occurrence of Banana streak virus (BSV) in improved banana and plantain interspecific hybrids produced during the past two decades. The discovery of a pararetrovirus in Rudbeckia that is seed-transmitted but does not occur as an EPRV indicates that vertical transmission of pararetroviruses in breeding programs can occur via mechanisms other than activation of integrated viral genomic sequences.

Publications

  • 1. Lockhart, B., Olzewski, N., and Mason, S. 2009. Genomic characterization of a seed-borne caulimovirus associated with flower distortion in Rudbeckia hirta. Phytopathology 99:576.
  • 2. Machado-Caballero, J.E., Lockhart, B.E.L., Mason, S.L., and Daughtry, M. 2009. Identification and properties of a caulimovirus causing chlorotic mottle of florists' hydrangea (Hydrangea macrophylla) in the United States. Plant Dis. 93:891-895.
  • 3. Mollov, D., Lockhart, B., and Zlesak, D. 2009. Genome characterization and transmission of Rose yellow vein virus, a new caulimovirus occurring in garden rose. Phytopathology 99:587.
  • 4. Staginnus, C., Iskra-Caruana, M.L., Lockhart, B., Hohn, T., and Richert-Poggeler, K.R. 2009. Suggestions for a nomenclature of endogenous pararetroviral sequences in plants. Archives of Virology 154:1189-1193.


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Outputs of this project have been identification and characterization of additional pararetroviruses infecting plants, and new information on the occurrence of activatable endogenous pararetroviruses (EPRVs) and their current and potential impact on plant breeding programs. Results have been disseminated through publications and presentations at conferences. PARTICIPANTS: Collaborators included: -Department of Plant Biology, University of Minnesota, Saint Paul MN. Dr Neil Olszewski -Centre International pour la Recherche en Development (CIRAD) France. Dr.Marie Line Caruana, Montpellier and Dr. Pierre Yves Tecycheney, Guadeloupe. Department of Primary Industries, Queensland, Australia. Dr. Andrew Geereing. Washington State University, Prosser WA. Dr. Naidu Rayapati. Short term training in detection and characterization of plant pararetroviruses was provided to Fei-Ling Zhang ( Beijing Agricultural University, China) and Femi Alebi (IITA, Nigeria) TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Data from studies of virion characterization and virus genomic analysis were used to develop immunological and PCR-based methods for virus detection in germplasm. These detection techniques have been used in the US and other countries throughout the world for quarantine purposes, plant health certification and germplasm testing in a number of commercially important crop species including bananas, sugar cane and cacas.

Publications

  • Gayral, P., Noa-Carrazana, J., Lescot, F., Lheureux, F., Lockhart, B., Matasumoto, T., Piffanelli, P., and Iskra-Caruana, M. 2008. A single Banana streak virus integration event in the banana genome as the origin of infectious endogenous pararetrovirus (EPRV). Journal of Virology 82: 6697-6710.
  • Zhang, L., Lockhart, B., Dahal, G., and Olszewski. N. 2008. Studies on Biology and genomic characterization of a caulimo-like virus associated with a leaf distortion disease of LAMIUN MACULATURM. Archives of Virology 153:1181-1184.


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: Retroviruses (e.g., HIV), have SSRNA genomes, and replicate by reverse transcription using a dsDNA intermediate that integrates into the host genome as an essential stage in the viral life cycle. Pararetroviruses (e.g., animal hepadnaviruses, plant badnaviruses and caulimoviruses have ds DNA genomes, and replicate by reverse transcription using a SSRNA intermediate. Integration into the host genome is not an essential function in their life cycle. Our ongoing research on the biology, molecular biology and molecular genetics of plant pararetroviruses (badnaviruses and caulimoviruses) has produced significant new data on the biological, molecular and evolutionary relationships between these pararetroviruses and their host plants. One such finding is that plant pararetroviruses, contrary to expectations, integrate into the host genome, and that de novo infections can arise from these integrated viral sequences. These, and other related findings, challenge conventional notions of the occurrence, molecular biology and potential importance of pararetroviruses in plant, animal and human hosts. 1. It was shown that in the case of Musa (banana and plantain) activatable pararetroviral integrants present in the host genome can confer resistance to infection by the homologous episomal virus, but not to unrelated viruses. 2. The genomes of two new badnaviruses were sequenced. These are Cycad leaf necrosis virus, the first badnavirus identified in a gymnosperm, and Aglaonema yellow leaf virus. 3. Studies were initiated to determine the number and taxonomic relationship of Nicotiana spp. containing integrated tobacco vein-clearing virus (TVCV). 4. New pararetroviruses belonging to the genus Caulimovirus were identified in roses, geranium and phlox. PARTICIPANTS: Mollov, D., Almeyda, Becera. C. TARGET AUDIENCES: Plant breeders throughout the world involved in breeding improved banana and plantain varieties. Researchers interested in the biology and molecular biology of plant, animal and human pararetrovirus. Plant systematists interested in using integrated pararetroviral sequences (e.g., in Nicotiana spp.) evolutionary and phylogenetic relationships between plant taxa. PROJECT MODIFICATIONS: None

Impacts
The strongest impact to date of this research project has been on conventional breeding programs designed to produce higher-yielding, disease resistant banana and plantain (Musa. sp). hybrids needed to meet current and future food security needs for millions of inhabitants of the world's humid tropical zones in sub-saharan Africa, Central and Latin America, Southeast Asia and Australia. We have shown that all improved banana and plantain hybrids produced in the principal breeding programs (France, Honduras, Brazil, Nigeria) during the past three decades are infected with a pararetrovirus (Banana streak virus) that originates from viral sequences integrated in the host genome. The impact of these findings has been to effectively suspend the deployment of improved Musa germplasm for fear of spreading BSV.

Publications

  • Lockhart, B.E.L., and Carvana, M.L. 2007. Activatable badnaviral integrants may confer resistance to viral infection in Musa. Phytopathology 97:S67.


Progress 01/01/06 to 12/31/06

Outputs
Virus transmission experiments demonstrated that integrated pararetroviral sequences confer protection or immunity against episomal virus infection. This was shown for Banana streak virus (BSV) in banana, and for Canna yellow mottle virus (CaYMV) in canna. However, not all integrated pararetroviral sequences conferred resistance to episomal viral infection. A novel badnavirus was identified in cycads (Ceratozamia, Zamia, and Stangeria spp.) The virus was named Cycad leaf necrosis virus and is the first badnavirus identified in gymnosperm. Two new caulimovirus-like pararetroviruses were discovered in phlox and roses.

Impacts
The results reported above shed new light on the role of integrated pararetroviral sequences in conferring resistance to external viral infection. The ultimate object of this line of research is the practical utilization of this phenomenon to develop disease-resistant crops of economic importance, i.e banana.

Publications

  • Lockhart, B.E., Fetzer, J., Zlesak, D. 2006. Association of a previously undescribed filamentous virus with a yellow mosaic disease of rose. Phytopathology 96:S70
  • Lockhart, B.E., Fetzer, J., Olszewski, N.E. 2006. Preliminary characterization of Cycad leaf necrosis virus, the first badnavirus identified in a gymnosperm. Phytopathology 96:S70
  • Lockhart, B. E. 2006. Occurrence of Arabis mosaic virus in Hostas in the United States. Plant Disease 90: 834.
  • Machado, J., Lockhart, B.E., Smith, J. 2006. White ash mosaic virus, a previously undescribed flexivirus occurring in Fraxinus spp. in North America. Phytopathology 96: S67
  • Voth, P.D., Mairura, L., Lockhart, B.E., May, G. 2006. Phylogeography of Ustilago maydis virus H1 in the USA and Mexico. Journal of General Virology 87:3433-3441.


Progress 01/01/05 to 12/31/05

Outputs
We completed the characterization of the locus containing a second Banana streak virus (BSV-GF) integrant present in the banana genome. A complete viral genome was found to occur in two non-contiguos segments both terminating in inverted repeats which could permit the creation of an infectious viral genome by two recombination events. Evidence for additional badnaviral integrated sequences in Musa and Kelanchoe was also obtained.

Impacts
We have established that pararetroviral sequences integrated into plant genomes fall into two categories: and those which are capable of giving rise to de novo viral infection, and those which are incapable of doing so. Numerous examples of the latter type were identified. Their presence can lead to false positives in PCR-based methods for screening germplasm for viruses, and this needs to be taken into account in plant breeding and selection programs.

Publications

  • Geering, A.D.W., Olszewski, N.E., Harper, G., Lockhart, B.E.L., Hull, R. and Thomas, J.E. 2005. Banana contains a diverse array of endogenous badnaviruses. J. Gen. Virol. 86: 511.520.
  • Geering, A.D.W., Pooggin, M.M., Olszewski, N.E., Lockhart, B.E.L. and Thomas, J.E. 2005. Characterization of Banana streak Mysore virus and evidence that its DNA is integrated in the B genome of cultivated Musa. Arch. Virol. 150: 787-796.
  • Yang, Z. Nicolaisen, M., Olszewski, N. and Lockhart, B.E.L. 2005. Sequencing improved detection and a novel form of Kalanchoe top-spotting virus. Plant Disease 89:298-302.


Progress 01/01/04 to 12/31/04

Outputs
Work was continued in three areas: (1) characterization of loci containing Banana streak virus (BSV) GF isolate in the Musa balbisiana genome. (2.) Biological function of episomally-expressable BSV integrants. (3.) Identification and characterization of new badnaviruses. BAC library clones from M. balbisana were successfully sub-cloned and sequenced. The complete BSV-GF genome was identified in two separate BAC library clones. These data are now being analyzed, with the objective of proposing a mechanism by which the viral integrant could escape from the Musa genome and initiate an episomal infection. (2) Virus transmission tests using mealybugs are in progress in the greenhouse. The object of these experiments is to confirm initial results suggesting that episomally-expressable BSV integrants may protect plants against external infection. (3) A new badnavirus was identified in a cyad. This is the first instance of a badnavirus infecting a gymnosperm.

Impacts
These results provide a basis for the possible marker-based selection of parental genotypes whose progeny are less prone to develop disease due to activation of integrated plant pararetroviral sequences. Results obtained so far have shown that integrated pararetroviral sequences occur in a wide range of plant species. Although we have not been able to propose a feasible virus disease control strategy based on our knowledge of the nature of pararetroviral integrants in Musa, we have uncovered evidence of a dynamic molecular-genetic interaction between virus and host plant.

Publications

  • Thomas, J.E., Geering,A.D.W.,Dahal, G.,Lockhart, B.E.L., and Thottappilly, G. 2004. Banana and plantain. In. G. Loebenstien and G. Thottappilly, (eds). Virus and Virus-Like Diseases of Major Crops in Developing Countries. Kluwer Academic Publishers, 800pp
  • Rassaby, L., Girard, J-C., Lemaire, O., Costet, L.,Irey, M.S.., Kodja, H., Lockhart, B.E.L., and Rott, P. 2004. Spread of Sugarcane Virus in Sugarcane Plants and Feilds on the Island of Reunion. Plant Pathology 53:117-125
  • Momol, M.T., Lockhart, B.E.L., Dankers, H., and Adkins, S. 2004 Canna Yellow Mottle Virus Detected in Canna in Florida. Plant Health Progress, Aug. 2004


Progress 01/01/03 to 12/31/03

Outputs
Collaborative research with CIRAD (Centre de Cooperation Internationale en Recherche Agronomique pour le Developpement) in Montpellier, France on a genetic approach to the understanding of the activation of integrated plant pararetroviruses led to the identification of genetic markers linked to episomal expression of Banana streak virus (BSV) in interspecific Musa hybrids.

Impacts
These results provide a basis for the possible marker-based selection of parental genotypes whose progeny are less prone to develop disease due to activation of integrated plant pararetroviral sequences.

Publications

  • Lhereux, F., Carreil, F., Jenny, C., Lockhart, B.E.L. and Iskra-Caruana, M.L. 2003. Identification of genetic markers linked to banana streak disease expression in inter-specific Musa hybrids. Theor. Appl. Genet. 106:594-598.


Progress 01/01/02 to 12/31/02

Outputs
A second locus containing a new Banana streak virus (BSV) integrant was identified in a genomic library of Musa BB Pisang klutuk wulung. Two significant discoveries were made: 1) BSV sequences integrated in the Musa B genome are activated only in interspecific hybrids with the Musa A genome; and 2) viral sequences integrated in the Musa B genome confer immunity to infection by the homologous episomal virus.

Impacts
Discovery and characterization of new badnaviruses in citrus and temperate berry and ornamental crops make these studies more directly relevant to US agriculture. Discovery of immunity conferred by integrated pararetroviral sequences opens interesting new avenues of investigation of these viruses in both plants and animals.

Publications

  • Lockhart, B.E.L. and Geering, A.D.W. 2002. Partial characterization of two aphid-transmitted viruses associated with yellow leafspot of spiraea. Acta Hort. 568:163-168.
  • Harper, G., Hull, R., Lockhart, B. and Olszewski, N. 2002. Viral sequences integrated into plant genomes. Annu. Rev. Phytopathol. 40:119-136.
  • Jones, A.T., McGavin, W.J., Geering, A.D.W. and Lockhart, B.E.L. 2002. Identification of Rubus yellow net virus as a distinct badnavirus and its detection by PCR in Rubus species and in aphids. Ann. Appl. Biol. 141:1-10.


Progress 01/01/01 to 12/31/01

Outputs
We decided to focus on the banana streak virus (BSV) - Musa system to study episomal expression of integrated plant pararetroviral sequences because it seemed the most likely to yield useful results. Significant results obtained were: 1) at lease four episomally-expressible BSV integrants occur in Musa. 2) We have cloned and sequenced one of the new integrants, BSV-GF. 3) Episomally-expressible BSV integrants are associated with the Musa B genome but not the Musa A genome. A group of badnaviruses, transmitted by aphids rather than mealybugs, were identified in temperate crop species (Rubus, Ribes and Spiraea).

Impacts
The discovery that episomally-expressible BSV mutants are associated exclusively with the Musa B genome provides a vital tool for the genetic analysis of the phenomenon of de novo viral infection arising from integrated, pararetroviral sequences. Detection of badnavirus infection in the three temperate crop species has been greatly improved by the use of PCR amplification.

Publications

  • Geering, A.D.W., Olszewski, N.E., Dahal, G., Thomas, J.E. and Lockhart, B.E.L. 2001. Analysis of the distribution and structure of integrated Banana streak virus DNA in a range of Musa cultivars. Molecular Plant Path. 2:207-213.
  • Dallot, S., Acuna, P., Rivera, C., Ramirez, P., Cote, F., Lockhart, B.E.L., and Caruana, M.L. 2001. Evidence that the proliferation stage of micropropagation procedure id determinant in the expression of Banana streat virus integrated into the genome of the FHIA 21 hybrid (Musa AAAB).
  • Jones, A.T., McGavin, W.J., Geering, A.D.W., and Lockhart. B.E.L. 2001. A new badnavirus in Ribes species, its detection by PCR and its close association with gooseberry vein-banding disease. Plant Dis. 85:417-420.
  • Jones, A.T., McGavin, W.J., Geering, A.D.W., and Lockhart, B.E.L. 2001. Detection and relationships of two new badnaviruses from temperate berryfruit plants Phytopathology 91:S45.


Progress 01/01/00 to 12/31/00

Outputs
We continued research on the episomal expression of plant pararetroviral sequences integrated in the plant host genome. A new virus with these properties (Tobacco vein-clearing virus) was described. Two new banana streak badnavirus (BSV) integrants were discovered in Musa (banana and plantain). Field studies indicated that episomal virus isolates originating from viral sequences integrated in plantains (cooking bananas) were spreading to, and occurred frequently in, bananas grown for export. Episomally-expressible BSV integrants were shown to be associated with the Musa balbisiana (B) genome and not with the Mus acuminata (A) geonome.

Impacts
The research result of greatest practical significance was the discovery that episomally-expressible BSV integrants are associated with the B but not the A Musa genoma. This finding will have a profound effect on future strategies for disease and pest resistance breeding in banana and plantain.

Publications

  • Lockhart, B.E., Menke, J., Dahal, G., and Olszewski, N.E. 2000. Characterization and genomic analysis of tobacco vein-clearing virus, a plant pararetrovirus that is transmitted vertically and is related to sequences integrated in host genome. J. Gen. Virol. 81:1579-1585.
  • Hull, R., Lockhart, B. and Harpa, G. 2000. Viral sequences integrated into plant genomes. Trends in Plant Science. 5:362-365.
  • Ndowora, T.C.R. and Lockhart, B.E.L. 2000. Developoment of a serological assay for detecting serologically diverse banana streak virus isolates. Acta Hort. 540:377-388.


Progress 01/01/99 to 12/31/99

Outputs
The research focus has shifted to elucidating the molecular mechanisms by which pararetroviral sequences integrated into genomic DNA of plants give rise to episomal viral infection, as a similar mechanism may eventually be found to operate in animal systems. This phenomenon was first described for banana streak badnavirus (BSV) (Ndowora, et al, 1999). Subsequent studies have shown that analogous phenomena occur in tobacco (Lockhart, et al, 2000) and several other plant species, involving caulimo-like plant pararetroviruses (Lockhart, et al, in preparation).

Impacts
Twenty years of work to breed higher-yielding, pest-and disease-resistant cooking bananas for developing countries have been geopardized by the discovery that most of the improved varieties are infected with BSV. We have shown that this infection arises from viral sequences integrated into the host genome. It is important to understand the nature of this phenomenon in order to avoid a similar situation in future breeding programs.

Publications

  • Ndowora, T., Dahal, G., LaFleur, D., Harper, G., Hull, R., Olszewski, N., and Lockhart, B. 1999. Evidence that badnavirus infection in Musa can originate from integrated pararetroviral sequences. Virology. 255:214-220.
  • Gauhl, F., Pasberg-Gauhl, C., Lockhart, B.E.L., Hughes, J.d'A., and Dahal, G. 1999. Incidence and distribution of banana streak badnavirus in the plantain production region of southern Nigeria. Intl. J. Pest Management 45:167-171.
  • Lockhart, B.E. and Olszewski, N.E. 1999. Badnaviruses. In: Encyclopedia of Virology, 2nd Edition, pp. 1296-1300. A. Ganoff and G. Webster, eds., Academic Press, N.Y.


Progress 01/01/98 to 12/31/98

Outputs
A. Identification of badnaviruses of economic importance. 1. Spiraea yellow leafspot virus, a previously undescribed aphid-transmitted badnavirus was found to occur widely in spiraea, and is of serious concern to the nursery industry. A method for detection of this virus by PCR was developed. A similar disease in spiraea was associated with a spherical virus containing double-stranded RNA. B. Molecular biology/molecular genetics of badnaviruses. 1. Results were obtained to support the hypothesis that integrated plant pararetroviral sequences can give rise to de novo episomal virus infection. Genomic sequences of banana streak badnavirus integrated into the genome of Musa spp. were characterized. One such viral integrant was shown to be capable of giving rise to a replication-competent BSV genome by a process of homologous recombination.

Impacts
(N/A)

Publications

  • Cheng, C. P., Tzafrir, I., Lockhart, B.E.L., and Olszewski, N.E. 1998. Tubules containing virioins are present in plant tissues infected with Commelina yellow mottle badnavirus. J. Ge. Virol. 79:925-929.
  • Thottappilly, G., Dahal, G., and Lockhart, B.E.L. 1998. Studies on a Nigerian isolate of banana streak badnavirus. I. Purification and enzyme-linked immunosorbent assay. Ann. Appl. Biol. 132:253-261.
  • Dahal, G., Hughes, J. d'A. and Lockhart, B.E.L. 1998. Status of banana streak disease in Africa: problems and future research needs. Integrated Pest Management Reviews 3:85-98.
  • Garnsey, S.G., Bene, C.G. and Lockhart, E.E. 1998. Transmission of citrus yellow mosaic badnavirus by the citrus mealybug. Phytopathology 88:531.
  • Dahal, G., Ndowora, T.C.R., Olszewski, N.E. and Lockhart, B.E. 1998. PCR assay for detection of episomally-expressible integrated banana streak badnavirus sequences in Musa germplasm. Phytopathology 88:520.


Progress 01/01/97 to 12/31/97

Outputs
A. Identification of new badnaviruses of economic importance. 1. Piper yellow mottle virus, a mealybug-transmitted badnavirus occurring throughout southeast Asia, was identified and characterized. This virus is considered to be one of the factors causing decline in black pepper production in many areas of Southeast Asia. 2. An aphid-transmitted badnavirus infecting Spiraea, a widely-grown landscape plant, was identified. B. Improving understanding of the molecular biology of badnaviruses. 1. Research results have confirmed the hypothesis that badnaviruses are capable of integrating into the host plant genome and may be subsequently expressed under certain conditions of stress.

Impacts
(N/A)

Publications

  • Lockhart, B.E.L., Kiratiya-angul, K., Jones, P., Eng, L., DeSilva, P., Olszewski, N.E., Lockhart, N., Deema, N., and Sangalang, J. 1997. Identification of Piper yellow mottle virus, a mealybug-transmitted badnavirus infecting Piper spp. in Southeast Asia. European J. Plant Pathology 103:303-311.


Progress 01/01/96 to 12/30/96

Outputs
A new badnavirus infecting citrus in India was described. Because this virus is a potential pathogen of citrus in the U.S., collaboration was established with the USDA Hort. Res. Lab in Orlando, FL. to provide materials for virus indexing. It was demonstrated that genomic DNA of badnaviruses are integrated into the DNA of their plant hosts. Experimental evidence for this was obtained for Musa spp. (banana & plantain), and there was evidence that a similar phenomenon occurs in the case of sugarcane. There was also evidence that in vitro propagation of Musa led to the episomal expression of integrated badnavirus sequences. These findings have far-reaching implications for breeding & improvement of Musa.

Impacts
(N/A)

Publications

  • Ahlawat, Y.S., Pant, R.P., Lockhart, B.E.L., Srivastava, M., Chakraborty, N.K., & Varma, A. 1996. Assoc. of a badnavirus with citrus mosaic disease in India.
  • Lockhart, B.E.L., & Olszeski, N.E. 1996. Schefflera ringspot virus, a widely distributed mealybug-transmitted badnavirus occurring in Schefflera & Aralia. Acta Hort. 432:196-202.
  • Cheng, Chiu-Ping, Lockhart, B.E.L., & Olszewski, N.E. 1996. The ORFI and II proteins of Commelina yellow mottle virus are virion-associated. Virology 223:263-271.
  • LaFleur, D.A., Lockhart, B.E.L., & Olszewski, N.E. 1996. Portions of the banana streak badnavirus genome are integrated in the genome of its host Musa sp. Phytopathology 86:S100.


Progress 01/01/95 to 12/30/95

Outputs
In a world-wide survey conducted in collaboration with the International Networkfor Banana and Plaintain (INIBAP), banana streak badnavirus (BSV) was found to occur in 39 countries, and appears to occur in all banana and plantain-producing areas of the world. Particularly, troubling was the occurrence of BSV infrection in a large percentage of recently bred tetraploid hybrids which have good resistance to fungal diseases and nematode infestation. A study has been undertaken to determine the reason for BSV infection in these new Musa clones. A third-generation polyclonal rabbit antiserum capable of detecting almost all BSV isolates by ELISA was produced. Production of mouse monoclonal and chicken polyclonal antisera are now in progress to further improve ELISA detection of BSV. Evidence was obtained that segments of badnavirus genomic DNA are integrated into the host plant genome and experiments are in progress to confirm this. A new badnavirus infecting citrus in India was identified and named citrus mosaic badnavirus (CMBV).

Impacts
(N/A)

Publications


    Progress 01/01/94 to 12/30/94

    Outputs
    1) Improved serological detection of banana streak (BSV) and sugarcane bacilliform (ScBV) badnaviruses. A new polyclonal rabbit antiserum, capable of detecting a wider range of BSV and ScBV strains in Musa and sugarcane germplasm, was produced and tested. The antigen mixture used for immunization consisted of twenty-six virus isolates which showed serological relatedness and unrelatedness to the initial antiserum. Tests with the new antiserum detected a wider range of virus isolates in F(ab) ELISA than in DAS-ELISA. 2) PCR detection of badnaviruses. Immunocapture (IC) PCR was used in an attempt to improve detection of badnaviruses in crude leaf extracts. Although the immunocapture was successful, PCR amplification failed in many cases. Test results indicated that failure of PCR amplification was not due to the presence of enzyme inhibitors, but to wide genomic heterogeneity among virus isolates. Modifications of the thermal cycling program have shown promise in compensating for this factor. 3) Identification of virus associated with yellow leaf syndrom of sugarcane. Luteovirus-like particles were isolates from sugarcane infected with yellow leaf syndrome (YLS), a disease of previously unknown etiology. Isometnic virus particles and virus-associated dsRNA were isolated from YLS-infected sugarcane from Brazil and Florida. The virus was transmitted by Melanaphis sacchari, and was serologically unrelated to barley yellow dwarf virus (BYDV).

    Impacts
    (N/A)

    Publications


      Progress 01/01/93 to 12/30/93

      Outputs
      Research efforts during this year were concentrated on developing methods for reliable detection of badnavirus infection in sugarcane and Musa (banana and plantain) germplasm using PCR-amplification and serological methods, mainly amplified immunoblotting (western blotting) and modified immunoassay (EIA) procedures. Development of reliable diagnostic techniques for badnaviruses infecting these crops is of particular importance because it has now been found that a high proportion of Saccharum and Musa germplasm available for breeding programs worldwide is infected by these viruses. Results using PCR have been encouraging when partially purified extracts are used, but field-scale screening using crude extracts or leaf pieces is hindered by problems of inhibitory compounds which block amplification. Efforts are now underway to circumvent the problem of these inhibitory substances by modification of extraction procedures. A collaborative project with the USDA Sugarcane Field Station at Canal Point, Florida, to determine the effect of badnavirus infection on sugarcane yield has ended its first year, and plant data are currently being analyzed.

      Impacts
      (N/A)

      Publications


        Progress 01/01/92 to 12/30/92

        Outputs
        Attempts to develop sensitive indexing techniques to index germplasm for badnavirus infection revealed that individual badnavirus species (e.g., sugarcane bacilliform badnavirus) display a large degree of serological and genomic heterogeneity, thus, limiting the usefulness of immunological and nucleic acid hybridization assays. An alternative strategy, based on PCR amplification of conserved badnavirus genomic sequences, was successfully developed and tested. Current research now focuses on refining this detection method by optimizing the PCR primer sequences based on sequence data from a wider range of badnaviruses.

        Impacts
        (N/A)

        Publications


          Progress 01/01/91 to 12/30/91

          Outputs
          A major new member of the plant badnavirus virus was identified. The virus, named Piper yellow mottle virus (PYMV) was found in black pepper (Piper nignum) and betel (Piper betel) in Thailand, Sri Lanka and Malaysia. A similar disease has been reported from Brazil. Literature surveys indicate that this virus played a role in the decline and collapse of the black pepper industry in Indochina in the 1950's. Research was continued on badnaviruses in sugarcane and banana, particularly with respect to vector relationships and genomic variation and the implication of this latter phenomenon with respect to the development of nucleic acid probes for virus detection in planting stock.

          Impacts
          (N/A)

          Publications


            Progress 01/01/90 to 12/30/90

            Outputs
            Research was continued on developing methods for effective and reliable diagnosis of dsDNA viruses, which occur usually in very low concentration in plant tissue. During this year it was found that these viruses may exhibit both genomic and serological variability, even within a given virion population obtained from a single plant. This finding has led us to question the reliability of blot-hybridization assays using genomic clones. A project is underway to generate a series of monoclonal antibodies to sugarcane bacilliform virus in the hope of finding one or more which will detect a range of variants of that virus, as well as other members of the group. An alternative approach will be to attempt to produce RT (reverse-transcriptase)-specific probes, as this is the region of the genome which appears to be most conserved among the dsDNA viruses so far sequenced. ELISA and ISEM were used to identify the presence of maize chlorotic mottle virus (MCMV) for the first time in Hawaii. Serological comparisons revealed that the Hawaiian MCMV isolate differs from the U.S. mainland and original Peruvian strains of MCMV.

            Impacts
            (N/A)

            Publications


              Progress 01/01/89 to 12/30/89

              Outputs
              Major emphasis was placed on isolation and characterization of ds DNA bacilliform plant viruses, with one ultimate objective being the use of P-labelled cloned viral DNA as probes to detect these viruses in commercial crops. This approach is especially important because these viruses (I have proposed the name badnaviruses for this new group) occur in vegetatively propagated crops, and at certain periods virion concentration in infected plants falls beyond the range of detection by routine immunoenzymatic methods. Genomic DNA from two badnaviruses, sugarcane bacilliform virus (SCBV) and Kalanchoe top-spotting virus (KTSV) has been cloned. Work will continue on virus detection by blot-hybridization.

              Impacts
              (N/A)

              Publications


                Progress 01/01/88 to 12/30/88

                Outputs
                Tomato spotted wilt virus (TSWV) infectioin was identified as a major disease incommercial greenhouses throughout the year. This virus was identified in several new host plants including Kalanchoe, gloxinia, chrysanthemum, Exacum and Impatiens. The commercially available ELISA kit was unable to detect all strains of TSWV. A grant proposal has been submitted to the American Florists' Association (co-investigator, F. Pfleger) to develop serological detection methods for all isolates of this virus, which has caused significant economic losses throughout the U.S. during the past year. In keeping with the original aims of the project, techniques for virus detection have been expanded beyond ELISA and ISEM. Western blotting is now being used to detect rhabdoviruses occurring in cereals and potatoes. Although more lenghty, this immunoblotting procedure offers great sensitivity and nick-translated radiolabelled ( P) probes. Ash yellows was identified for the first time in Minnesota and a project designed to provide a serological detection method for ash yellows mycoplasma infectioin was initiated.

                Impacts
                (N/A)

                Publications


                  Progress 01/01/87 to 12/30/87

                  Outputs
                  In cooperation with the Pillsbury Company, antisera were prepared for ELISA testing of peas for pea seed-borne mosaic virus (PSBmv) in peas and for barley yellow dwarf virus (BYDV) in sweet corn. An employee of the company was trained in serological assay techniques, which are now done routinely by the company. The genotype-environment interactions of the BYDV-induced red-leaf disease of sweet corn were identified. The disease was produced under conditions of moderate temperatures when a particular inbred line was used as parent in crosses to produce hybrids. Potato yellow dwarf virus was identified in ornamental plants for the second successive year. There had been no reports of this virus in the Midwest since 1942. More than a dozen new plant virus antisera were fabricated or purchased in order to expand the range of antisera available for testing ornamental and field crops. Broad bean wilt virus (BBWV) was identified as the cause of a widespread disease of Spathyphyllum and Schefflera in commercial greenhouses in Minnesota. A similar disease of Gloxinia is under study. Collaboration with colleagues overseas has continued on viruses that are of potential interest.

                  Impacts
                  (N/A)

                  Publications


                    Progress 01/01/86 to 12/30/86

                    Outputs
                    This project has been active for approximately 8 months. Much of that time has been spent setting up laboratory equipment and facilities. Elucidation of the virus disease situation in canning peas. This involves clearly defining the role of pea seed-borne mosaic virus (PSBMV) and development of a sensitive detection system for this virus. The role of non-seed transmitted viruses (e.g. clover yellow-vein virus) is also being studied. Barley yellow dwarf virus (BYDV) in sweet corn. BYVD was identified as the cause of the red-leaf/stunt syndrome that occurred in near-epidemic proportions during summer of 1986. Virus-host genotype interactions and serological detection methods are being studied. Potato yellow dwarf virus (PYDV) was identified for the first time in the Midwest. The epidemiology of the virus disease is being studied. Immunoenzymatic detection of ornamental plant viruses, principally of bulbous crops, has been started.

                    Impacts
                    (N/A)

                    Publications


                      Progress 01/01/81 to 12/30/81

                      Outputs
                      Activities dealt mainly with investigation ornamental virus diseases. Leaf-dropnecrosis of peperomia in commercial greenhouses identified as caused by tomato aspermy virus. Yellow stunting and mosaic of begonia found due to virus related to clover yellow mosaic virus. Virus disease of impatiens found caused by virus related to heracleum lantent virus. All previously undescribed diseases. Publications in preparation for all three. Research also done on aphid transmission of tomato aspermy virus in tomato in Minnesota. No aphids found able to transmit virus in spite of natural spread.

                      Impacts
                      (N/A)

                      Publications


                        Progress 01/01/80 to 12/30/80

                        Outputs
                        The major emphasis during this year was the identification of viruses of economic importance in ornamentals and vegetables. This work included, besides identification of viruses, a study of the means of spread and methods of control. Viruses causing significant damage were found in pepperomia, begonia, fuschia and several other ornamentals. Two viruses from begonia were found to be highly virulent on pea, an important legume crop in Minnesota, and these and other ornamental viruses are therefore established as potential pathogens to other crops in the state. One of those viruses, a new strain of clover yellow mosaic virus, has been reported previously only from the western United States, and the other, broad bean wilt virus, has been reported only from upstate New York and from South Carolina. This finding emphasizes the role of ornamentals in the spread of pathogens from one area to another, and points out the need for more stringent regulations on interstate movement of plant materials. Additional work was carried out in cooperation with the Department of Horticulture for the serological detection of pea seedborne mosaic virus in pea breeding lines.

                        Impacts
                        (N/A)

                        Publications


                          Progress 01/01/79 to 12/30/79

                          Outputs
                          It was concluded that virus diseases in Minnesota were of greatest economic importance on ornamental plants in commercial greenhouses. This is because a) many of these plants are vegetatively propagated; b) viruses are being imported in infected planting stock from other states or even overseas, and c) the protected environment allows virus spread and insect vector activity throughout the winter. Virus diseases identified as being of economic importance were caused by tobacco ringspot virus in Impatiens and Begonia, tobacco streak in Impatiens, a newly described Impatiens mild mottle virus and a newly described Tradescantia mosaic virus. Watermelon mosaic virus was found to be capable of causing severe damage in cucurbits in home gardens, but that the incidence of the disease was related to insect vector populations and stage of plant growth. Tomato aspermy virus, widespread in Chrysanthemum but not reported from tomato, was found frequently in tomatoes in 1979. The virus was not transmissible by aphids (Myzus persicae, Macrosiphum euphorbiae) commonly found on tomato and the reason for this outbreak is under study. A disease of ornamental buckthorn (Rhamnus sp.) was found to be caused by tomato ringspot virus infection. An epiphytotic of cucumber mosaic on peppers in commercial pepper fields, which caused almost total yield losses in many cases, was found to be probably due to importation of infected seedling transplants from Georgia.

                          Impacts
                          (N/A)

                          Publications