Location: Floral and Nursery Plants Research2014 Annual Report
The objectives of this project are 1) Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops and develop corresponding diagnostic testing methods; 2) Determine the genome organization of selected important ornamental viruses and utilize full-length infectious clones to determine the genes or gene products involved in replication, systemic movements, and pathogenicity; and 3) identify, detect, and gain a better understanding of genetic relationships and molecular basis of pathogenicity to facilitate effective control of bacterial diseases of major significance to ornamental and agronomic crops. The long-term objective of this project is to develop effective means for the detection and identification of new and emerging plant viral and bacterial diseases of ornamentals, thus allowing growers to select pathogen-free or pathogen-indexed plants for propagation. Improved detection and differentiation methods for these pathogens will enable state and federal regulatory officials to make timely and appropriate recommendations in safeguarding the movement of horticultural and agricultural products into the United States. Understanding viral and bacterial genome structures and functions, their mechanisms of pathogenicity and resistance, and conferring virus and bacterial resistance in plants will lead to the development of better disease control measures and increases in both productivity and quality of ornamental plants for industry and the consumer.
Characterize viruses of major significance to ornamental and nursery crops, including uncharacterized or emerging viruses affecting key ornamental crops, and develop corresponding diagnostic testing methods. The overall approach is to develop knowledge, tools, and reagents that will aid U.S. floriculture companies to establish effective virus testing protocols and improve clean stock production for new vegetatively propagated annuals and perennials. Based on the knowledge and tools developed while identifying and characterizing new viruses, new virus-specific and broad-spectrum polyclonal and/or monoclonal antibody reagents, purification protocols, molecular nucleic acid hybridization probes, PCR primers, and improved associated protocols will be developed. A Universal Plant Virus Microarray, currently being developed cooperatively under an NRI grant, will add new capabilities in identifying newly emerging viruses to the appropriate family and genus, thus significantly aiding selection of appropriate methods for further characterization. Research will initially focus on those "new" currently uncharacterized or emerging viruses affecting key ornamental crops recently identified as significant to the floral and nursery industry. Determine the genome organization of selected important ornamental viruses and utilize full-length infectious clones to determine the genes or gene products involved in replication, systemic movement, and pathogenicity. Infectious clones of selected viruses will be modified by gene exchange and site-directed mutagenesis. Interactions between viral gene products, and between viral and host proteins, using yeast two-hybrid, bimolecular fluorescence complementation, and GST-pull down assays will be examined. Virus-induced gene silencing (VIGS) and/or protein over-expression will also be utilized. Identify, detect, and gain a better understanding of genetic relationships and molecular basis of pathogenicity to facilitate effective control of bacterial diseases of major significance to ornamental and agronomic crops. Research will be conducted on Xylella fastidiosa genome characterization, and specific detection and identification of ornamental strains, as well as genetic relationships among ornamental and non-ornamental strains of X. fastidiosa. Using high throughput sequencing and comparative genomics, a better understanding of their molecular basis of pathogenicity will be retained. Current detection and identification methods for the select agent pathogen Ralstonia solanacearum race 3 biovar 2 will be improved using comparative genomics to develop, for example, a multiplex PCR.
Progress was made on all three objectives, which fall under NP303. Under Objective 1a: Additional isolates of Alternanthera mosaic virus (AltMV) were detected in Phlox stolonifera and P. divaricata, and in Mazus reptans; detection in M. reptans constitutes the first report for this host, and in mixed infection with Ligustrum necrotic ringspot virus. Plantago asiatica mosaic virus (PlAMV-MD) was detected for the first time in the United States in lily bulbs of several cultivars, all imported from the Netherlands. Although Nandina mosaic isolates of PlAMV have been known in the U.S. since 1978, Nandina mosaic isolates have never been reported to infect lilies. As PlAMV is known to cause significant losses in cut-flower lilies in both Europe and Asia, there is potential for economic loss to lily growers in the U.S. This work complements a collaboration with Dutch and Italian scientists to generate an infectious clone (Objective 2) of PlAMV to investigate transmission and movement of PlAMV within lilies. Thirteen species of ornamental Allium are being investigated for the presence of virus in collaborative research with a graduate student from Brazil. At least five viruses have been identified to date, including one potyvirus not previously reported to infect Allium species, as well as one carlavirus and three allexiviruses previously known to infect garlic, onion, or leeks. Two distinct sequences of Butterbur mosaic virus and one sequence of Helenium virus S (both carlaviruses) were obtained from a single plant of veronica showing mosaic symptoms (collaboration with National Germplasm Resources Laboratory). A new potyvirus, Shamrock chlorotic ringspot virus, was detected in ornamental Oxalis. At least one of these plants was also infected with a nepovirus, Beet ringspot virus; this is the first report of this virus in Oxalis. A U.S. isolate of Catharanthus mosaic virus was detected in Mandevilla; this is the first report of this virus in this host. All three of these viruses have been partially to fully sequenced. (Collaboration with National Germplasm Resources Laboratory and Michigan Dept. of Agriculture). Kalanchoe latent virus (carlavirus) and Kalanchoe mosaic virus (potyvirus) were detected in a Sedum plant with ringspots (collaboration with Michigan Dept. of Agriculture). Under Objective 1b: Additional members of the Alphaflexiviridae and Betaflexiviridae were successfully amplified using generic primers, and sequences obtained. Virus-specific primers were developed and tested for several species, including Ligustrum necrotic ringspot virus, Narcissus yellow stripe virus, Shamrock chlorotic ringspot virus, Catharanthus mosaic virus, and Beet ringspot virus. Several viruses detected under Objectives 1a and 1b were further characterized and partially sequenced. Under Objective 1c: Additional viruses, including some mixed infections, were successfully detected with the Universal Plant Virus Microarray (UPVM). In collaboration with scientists at the Danforth Center and the University of Utah, more than 100 additional UPVM slides were hybridized with test samples obtained from the DSMZ (Germany); the International Potato center (Peru); the International Center for Tropical Agriculture, Colombia; Washington State University, Pullman and Prosser, WA; from a USDA-ARS lab in Wooster, OH; and from our own local collections, and used to further validate the UPVM. Including these samples, the UPVM was validated for several additional viruses of different taxonomic groups, expanding the number of genera from which viral species have been correctly detected and identified to at least 44 genera and recognized taxonomic groups representing at least 15 families (with two genera unassigned to families). Two previously unassigned viruses were correctly identified to appropriate taxonomic groups. In addition, two recently-described viruses not represented by species-specific probes were identified to the correct genus. Nucleic acid extracts of several symptomatic samples (including ‘unknown’ field samples from the International Potato Center), and previously characterized samples either treated by subtractive hybridization or untreated, were submitted for next generation sequencing (NGS) as well as being hybridized to the UPVM. The raw NGS results have been received, but not yet analyzed. Comparison of NGS results to UPVM results may reveal viruses not detected by the UPVM, due to either low titer (potentially including secondary components of mixed infections) or possibly novel viruses without representation among the 9556 virus-specific probes of the UPVM. Comparison of virus detection and identification in parallel samples either treated by subtractive hybridization, or not treated, will aid in determination of the cost-effectiveness of this treatment for both the UPVM and NGS approaches. Under Objective 2.1: Polyclonal and monoclonal antibodies to movement protein 1, MP-1 of various pelarspoviruses (using synthetic peptides containing species unique or genus conserved amino acid sequences) that are either specific to one species or cross-reactive to all species, respectively were developed. These antibodies will be useful in confirming the gene exchange of the MP-1 gene between infectious clones of these viruses as well as in experiments to determine the expression, localition, and functionality of the protein in viral pathogenesis. Under Objective 3.1: Sequence gaps that were lacking in our draft genome sequence of the mulberry strain of X. fastidiosa have been closed. Some open reading frames were manually annotated and the draft genome of this mulberry strain published. This mulberry strain was compared with other strains of X. fastidiosa, its unique open reading frames and designed primers identified. Those primers are now being tested for specific detection of this particular strain. The genes used in MLST in the mulberry strain were identified and used in establishing phylogenetic relationships between the mulberry strain and other X. fastidiosa strains. Under Objective 3.2: A multiplex PCR assay that includes using a host plant primer pair as an internal control was developed and tested, as well as R. solanacearum species- and r3b2-specific primer pairs, on 90 target and non-target R. solanacearum strains, including samples from soil and from different artificially infected plants including potato, geranium, and tomato to insure its specificity. Using the unique sequences that were previously identified, R. solanacearum species- and r3b2-specific, as well as plant-specific primers and probes were designed in order to develop a multiplex qPCR assay for R. solanacearum.
1. Determination of the complete genome sequence of a Xylella fastidiosa strain causing mulberry leaf scorch disease in Maryland. Xylella fastidiosa is a Gram-negative, nutritionally fastidious, xylem-inhabiting and insect-transmitted bacterium affecting over 30 plant families and causes bacterial leaf scorch and decline in many important woody ornamentals, including mulberry. ARS researchers in Beltsville, Maryland determined the draft genome of the mulberry strain (Mul-MD) in order to gain a better understanding of the molecular basis of strain divergence, host specificity, nutrient requirements, and pathogenicity, as well as to develop genome-based specific detection methods for this important plant pathogen. Understanding bacterial genome structures and functions, and the mechanisms of bacterial pathogenicity, will lead to a better understanding of host-pathogen interactions and mechanisms of plant disease resistance. This, coupled with more sensitive pathogen detection methods, will lead to the development of better, more environmentally friendly, disease control measures and increases in both productivity and quality of ornamental plants for industry and the consumer.
Seo, E., Nam, J., Kim, H., Lakshman, D.K., Bae, H., Hammond, J., Lim, H. 2014. Selective interaction between Chloroplast B ATPase and TGB1 retards severe symptoms caused by Alternanthera mosaic virus infection. Plant Pathology Journal. 11736-11743.
Bobev, S.G., Taphradjiiski, O.I., Hammond, J., Vaira, A. 2013. First report of Freesia sneak virus associated with foliar necrosis of Freesia refracto in Bulgaria. Plant Disease. 97:1514.
Nam, J., Nam, M., Bae, H., Lee, C., Lee, B., Hammond, J., Lim, H. 2013. AltMV TGB1 nucleolar localization requires homologous interaction and correlates with cell wall localization associated with cell-to-cell movement. Plant Pathology Journal . 29(4):454-459.
Cho, S., Kim, J., Li, M., Seo, E., Lim, S., Moon, J., Hammond, J., Lim, H. 2013. Occurrance in Korea of three major soybean viruses, Soybean mosaic virus (SMV), Soybean yellow mottle mosaic virus (SYCMV), and Soybean yellow common mosaic virus (SYCMV) revealed by a nationwide survey of soybean fields. Research in Plant Disease. 19(4):313-325.
Li, M., Kim, J., Seo, E., Hwang, E., Domier, L.L., Hammond, J., Youn, Y., Lim, H. 2014. Sequence variability in HC-Pro genes of Korean Soybean mosaic virus isolates is associated with differences in gene silencing suppression. Archives of Virology. 159(6):1373-1383.
Lim, H.S., Vaira, A., Bae, H., Jang, C., Nam, J., Lee, C., Lee, Z., Hwang, J., Kim, H., Roh, M.S., Hammond, J. 2014. The coat protein of Alternanthera mosaic virus is the elicitor of a temperature-sensitive systemic necrosis in Nicotiana benthamiana, and interacts with a host boron transporter protein. Virology. http://dx.doi.org/10.1016/j.virol.2014.01.021.