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ARS Home » Northeast Area » Washington, D.C. » National Arboretum » Floral and Nursery Plants Research » Research » Research Project #423161

Research Project: NEW AND EMERGING VIRAL AND BACTERIAL DISEASES OF ORNAMENTAL PLANTS: DETECTION, IDENTIFICATION, AND CHARACTERIZATION

Location: Floral and Nursery Plants Research

2015 Annual Report


Objectives
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.


Approach
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 Report
Under Objective 1a: Additional imported Asiatic and Oriental lily bulbs were found to be infected with Plantago asiatica mosaic virus (PlAMV); in 2015 every single bulb (four bulbs each of four cultivars of Asiatic lilies, and four bulbs each of three Oriental lily cultivars) were found to be infected by PlAMV. The virus was also detected in the packing material around the bulbs, suggesting exudation of PlAMV from the developing roots of the bulbs. A method using agarose to stabilize the root:growing media matrix prior to fixation for electron microscopy of root tissue of lilies and Nicotiana benthamiana infected with PlAMV was developed to examine the possible presence of exuded virus at the root surface. Virus-infected root cells were identified, but no virus has been detected external to the root so far. A survey of viruses present in ornamental Allium species (collaboration with a graduate student from Brazil) identified Leek yellow stripe virus, Onion yellow dwarf virus, and Narcissus yellow stripe virus (NYSV), all potyviruses. NYSV has not previously been reported from any Allium species. Also detected were: Shallot latent virus and Garlic common latent virus (carlaviruses), the allexiviruses Garlic virus A, Garlic virus B, Garlic virus C, Garlic virus D, Garlic virus E, the Garlic mite-borne filamentous virus, and Shallot virus X. Identification of all viruses was by PCR with genus-generic and virus-specific primers; and, for several viruses, was also confirmed by ELISA with virus-specific antibodies, or by sequencing of cloned PCR products. Two distinct viruses, a new potyvirus (Shamrock chlorotic ringspot virus; SCRV) and a nepovirus, Beet ringspot virus (BRV), found in a mixed infection of ornamental Oxalis exhibiting chlorotic ringspot symptoms, were studied further. The full length genomes of both viruses were determined. Preliminary results indicate that the symptoms present in dually-infected plants are caused solely by SCRV. The full length genome of the United States isolate of Catharanthus mosaic virus was also determined. (Collaboration with National Germplasm Resources Laboratory and Michigan Department of Agriculture personnel). A mixed infection of Veronica with two distinct isolates of Butterbur mosaic virus (ButMV) and an isolate of Helenium virus S (HelVS) has been examined in greater detail. The two ButMV isolates have distinct sequences across the regions that have been sequenced to date (>3 kb from the 3' end). One of these ButMV isolates has been determined to be defective, having a major deletion in one gene; the defective isolate is thus presumed to be dependent on the other ButMV isolate for functions encoded by the deleted gene. Approximately 3.1 kb of the 3' end of the HelVS genome has also been determined, and shows significant levels of identity to partial sequences of isolates from Helenium and Impatiens (collaboration with National Germplasm Resources Laboratory). Under Objective 1b: Garden roses, which form the cornerstone of the multi-billion dollar landscape industry, annually generate wholesale U.S. domestic production valued at ~ $400 million. Over the past few decades Rose Rosette Disease has become very serious and threatens to decimate the United States Rose industry. The causal agent, Rose rosette virus (RRV), is transmitted by wind-blown eriophyid mites, and can kill a rose within 2-3 years of infection. The nucleoprotein (NP) from several Maryland isolates of RRV have been cloned and sequenced and shown to have high sequence homology with 22 other U.S. isolates. In collaborative research (a USDA-NIFA-SCRI Project) to develop efficient serological diagnostic tools to enable the rapid, user-friendly and accurate detection of the virus, the NP of RRV has also been cloned into a bacterial expression vector. Purified expressed protein will be used as an immunogen for production of polyclonal, monoclonal and/or single-chain antibodies. Synthetic peptides from highly conserved regions of the NP have also been developed to be used as immunogen and selecting antigen. Specific primers were developed for the detection and identification of several viruses, including: Plantago asiatica mosaic virus, Helenium virus S, Butterbur mosaic virus, Shamrock chlorotic ringspot virus, Catharanthus mosaic virus, and Beet ringspot virus. Under Objective 2a: The complete sequence of an isolate of Plantago asiatica mosaic virus (PlAMV) was determined, and two infectious clones of PlAMV differing in symptom severity were generated (collaboration with Italian and Dutch scientists). A major portion of the genome of two distinct isolates of NYSV from ornamental Allium has been determined (collaboration with a graduate student from Brazil). Partial sequences of additional isolates of Alternanthera mosaic virus were determined. The P1 genes of two distinct (N- and S-type) isolates of Leek yellow stripe virus were separately expressed in Nicotiana benthamiana as an additional gene from an Alternanthera mosaic virus protein expression vector. The P1 proteins were also expressed in Nicotiana benthamiana as GFP fusion proteins by agro-infiltration to examine sub-cellular localization. Some increase in symptom severity was observed when the P1 protein of the S-type isolate was expressed from AltMV (collaboration with a graduate student from Brazil). Infectious clones of Alternanthera mosaic virus and Soybean yellow common mosaic virus have been adapted for use as virus-induced gene silencing vectors and applied to screen whitefly cDNA sequences for RNAi against whiteflies, and for identification of soybean gene functions, respectively (collaboration with Korean scientists). 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 are being used in ongoing experiments confirming the gene exchange of the MP-1 gene between infectious clones of these distinct viruses, as well as in experiments to determine the expression, location, and functionality of the MP-1 protein in viral pathogenesis. Under Objective 2b: A yeast two-hybrid cDNA library of Nicotiana benthamiana was generated for examination of further host proteins interacting with the proteins of Alternanthera mosaic virus, Lolium latent virus, Plantago asiatica mosaic virus, and other viruses of interest. The sub-cellular localization of three proteins of several isolates of Turnip mosaic virus has been examined (as fusions to Green fluorescent protein) by confocal microscopy, with differences in localization associated with some amino acid variants between the equivalent proteins of different isolates. Sub-cellular localization of two proteins of each of two isolates of Barley stripe mosaic virus was also examined (collaboration with Korean scientists and graduate students). Under Objective 3.1: Several remaining ‘gaps’ in the genome of the sycamore strain of X. fastidiosa were determined. Some open reading frames were manually annotated and the draft genome of this sycamore strain was published. Based on our previously published genome sequence of the mulberry-infecting strain of X. fastidiosa, we identified its unique open reading frames. One of the unique gene sequences was also identified in the recently sequenced olive-associated strain CoDiRO isolated in Italy. Primers designed based on this unique sequence were successfully tested in both the U.S. and Italy against targeted and non-targeted X. fastidiosa strains in cultures and in naturally infected plant samples to ensure their specificity. Under Objective 3.2: A multiplex PCR assay was developed and tested that includes 1) a plant primer pair as an internal control; 2) R. solanacearum species complex; and 3) r3b2-specific primer pairs against 95 target and non-target R. solanacearum, as well as out-group bacterial strains. The assay was also tested against different plants including potato, geranium, tobacco and tomato artificially inoculated with r3b2 or non-r3b2 strains of R. solanacearum. Based on the unique r3b2 and plant mitochondrial cytochrome oxidase subunit 1 (cox1) sequences previously identified by us, we designed r3b2- and plant-specific primers and probes. We also designed phylotype II or the newly proposed R. solanacearum species-specific primers and a probe based on previously published phylotype II primers and their amplicon sequence. We tested the multiplex qPCR assay in vitro and in planta. We also developed a simple extraction method using an extraction buffer we designed to reduce PCR inhibition from geranium tissues for easy and fast processing of infected plant samples.


Accomplishments
1. A multiplex PCR assay to detect and differentiate select agent strains of Ralstonia solanacearum. The fungal pathogen R. solanacearum race 3 biovar 2 causes destructive brown rot of potato and is capable of surviving and infecting at lower temperatures. Current select agent regulations list all strains of R. solanacearum as select agents unless further testing can exclude them from race 3 biovar 2. ARS researchers at Beltsville, Maryland, developed a molecular assay that can identify whether a strain of R. solanacearum is race 3 biovar 2, and also exclude false negatives caused by unsuccessful DNA extraction or PCR inhibition. Our rapid, accurate, and reliable detection assay can help government officials make timely and appropriate recommendations to exclude this bacterium from the United States.

2. Specific detection and identification of American mulberry-infecting and Italian olive-associated strains of Xylella fastidiosa by polymerase chain reaction. X. fastidiosa causes bacterial leaf scorch in many important landscape trees and was also recently identified for the first time in Italy in olive trees affected by a devastating disease named “Olive Quick Decline Syndrome”. ARS researchers at Beltsville, Maryland, in collaboration with an Italian scientist have developed a PCR assay that can specifically detect and identify both the mulberry-infecting strains of X. fastidiosa strains in the U.S. and the olive-associated strains in Italy. The PCR assay will be valuable for disease diagnosis, studies of strain-specific infections in insects and plant hosts, and management of diseases caused by X. fastidiosa. It will also be useful for detection and identification of the new Italian group of X. fastidiosa strains to aid understanding of the occurrence, evolution, and biology of this new group.

3. Determination of the genome sequence of a Xylella fastidiosa strain causing sycamore leaf scorch disease in Virginia. X. fastidiosa causes bacterial leaf scorch in many important landscape trees, including sycamore. The disease is spread by insect vectors, with different strains of the bacteria infecting different plant species. ARS researchers at Beltsville, Maryland, determined the draft genome sequence of the sycamore strain ‘Sy-VA’ in order to gain a better understanding of the molecular basis of strain divergence, host specificity, nutrient requirements, and pathogenicity. This information will also be used to develop genome-based specific detection methods for this important plant pathogen.

4. Determination and recognition of a new genus, Pelarspovirus, in the plant virus family Tombusviridae. Plant viruses are responsible for millions of dollars of losses to agronomic crops, vegetables, fruits, and ornamentals. Understanding the relationships and identity of viruses is critical to pathogen detection, diagnosis, and control. By studying viruses in geranium, an ARS scientist in Beltsville, Maryland, and cooperators proposed that six virus species are distinct enough to be given their own genus, named Pelarspovirus, within the family Tombusviridae. This discovery was made by studying biochemical and genomic features that differentiate these species from other related species. Characterization and classification of these and other plant viruses will aid in the detection and identification of new and emerging viruses infecting ornamentals. This knowledge will be used by state and federal regulatory officials to make timely and appropriate recommendations in safeguarding the movement of horticultural and agricultural products into and through the United States.


Review Publications
Guan, W., Shao, J.Y., Davis, R.E., Zhao, T., Huang, Q. 2014. Genome sequence of a Xylella fastidiosa strain causing sycamore leaf scorch disease in Virginia. Genome Announcements. 2(4):e00773-14.
Hammond, J., Bampi, D., Reinsel, M.D. 2015. First report of Plantago asiatica mosaic virus in imported Asiatic and Oriental lilies (Lilium hybrids) in the United States. Plant Disease. 99:292.
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.
Seo, E., Cho, S., Moon, J.S., Gotoh, T., Kim, H., Domier, L.L., Hammond, J., Lim, H., Song, K. 2015. A high throughput soybean gene identification system developed using soybean yellow common mosaic virus (SYCMV). Journal of Faculty of Agriculture. 60:127-138.
Ko, N., Kim, H., Kim, J., Cho, S., Seo, E., Kwon, H., Yu, Y., Gotoh, T., Hammond, J., Youn, Y., Lim, H. 2015. Developing an Alternanthera mosaic virus vector for efficient clonging of Whitefly cDNA RNAi to screen gene function. Journal of Faculty of Agriculture. 60:139-149.
Guan, W., Shao, J.Y., Davis, R.E., Zhao, T., Huang, Q. 2015. Specific detection and identification of mulberry-infecting strains of Xylella fastidiosa by polymerase chain reaction. PLoS One. DOI: 10.1371/journal.pone.0129330.
Scheets, K., Jordan, R.L., White, A., Hernandez, C. 2015. Pelarspovirus, a proposed new genus in the family Tombusviridae. Archives of Virology. 160:2385-2393.
Scheets, K., Hernandez, C., Jordan, R.L., White, A. 2014. Create four new unassigned species in the family Tombusviridae. Archives of Virology. DOI:10.1007/s00705-015-2500-5.