Location: Floral and Nursery Plants Research2013 Annual Report
1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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.
3. Progress Report:
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 Celosia hybrids from different sources. Several viruses were detected for the first time in new hosts (Ornithogalum mosaic virus in Ixia and Crocosmia; Triteleia mosaic virus in Brodaiea; Ligustrum necrotic ringspot virus in P. divaricata). Potentially ‘new’ potyviruses were detected in Aconitum and Dichlostemma, and Aconitum latent virus carlavirus was detected in Aconitum (a new report for the U.S.). Parts of this work were carried out in collaboration with scientists at Agdia, Inc.; Michigan Dept. of Agriculture; and the Univ. Maryland. Under Objective 1b: Additional members of the Alphaflexiviridae and Betaflexiviridae were successfully amplified using generic primers, and sequences obtained. Under Objective 1c: Additional viruses, including some mixed infections, were successfully detected with the Universal Plant Virus Microarray (UPVM). Methods for normalizing amplification of viral and host RNAs were improved, allowing increased sensitivity of detection of low-titered. Several viruses detected under Objective 1a were further characterized and partially sequenced. Under Objective 2a: Interactions of several natural and mutant sequence variants of AltMV TGB1 were examined with a number of host proteins using Bimolecular Fluorescence Complementation (BiFC), in collaboration with a scientist at Chungnam National University (Korea) and at Beltsville using laser scanning confocal microscopy (LSCM). Under Objective 2b: Interactions of AltMV CP and TGB3 with plant host proteins were examined by LSCM, BiFC, and yeast two-hybrid analysis. Monoclonal antibodies were developed to several unique and conserved regions of the predicted movement protein of several pelarspoviruses. Under Objective 3.1: We obtained 20x draft genomes of four landscape strains of X. fastidiosa and are almost done with a fifth strain. We examined published MLST primers in the sequenced strains of X. fastidiosa. Under Objective 3.2: By using in silico genome subtraction, we identified 76 non-phage related fragments unique only to the r3b2 select agent strains of R. solanacearum. Similarly, we also identified sequences conserved among strains belonging to Ralstonia species complex, but not in other closely related species, as well as other plant pathogenic bacteria including Xanthomonas campestris and Pseudomonas syringii. With this information we designed and tested primers that are specific to R. solanacearum species complex, and to r3b2, respectively. We also developed a multiplex PCR assay by combining the species-, r3b2-, and plant-specific primers in a single reaction to allow fast and reliable detection and differentiation of r3b2 strains in plant extracts. Summaries to document research conducted under nine Specific Cooperative Agreements or Reimbursable Agreements between the Floral and Nursery Plants Research Unit and others can be found in those specific annual reports.
1. Improvement of current detection and identification methods for select agent strains of Ralstonia solanacearum via a multiplex PCR assay. R. solanacearum is a species complex that is pathogenic to a wide range of plant species in many regions throughout the world. Specifically detecting one subgroup of strains, the race 3 biovar 2 (r3b2) strains, is of utmost importance, since they are adapted to temperate climates and are listed as select agents due to their potential threat to US agriculture. ARS scientists at Beltsville, MD have designed R. solanacearum species- and r3b2-specific primers in a multiplex PCR assay, allowing for simultaneous detection and differentiation of r3b2 from non-r3b2 strains of R. solanacearum in a single reaction. An internal plant DNA control primer pair was also developed and included in the multiplex assays to improve the confidence and reliability of r3b2 detection in plant extracts by validating the plant extracts and excluding false negative results. Rapid, accurate, sensitive, and reliable detection and identification of r3b2 are critical for state and government officials to make timely and appropriate recommendations in safeguarding the movement of agricultural and horticultural products into the United States.
2. Critical interactions between viral proteins and plant host proteins. Plant viruses of the genus Potexvirus cause disease in a wide variety of crops; among these viruses, Alternanthera mosaic virus (AltMV) infects many taxonomically diverse ornamental crops. ARS scientists in Beltsville, MD, in collaboration with a scientist from Chungnam National University, Korea, have identified protein interactions between AltMV and the chloroplast of host plants that are critical in disease development. Mutation and gene expression studies revealed the role that these proteins may play in viral replication efficiency and movement, as well as the importance of chloroplast interactions in diseases caused by AltMV. This discovery will potentially allow disruption of the virus infection process through selection of plants that have protein variants that do not allow successful virus interactions, thereby leading to the development of virus-resistant plants.
3. First Report of Nerine yellow stripe virus in Amaryllis in the United States. Ornamental flower bulbs, including the well-known amaryllis cultivars, are increasingly important floriculture crops. A home gardener in California observed mosaic symptoms on the leaves of an Amaryllis belladonna plant growing in her garden. ARS Researchers in Beltsville, MD tested these leaves using PCR and electron microscopy and found that they contained a type of Potyvirus. Further analysis of DNA sequences and serological testing revealed the virus to be a unique US isolate of Nerine yellow stripe virus (NeYSV) that we designated NeYSV-US. NeYSV has previously been reported in the United Kingdom, the Netherlands, Australia, and New Zealand, but to our knowledge, this is the first report of Nerine yellow stripe virus in the United States.
Damsteegt, V.D., Stone, A.L., Smith, O.P., Mcdaniel, L., Sherman, D.J., Dardick, C.D., Hammond, J., Jordan, R.L., Schneider, W.L. 2013. A previously undescribed potyvirus isolated and characterized from arborescent Brugmansia. Archives of Virology. 158:1235-1244.