Location: Floral and Nursery Plants Research2012 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:
This report documents progress for Project Number 1230-22000-032-00D, which started in May 2012 and continues research from Project Number 1230-22000-022-00D, entitled “Detection, identification and characterization of new and emerging viral and bacterial diseases of ornamental plants" which terminated in April 2012. Ligustrum necrotic ringspot virus was detected for the first time in plants of Mazus radicans, in addition to further plants of Mazus reptans; this virus has previously been reported only in Ligustrum (privet), apart from our first identification and reports in New Guinea Impatiens, Phlox stolonifera and Mazus reptans. Nerine yellow stripe virus (NeYSV) was detected for the first time in Amaryllis in California. NeYSV has previously only been reported in the United Kingdom, the Netherlands, Australia, and New Zealand. A putative carlavirus has been identified by electron microscopy and a generic polymerase chain reaction (PCR) assay in Magnolia tripetala showing significant mosaic and ringspot symptoms from two locations; cloning and sequencing of the PCR product is in progress to further identify the virus. In collaboration with a plant breeder, we have previously identified the causal agent of a mosaic disease of peppers and tomatoes as a rhabdovirus. Partial sequence data indicates that the virus is most closely related to, but distinct from, Potato yellow dwarf virus. We have now sequenced approximately 25% of the viral genome by PCR extension from previously determined sequences. Samples of infected tissue are currently being analyzed by high-throughput ‘deep’ sequencing. In collaboration with a graduate student at the University of Maryland, a hemi-nested PCR assay was developed to increase the sensitivity of detection of Cymbidium mosaic virus (CymMV). The new method yielded an essentially saturated signal, even for samples of the initial PCR product diluted 1:312,500. This assay is capable of detecting most, if not all, known isolates of CymMV, including recently-identified isolates that do not react with some CymMV-specific antisera. Several plant virus genes (coat protein genes of Nerine yellow stripe virus and Pelargonium chlorotic ringspot virus) and one modified Bacillus thuringiensis Cry6A protein gene (demonstrated to confer plant resistance to an endoparasitic nematode) were cloned into bacterial expression vectors. The expressed proteins were isolated, purified and used as immunogens for the production of antibodies that will be useful in detecting the respective proteins in infected or transgenic plants. Three landscape strains of Xylella fastidiosa (Xf) were isolated, verified, and propagated. One tree strain of Xf was obtained from ATCC and propagated and verified. Genomic DNAs for all four landscape tree strains were prepared for large-scale sequencing. GenBank sequences and contigs from NCBI’s whole genome and shotgun reads database for Ralstonia solanacearum have been downloaded and compared.
1. A PCR assay developed for the specific detection and quantification of Xylella fastidiosa strains causing bacterial leaf scorch in oleander. Oleander leaf scorch caused by X. fastidiosa has become a serious problem in recent years throughout the American west and southwest including California, Arizona and Texas. A sensitive and specific detection and quantification of oleander strains of X. fastidiosa is greatly needed in order to facilitate epidemiological and etiological studies and control of this disease. In collaborative research with scientists from Louisiana State University and the Chinese Academy of Agricultural Sciences, ARS scientists at Beltsville, MD developed a new PCR assay that allows specific and sensitive detection and quantification of X. fastidiosa strains causing oleander leaf scorch disease, and differentiation of oleander strains from other strains of X. fastidiosa. This method will be useful for disease diagnosis, epidemiological studies, management of oleander leaf scorch disease, and resistance screening for oleander shrubs.