Location: Floral and Nursery Plants Research Unit
2012 Annual Report
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.
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.
Huang, Q., Yan, X., Wang, J. 2011. Improved biovar test for Ralstonia solanacearum. Journal of Microbiological Methods. 88:271-274.