Peronospora tabacina Adam. is the causal agent of blue mold or downy mildew of tobacco. The pathogen is a fungus-like organism and is a member of the Oomycota. P. tabacina is an obligate parasite restricted to species in the genus Nicotiana. Identification of the pathogen is difficult since symptoms and signs generally are found 6--12 days post inoculation if artificial or post infection if natural. The spread of the pathogen occurs through aerial long distance dispersal of inoculum and severe epidemics occur yearly in tobacco growing areas of the world. The first objective of this work was to develop a real-time Taq Man assay for the detection and quantification of P. tabacina. Optimization of the assay was established at a final concentration of 450nM of primers and 125nM of probe. The assay was useful for detection of the pathogen down to a lower limit of 1 fg of DNA. The pathogen could be detected after 4 days post inoculation. The real-time PCR assay was useful for the specific detection of P. tabacina in field samples, artificially inoculated leaves, roots, and systemically infected tobacco seedlings and could be used as a tool for regulatory agencies interested in the detection of the pathogen. A second objective was to examine the genetic structure of the pathogen in North America, Central America, the Caribbean and Europe and determine the direction of migration of the pathogen. The intergenic spacer Igs2 region of the nuclear ribosomal DNA (rDNA) and the Ras-related protein (Ypt1) gene, and the mitochondrial cytochrome c oxidase subunit 2 (cox2 gene) were sequenced. Populations of P. tabacina were characterized by high nuclear diversity, low population division and a possible mixed sexual and asexual reproductive system. Subpopulations from CCAM and EULE had the highest estimates for nucleotide diversity and mean mutation rate. Neutrality tests were significant and negative for all the subpopulations and the equilibrium model of neutral evolution was rejected. Large population size, the mechanism of dispersal, the parameters of mutation rate and genetic diversity found for the whole population indicate that this pathogen is a high evolutionary risk plant pathogen. Isolation with Migration (IM) model was used to study genetic diversity in the U.S./Central America and the Caribbean (CCAM) and the European subpopulations. Results support migration from the CCAM region, Florida and Texas into north states further in the U.S. including North Carolina. These data validate previous migration reports of the pathogen by the North American Plant Disease Forecasting Center at NCSU. In Europe estimates for the migration of the pathogen from North Central to Western Europe and both these regions to Lebanon support migration reports for the first introductions of the pathogen into Europe. Mitochondrial sequences of P tabacina and the Hyaloperonspora parasitica genome were generated using bioinforrmatics approaches and PCR methodology. One quarter of the mitochondrial genome of P. tabacina has been annotated and compared with that of Phytophthora infestans and Hyaloperonospora parasitica. Similarities in direction, arrangement and number of genes and regions have been found. H. parasitica mitochondrial genome exhibited higher similarities with P. ramorum and P. sojae genomes than with P. infestans. Results from this research will be useful in understanding the evolutionary history, epidemiology and population genetics of this important plant pathogen.
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