DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer

Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes

Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire

Background: Pythium ultimum (P. ultimum) is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species. Results: The P. ultimum genome (42.8 Mb) encodes 15,290 genes and has extensive sequence similarity and synteny with related Phytophthora species, including the potato blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86% of genes, with detectable differential expression of suites of genes under abiotic stress and in the presence of a host. The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions although surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes. A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host specific oomycete species. Although we observed a high degree of orthology with Phytophthora genomes, there were novel features of the P. ultimum proteome including an expansion of genes involved in proteolysis and genes unique to Pythium. We identified a small gene family of cadherins, proteins involved in cell adhesion, the first report in a genome outside the metazoans. Conclusions: Access to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae

Integrated air stream micromixer for performing bioanalytical assays on a plastic chip

This paper describes the design, functioning and use of an integrated mixer that relies on air flux to agitate microliter entities of fluid in an embedded microfluidic cavity. The system was fabricated from multiple layers of a thermoplastic elastomer and features circuits for both liquid and air supply along with pneumatic valves for process control. Internally-dyed polymer particles have been used to visualize flow within the fluid phase during agitation. Numerical modelling of the micromixer revealed an overall efficacy of 10-1 to 10-2 for momentum transfer at the air-water interface. Simulation of air vortex dynamics showed dependency of the flow pattern on the velocity of the flux entering the cavity. Three bioanalytical assays have been performed as proof-of-concept demonstrations. In a first assay, cells of Listeria monocytogenes were combined with magnetic nanoparticles (NPs), resulting in high-density coverage of the bacteria's surface with NPs after 1 min of agitation. This finding is contrasted by a control experiment without agitation for which interaction between bacteria and NPs remains low. In a second one, capture and release of genomic DNA from fungi through adsorption onto magnetic beads was tested and shown to be improved by agitation compared to non-agitated controls. A third assay finally involved fluorescently-labelled target oligonucleotide strands and polystyrene particles modified with DNA capture probes to perform detection of nucleic acids on beads. Excellent selectivity was obtained in a competitive hybridization process using a multiplexed micromixer chip design.Peer reviewed: YesNRC publication: Ye