UBC Theses and Dissertations
A targeted sequencing approach for viability assessment and management of invasive Phytophthoras Capron, Resmi
Members of the Phytophthora genus are among the most serious invasive pathogens of forests and cause devastating ecological and economic impacts. Epidemics caused by the sudden oak death pathogen, Phytophthora ramorum, and root rot of Christmas trees caused by multiple Phytophthora species are among the many causing loss of millions of dollars to the nursery industry. In addition, a large volume of manufactured products is traded in packages made of wood and wood products globally raising phytosanitary concerns for global trade. Rigorous inspections and biosurveillance involving the monitoring of pathogens before and after applying phytosanitary measures are essential to keep these organisms under control. Despite technological advances in diagnostic assays based on genomics, assessment of treatment efficacy remains challenging for these pathogens. DNA remains stable during heat treatments and can remain as a relic in environments long after the pathogen is dead. Culture methods used for viability testing are time consuming and often report false negatives. We propose a solution to the problem using a transcriptomics approach applying the heat lability of RNA for the prediction of viability and sequence variability for taxonomic identity. We measure levels of RNA degradation by simulating phytosanitary treatment and show that RNA degradation by heat can be assessed from the length of the RNA transcript transcribed and amplified by RT-PCR. Phytophthora transcript sequences identified from an RNAseq study and reference genomes of eight clades of Phytophthora are used to create a panel of oligonucleotides that can target multiple regions along the length of several transcripts commonly expressed during host infection. Significant reduction (60-99%) in mean coverage within transcripts is observed after heat treatments for the 18 genes targeted by the panel. We present the design and testing of a rapid and high-throughput transcriptomics assay for viability assessment of Phytophthoras using the power of targeted next generation sequencing. The assay can be applied for implementation of trade regulations and management of Phytophthora infections in nurseries by regulatory agencies.
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