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UBC Theses and Dissertations

Development of molecular assays to detect the presence and viability of Phytophthora ramorum and Grosmannia clavigera Wong, Barbara


In order to determine if living fungi of phytosanitary concern are present in wood or to evaluate the efficacy of treatments, the method of choice is to grow microbes in petri dishes for subsequent identification. However, some fungi are difficult or impossible to grow in cultures, and thus, to validate the effectiveness of existing and emerging wood treatments, a molecular methodology that can detect living fungi and fungus-like organisms is required. RNA-based molecular diagnostic assays were developed to detect the presence of living fungi and fungi-like organisms of phytosanitary concern. Since RNA represents the transcription of genes and can therefore only be produced by living organisms, it provides a marker to determine if an organism is alive. The assays were designed to target genes that are essential to vital processes, then used to assess their presence and abundance through real-time reverse transcription polymerase chain reaction (PCR). A stability analysis was conducted by comparing the RNA to DNA ratio over treatment time. The results illustrated that for treated samples, DNA remained stable over a period of 10 days post treatment, whereas RNA could not be detected after 24 hours for Phytophthora ramorum or 96 hours for Grosmannia clavigera. Therefore, this method provides a reliable way to evaluate viability of organisms following treatments and can have profound impacts on assessing both timber and non-timber forest products of commercial value.

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