Invasive alien tree pathogens can cause significant economic losses as well as large-scale damage to natural ecosystems. Early detection to prevent their establishment and spread is an important approach used by several national plant protection organizations (NPPOs). Molecular detection tools targeting 10 of the most unwanted alien forest pathogens in Canada were developed as part of the TAIGA project (http://taigaforesthealth.com/). Forest pathogens were selected following an independent prioritization. Specific TaqMan real-time PCR detection assays were designed to function under homogeneous conditions so that they may be used in 96- or 384-well plate format arrays for high-throughput testing of large numbers of samples against multiple targets. Assays were validated for 1) specificity, 2) sensitivity, 3) precision, and 4) robustness on environmental samples. All assays were highly specific when evaluated against a panel of pure cultures of target and phylogenetically closely-related species. Sensitivity, evaluated by assessing the limit of detection (with a threshold of 95% of positive samples), was found to be between one and ten target gene region copies. Precision or repeatability of each assay revealed a mean coefficient of variation of 3.4%. All assays successfully allowed detection of target pathogen on positive environmental samples, without any non-specific amplification. These molecular detection tools will allow for rapid and reliable detection of 10 of the most unwanted alien forest pathogens in Canada.
We evaluated radio frequency (RF) dielectric heating for eradication of pinewood nematodes (PWN) in infested wood. Thirteen temperatures were tested (from ambient to 70 °C) on small wood samples (2.5 9 3.8 9 0.64 cm) to determine the minimum lethal temperature (100 % mortality), which was 56 °C [based on infra-red (IR) thermal images data 55.5-57.4 °C] with a 1 min hold time. We also used thermal probes inside the wood to confirm that temperatures were C56 °C. Thirty additional samples were tested bracketing the minimum lethal temperature using 54, 56 and 58 °C with additional replications to produce the minimum sample size equivalent of 100 % mortality of at least 93,616 nematodes to satisfy the Probit 9 efficacy requirement. This minimum lethal temperature was further verified by treating infested large wood blocks (10.2 9 10.2 9 25.4 cm). All samples that met or exceeded the 56 °C lethal temperature for the required 1 min hold time (as measured by probes inserted in the wood and on the wood surface by IR) produced 100 % mortality. The sample size required to show Probit 9 efficacy was also satisfied. This study supports the consideration of RF in addition to microwave (MW) dielectric heating as alternative treatments of wood packaging material for inclusion in ISPM No. 15, provided the treatment delivers the target lethal temperature throughout the profile of the material in industrial scale operations.
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