Faculté de foresterie et géomatique, Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, Québec, QC, Canada.
Department of Forest and Conservation Sciences, University of British Columbia, Vancouver, BC, Canada.
PLoS One. 2020 Feb 5;15(2):e0221742. doi: 10.1371/journal.pone.0221742. eCollection 2020.
Wood and wood products can harbor microorganisms that can raise phytosanitary concerns in countries importing or exporting these products. To evaluate the efficacy of wood treatment on the survival of microorganisms of phytosanitary concern the method of choice is to grow microbes in petri dishes for subsequent identification. However, some plant pathogens are difficult or impossible to grow in axenic cultures. A molecular methodology capable of detecting living fungi and fungus-like organisms in situ can provide a solution. RNA represents the transcription of genes and can become rapidly unstable after cell death, providing a proxy measure of viability. We designed and used RNA-based molecular diagnostic assays targeting genes essential to vital processes and assessed their presence in wood colonized by fungi and oomycetes through reverse transcription and real-time polymerase chain reaction (PCR). A stability analysis was conducted by comparing the ratio of mRNA to gDNA over time following heat treatment of mycelial cultures of the Oomycete Phytophthora ramorum and the fungus Grosmannia clavigera. The real-time PCR results indicated that the DNA remained stable over a period of 10 days post treatment in heat-treated samples, whereas mRNA could not be detected after 24 hours for P. ramorum or 96 hours for G. clavigera. Therefore, this method provides a reliable way to evaluate the viability of these pathogens and offers a potential way to assess the effectiveness of existing and emerging wood treatments. This can have important phytosanitary impacts on assessing both timber and non-timber forest products of commercial value in international wood trade.
木材及其制品可能藏匿微生物，而这些微生物会引发进出口这些产品的国家的植物检疫问题。为了评估木材处理对植物检疫关注微生物存活的效果，首选方法是在培养皿中培养微生物，以便随后进行鉴定。然而，一些植物病原体很难或不可能在无菌培养物中生长。一种能够原位检测活真菌和真菌类生物的分子方法可以提供一种解决方案。RNA 代表基因的转录，在细胞死亡后会迅速变得不稳定，从而提供了一种可行性的替代测量方法。我们设计并使用了基于 RNA 的分子诊断检测方法，针对对生命过程至关重要的基因，通过逆转录和实时聚合酶链反应 (PCR) 评估了它们在真菌和卵菌定殖的木材中的存在情况。通过比较疫霉菌 Phytophthora ramorum 和 Grosmannia clavigera 丝状真菌培养物的热疗后时间的 mRNA 与 gDNA 的比值进行了稳定性分析。实时 PCR 结果表明，在热处理样品中，DNA 在 10 天的时间内保持稳定，而对于 P. ramorum 或 G. clavigera，在 24 小时或 96 小时后，mRNA 则无法检测到。因此，这种方法为评估这些病原体的生存能力提供了一种可靠的方法，并为评估现有和新兴木材处理方法的有效性提供了一种潜在方法。这对于评估国际木材贸易中具有商业价值的木材和非木材林产品的植物检疫问题具有重要意义。
