Tremblay Émilie D, Bilodeau Guillaume J
Agriculture and Agri-Food Canada, Ottawa, ON, Canada.
Canadian Food Inspection Agency, Nepean, ON, Canada.
Methods Mol Biol. 2022;2536:309-346. doi: 10.1007/978-1-0716-2517-0_18.
Fungal and oomycete plant pathogens are responsible for the devastation of various ecosystems such as forest and crop species worldwide. In an effort to protect such natural resources for food, lumber, etc., early detection of non-indigenous phytopathogenic fungi in new areas is a key approach in managing threats at their source of introduction. A workflow was developed using high-throughput sequencing (HTS), more specifically metabarcoding, a method for rapid and higher throughput species screening near high-risk areas, and over larger geographical spaces. Biomonitoring of fungal and oomycete entities of plant pathogens (e.g., airborne spores) regained from environmental samples and their processing by metabarcoding is thoroughly described here. The amplicon-based approach goes from DNA and sequencing library preparation using custom-designed polymerase chain reaction (PCR) fusion primers that target the internal transcribed spacer 1 (ITS1) from fungi and oomycetes and extends to multiplex HTS with the Ion Torrent platform. In addition, a brief and simplified overview of the bioinformatics analysis pipeline and other available tools required to process amplicon sequences is also included. The raw data obtained and processed enable users to select a bioinformatics pipeline in order to directly perform biodiversity, presence/absence, geographical distribution, and abundance analyses through the tools suggested, which allows for accelerated identification of phytopathogens of interest.
真菌和卵菌植物病原体对全球范围内的各种生态系统（如森林和农作物物种）造成破坏。为了保护诸如食物、木材等自然资源，在新区域早期检测非本地植物致病真菌是从源头管理威胁的关键方法。利用高通量测序（HTS），更具体地说是元条形码技术，开发了一种工作流程，该技术是一种在高风险区域附近以及更大地理空间内进行快速且高通量物种筛选的方法。本文详细描述了从环境样本中回收植物病原体的真菌和卵菌实体（如空气传播的孢子）并通过元条形码技术进行处理的生物监测过程。基于扩增子的方法从使用针对真菌和卵菌的内部转录间隔区1（ITS1）的定制设计聚合酶链反应（PCR）融合引物进行DNA和测序文库制备开始，一直延伸到使用Ion Torrent平台进行多重高通量测序。此外，还包括生物信息学分析流程以及处理扩增子序列所需的其他可用工具的简要概述。所获得和处理的原始数据使用户能够选择生物信息学流程，以便通过所建议的工具直接进行生物多样性、存在/不存在、地理分布和丰度分析，从而加速鉴定感兴趣的植物病原体。
