First, second, and sixth authors: Canadian Food Inspection Agency (CFIA), 3851 Fallowfield Road, Ottawa, Ontario, K2H 8P9, Canada; third author: Natural Resources Canada, Laurentian Forestry Centre, 1055 Du P.E.P.S. Street, P.O. Box 10380 Québec, Québec, G1V 4C7, Canada; fourth author: CFIA, 4321 Still Creek Dr, Burnaby, British Columbia, V5C 6S7, Canada; and fifth author: Institut de biologie intégrative et des systèmes, 1030 avenue de la Médecine, Québec, Québec, G1V 0A6, Canada.
Phytopathology. 2018 Dec;108(12):1509-1521. doi: 10.1094/PHYTO-02-18-0028-R. Epub 2018 Nov 2.
Anthropogenic activities have a major impact on the global environment. Canada's natural resources are threatened by the spread of fungal pathogens, which is facilitated by agricultural practices and international trade. Fungi are introduced to new environments and sometimes become established, in which case they can cause disease outbreaks resulting in extensive forest decline. Here, we describe how a nationwide sample collection strategy coupled to next-generation sequencing (NGS) (i.e., metagenomics) can achieve fast and comprehensive screening for exotic invasive species. This methodology can help provide guidance to phytopathology stakeholders such as regulatory agencies. Several regulated invasive species were monitored by processing field samples collected over 3 years (2013 to 2015) near high-risk areas across Canada. Fifteen sequencing runs were required on the Ion Torrent platform to process 398 samples that yielded 45 million reads. High-throughput screening of fungal and oomycete operational taxonomic units using customized fungi-specific ribosomal internal transcribed spacer 1 barcoded primers was performed. Likewise, Phytophthora-specific barcoded primers were used to amplify the adenosine triphosphate synthase subunit 9-nicotinamide adenine dinucleotide dehydrogenase subunit 9 spacer. Several Phytophthora spp. were detected by NGS and confirmed by species-specific quantitative polymerase chain reaction (qPCR) assays. The target species Heterobasidion annosum sensu stricto could be detected only through metagenomics. We demonstrated that screening target species using a variety of sampling techniques and NGS-the results of which were validated by qPCR-has the potential to increase survey capacity and detection sensitivity, reduce hands-on time and costs, and assist regulatory agencies to identify ports of entry. Considering that early detection and prevention are the keys in mitigating invasive species damage, our method represents a substantial asset in plant pathology management.
人为活动对全球环境有重大影响。真菌病原体的传播威胁着加拿大的自然资源，而农业实践和国际贸易又促进了病原体的传播。真菌被引入新的环境中，有时会在那里建立起来，在这种情况下，它们会导致疾病爆发，导致广泛的森林衰退。在这里，我们描述了如何通过全国性样本采集策略与下一代测序（NGS）（即宏基因组学）相结合，实现对外来入侵物种的快速和全面筛选。这种方法可以帮助向植物病理学家等利益相关者提供指导。在加拿大各地高风险地区附近采集的 3 年（2013 年至 2015 年）野外样本，监测了几种受监管的入侵物种。在 Ion Torrent 平台上进行了 15 次测序运行，处理了 398 个样本，产生了 4500 万条读数。使用定制的真菌特异性核糖体内部转录间隔区 1 条形码引物对真菌和卵菌的操作分类单元进行高通量筛选。同样，使用 Phytophthora 特异性条形码引物扩增三磷酸腺苷合酶亚基 9-烟酰胺腺嘌呤二核苷酸脱氢酶亚基 9 间隔区。通过 NGS 检测到几种 Phytophthora spp.，并通过物种特异性定量聚合酶链反应（qPCR）检测进行了确认。通过宏基因组学只能检测到 Heterobasidion annosum sensu stricto 目标物种。我们证明，使用各种采样技术和 NGS 筛选目标物种（结果通过 qPCR 验证）有可能提高调查能力和检测灵敏度，减少手工时间和成本，并帮助监管机构识别入侵物种的入境口岸。考虑到早期发现和预防是减轻入侵物种危害的关键，我们的方法代表了植物病理学管理的重要资产。
