Al Rwahnih Maher, Klaassen Vicki, Erickson Teresa, Alabi Olufemi Joseph, Stevens Kristian, Hwang Min Sook, Port Lauren
Department of Plant Pathology, University of California Davis, Davis, CA 95616, U.S.A.
Foundation Plant Services, University of California Davis, Davis, CA 95616, U.S.A.
Plant Dis. 2025 Jul;109(7):1392-1403. doi: 10.1094/PDIS-10-24-2104-FE. Epub 2025 Jul 8.
Quarantine and certification programs exist to prevent the entry or spread of harmful pests and pathogens into agricultural systems. Their common objective is to identify pathogen-free source material through the application of validated testing methods for subsequent release for propagation. Tests must be accurate, efficient, and cost-effective. In recent decades, the best tests have been biological assays in conjunction with PCR testing. High-throughput sequencing (HTS) has now become a reliable and cost-effective diagnostic method having greater accuracy and efficiency than biological assays. In this article, we review the role of clean plant centers in quarantine and certification programs, as well as the process by which HTS was evaluated as a testing method to replace biological assays for screening source material. The data from this evaluation included a side-by-side comparison of HTS and biological assays for cultivars of grapevine, , and rose and intra- and interlaboratory validations of an HTS protocol. Based on the results of these evaluations, in 2021, the U.S. Department of Agriculture Animal and Plant Health Inspection Service and several state regulatory agencies accepted the use of HTS and quantitative PCR to test new introductions of source material, replacing biological indexing. This new protocol requires testing at two timepoints within at least a 6-month interval and a dormancy separating the two tests. Under ideal conditions, testing can be completed in 18 to 24 months with subsequent release from quarantine of plant material that has tested negative for regulated pathogens. This new testing protocol has a profound impact on quarantine and certification programs, facilitating quicker access of stakeholders to clean materials for propagation and increasing the number of pathogens that are detected, and even discovered, with reduced cost, effort, and time.
检疫和认证计划旨在防止有害害虫和病原体进入农业系统或在其中传播。其共同目标是通过应用经过验证的检测方法来识别无病原体的源材料，以便随后用于繁殖。检测必须准确、高效且具有成本效益。近几十年来，最佳检测方法是结合PCR检测的生物测定法。高通量测序（HTS）现已成为一种可靠且具有成本效益的诊断方法，其准确性和效率高于生物测定法。在本文中，我们回顾了清洁植物中心在检疫和认证计划中的作用，以及评估HTS作为一种检测方法以取代生物测定法来筛选源材料的过程。该评估数据包括对葡萄、 和玫瑰品种的HTS与生物测定法的并行比较，以及HTS方案的实验室内部和实验室间验证。基于这些评估结果，2021年，美国农业部动植物卫生检验局和几个州监管机构接受使用HTS和定量PCR来检测新引入的源材料，取代生物索引法。这个新方案要求在至少6个月的间隔内的两个时间点进行检测，并且两次检测之间要有休眠期。在理想条件下，检测可在18至24个月内完成，随后对经检测对受监管病原体呈阴性的植物材料解除检疫。这个新的检测方案对检疫和认证计划产生了深远影响，有助于利益相关者更快地获取用于繁殖的清洁材料，并增加了检测到甚至发现的病原体数量，同时降低了成本、工作量和时间。
