A New Era in Federal Quarantine and State Certification Diagnostics at Clean Plant Centers in the United States.

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, Prunus, 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.