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.

This is the first viral metagenomic analysis of grapevine conducted in Mexico. During the summer of 2021, 48 plants displaying virus-like symptoms were sampled in Queretaro, an important grapevine-producing area of Mexico, and analyzed for the presence of viruses via high-throughput sequencing (HTS). The results of HTS were verified by real-time RT-PCR following a standardized testing scheme (Protocol 2010). Fourteen different viruses were identified, including grapevine asteroid mosaic-associated virus (GAMaV), grapevine Cabernet Sauvignon reovirus (GCSV), grapevine fanleaf virus (GFLV), grapevine fleck virus (GFkV), grapevine Pinot gris virus (GPGV), grapevine red globe virus (GRGV), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine rupestris vein feathering virus (GRVFV), grapevine Syrah virus 1 (GSyV-1), grapevine virus B (GVB), and grapevine leafroll-associated viruses 1, 2, 3, 4 (GLRaV1, 2, 3, 4). Additionally, divergent variants of GLRaV4 and GFkV, and a novel Enamovirus-like virus were discovered. This is the first report of GAMaV, GCSV, GLRaV4, GPGV, GRGV, GRVFV, and GSyV-1 infecting grapevines in Mexico; the impact of these pathogens on production is unknown.

Worldwide, virus infections in grapevines are of concern due to the potential for economic loss. Although the grape industry in Mexico is relatively small and focused mainly on the local market, production dates back to the time of the Spanish colonization. This manuscript discusses the findings on grapevine viruses in Mexico. Nine viruses have been identified in the last fifty years, including grapevine red blotch virus (GRBV), grapevine leafroll-associated virus 3 (GLRaV-3), grapevine fanleaf virus (GFLV), and grapevine virus A (GVA). Important information is provided about these viruses and viral pathogens that have not yet been reported in Mexico, but represent an ongoing threat to plant health and grapevine production in other viticultural regions of the world. Strategies for virus control in vineyards are described. The information discussed here should be shared with growers and stakeholders to prevent future negative impacts on the Mexican grapevine industry and to save ancient grapevine accessions.

Grapevine leafroll-associated virus 3 (GLRaV3) and grapevine red blotch virus (GRBV) are two major pathogens associated with significant economic losses, requiring reliable and sensitive diagnostic tools for rapid identification. Current RT-qPCR and qPCR assays are widely used in certification programs; however, the risk of false-negative results due to the genetic diversity of plant viruses is always a concern. Here, we present a new targeted HiPlex assay designed for the detection of GLRaV3 and GRBV. A custom primer panel containing 98 oligonucleotides targeting GLRaV3 and 14 targeting GRBV was evaluated across 45 different grapevine accessions. HiPlex diagnostic performance metrics were evaluated up to the 1:1000 dilution level. The HiPlex assay detected both viruses in 30 ng of total nucleic acids (TNA) and at 1:10 and 1:100 dilutions, exhibiting a strong inverse correlation between read counts and Ct values. Analysis of reads from qPCR-negative samples allowed the determination of a positivity cutoff of 5000 reads, which provided high specificity while maintaining sensitivity. The results presented here support the application of HiPlex amplicon sequencing for the detection of GLRaV3 and GRBV and provides a foundation for future expansion of the primer panel to additional grapevine viruses. To our knowledge, this is the first study evaluating multiplex PCR-based amplicon sequencing specifically for grapevine virus diagnostics.

The grapevine fleck virus (GFkV) is a ubiquitous grapevine-infecting virus found worldwide, is associated with the grapevine fleck complex, and is often found in mixed infections with viruses of the grapevine leafroll complex and/or vitiviruses. Although GFkV has been studied for a long time, limited sequence information is available in the public databases. In this study, the GFkV sequence data available in GenBank and data generated at the Foundation Plant Services, University of California, Davis, were used to perform nucleotide sequence comparisons, construct a phylogenetic tree, and develop a new RT-qPCR assay. Sequence comparisons showed high genetic diversity among the GFkV isolates, and the phylogenetic analyses revealed a new group comprised of GFkV isolates identified in the present study. A new assay, referred to as GFkV-CP, was designed and validated using an existing GFkV positive control together with 11 samples known to be infected with combinations of different marafiviruses and maculaviruses but not GFkV. In addition, the newly designed assay was used in a field survey to screen grapevines from diverse geographical locations that are maintained at the United States Department of Agriculture (USDA) National Clonal Germplasm Repository (NCGR) in Winters, CA.