Development and Application of Next Generation Sequencing to Facilitate the Release of New Grapevine Accessions in Quaraions in Quarantine and Certification Programs

Recent developments in molecular technology have led to significant improvements in detection and control of many pathogens. The use of those techniques for pathogen detection in quarantine and certification programs has not yet been universally accepted. This is primarily because of the need to validate these techniques and determine their limitations. We are proposing to supply the data for that validation for the case of grapevine registration and certification. We will make a side-by-side comparison of 1) the classical, currently used technique for the analysis of viral pathogens of grapevine, with 2) the more recently developed technology of Next Generation. Sequencing (NGS). In both cases, we will test the same set of fifty selected grapevine accessions infected with one or multiple viruses of importance to the grape industry. In the first case, viral pathogens will be analyzed using biological assays on a standard herbaceous and woody index panel of host plants, as is required by APHIS and CDFA for certification. We will compare the results from that bioassay with a second analysis based on NGS of the total grapevine viruses in each of the selected accessions. The two tests will run concurrently. We expect to show that, for the evaluation of the disease status of grapevine stocks, NGS is superior to biological assay, as well as to ELISA, RT-PCR and real time RT-qPCR in sensitivity, reliability, speed and labor intensity, and cost. We will make the case for the replacement of biological assays with NGS for the certification of novel grapevine accessions. Our data will be useful to federal and state regulatory agencies as evidence supporting the revision of the existing mandated protocols for the testing and release of novel grapevine accessions from quarantine. The improvements brought with the up-date to NGS technology for this application will be of significant benefit to the grape growing industry.

The first year objectives of this project have been met. We have identified the first batch of 20 grapevine accessions that carry infections of agronomic importance, for use in the comparative demonstration of the effectiveness of the two techniques evaluated in this project. We have   chipbud grafted material from each of those to the standard four bioassay index hosts, and begun their two year incubation period toward symptom scoring. We have also made total RNA extractions from those infected plants and begun NGS analysis, using BLAST sequence comparisons to subtract the host coded sequences from those of the pathogens of interest. This progress will generate the data for the comparisons between the techniques, which will meet the subsequent objectives of this proposal.