Project Number: 2072-21000-060-003-G
Project Type: Grant
Start Date: Sep 1, 2021
End Date: Oct 31, 2024
1. Study viral genetic variability for a better understanding of the spectrum of viral populations circulating in vineyards; 2. Develop mutational fingerprints of the virus to discriminate between pathogenic and non-pathogenic strains that are otherwise indistinguishable; 3. Design virus-specific tracking system to elucidate spatial and temporal dynamics of genetic variants in epidemiological investigations on a local and regional scale.
With assistance from commercial growers, we will collect samples from both red and white-fruited wine grape (Vitis vinifera) cultivars from specific commercial vineyards in Washington, Oregon, and Idaho known to have GLD infections. The number of samples collected will be proportional to vineyard acreage in different American Viticultural Areas, such that higher number of samples will be from Washington followed by Oregon and Idaho vineyards. In red-fruited cultivars, leaf samples will be collected from individual grapevines showing GLD symptoms. Since white-fruited cultivars do not express symptoms similar to red-fruited cultivars, leaf samples will be collected from white-fruited vines at random. Total nucleic acids will be extracted from petiole and/or dormant cane samples and tested by single tube-one step reverse transcription-polymerase chain reaction (RT-PCR) with primers specific for the heat shock protein 70 homologue (Hsp70h) gene of GLRaV-3 (Adiputra et al., 2018). Samples tested positive for GLRaV-3 will be used subsequently for genetic diversity studies. Due to the unusually large genome size (~18,500 nucleotides) of GLRaV-3 (Jarugula et al., 2010a), high-throughput Illumina Hi-Seq 2500 platform followed by de novo assembly of quality reads will be adopted to generate a pool of viral contigs using CLC Genomics Workbench 8.0 with default parameters. The contigs will be subsequently annotated using BLASTX against the nonredundant plant virus database as a reference available from GenBank (http://www.ncbi.nlm.nih.gov/blast). Internal gaps in viral genome sequence will be filled by Sanger sequencing to generate complete genome sequence of virus isolates from individual vines. Phylogenetic analysis of GLRaV-3 strains generated in this study relative to those obtained from public databases will be conducted by Maximum-likelihood method using the best fit nucleotide substitution models in MEGA7. In addition, population genetics studies will be conducted using bioinformatics programs for analyzing the mutational landscape of GLRaV-3 in the region and to characterize emerging variants towards assessing their fitness advantage leading to higher transmission by vectors. Using metadata of vineyard location, cultivar and disease severity, date of sampling, and genome sequence and population genetics of GLRaV-3 generated in this project and available from other countries in public databases, a user-friendly and interactive mutation tracking system will be developed for real-time tracking of virus variants in vineyards and build location-specific information at regional scale to predict risk of disease outbreaks for implementing area-wide management of GLD.