Location: Molecular Plant Pathology Laboratory
Project Number: 8042-22000-320-006-I
Project Type: Interagency Reimbursable Agreement
Start Date: Aug 1, 2023
End Date: Aug 1, 2024
The research aims to identify specific housekeeping genes of grapevine flavescence dorée (FD) phytoplasma, optimize PCR primers, evaluate MinION HTS sequencing, develop a bioinformatic analysis pipeline, and test its performance using real field samples for the rapid detection and differentiation of FD phytoplasma and closely related phytoplasma strains in the 16SrV group. Objective 1: Screen a minimal set of housekeeping genes that can differentiate ‘Ca. P. vitis’ and other closely related phytoplasma strains in 16SrV group using the reference available genome information and gene information. Select and optimize PCR primers for the specific amplification of three to five genes (e.g., 16S rRNA; secY; map; vmpA; vmpB) only from phytoplasmas of group 16SrV and excluding other bacteria. Objective 2: Collect field DNA samples of phytoplasma strains in 16SrV group from collaborators and generate a library of the selected housekeeping gene fragments (with known sequences) to assess the resolution, consistency, and accuracy of MinION HTS sequencing as a tool to distinguish closely related 16SrV phytoplasma strains. Objective 3: Establish a curated database of DNA sequences from these three to five phylogenetically relevant targets and develop a bioinformatic analysis pipeline that can rapidly detect phytoplasma of group 16SrV and specifically grapevine flavescence dorée phytoplasma and related strains. Lastly, test the performance of this diagnostic tool with real field samples and report experimental results.
To achieve the objectives, the following experimental approaches will be utilized: 1. Compare three DNA isolation methods on field-collected tissue samples, evaluating their ability to produce quality genomic DNA with high molecular weight. Identify or design PCR primers for the specific amplification of these three to five genes only from phytoplasmas of group 16SrV and excluding other bacteria. 2. Amplify the targeted genes using Nanopore sequencing adapter linked primers of 16S rRNA, secY; map; vmpA, and vmpB genes. The resulting amplicons will be pooled together and sequenced. via MinION. During the planned experiments, sequencing libraries will be prepared and sequenced via MinION following the MinION library loading protocol. 3. Establish a curated database of DNA sequences from these three to five phylogenetically relevant targets (e.g., 16S rRNA; secY; map; vmpA; vmpB) and develop a bioinformatic analysis pipeline that can detect phytoplasma of group 16SrV and specifically grapevine flavescence dorée phytoplasma strains. We will examine bulk DNA extracts from infected samples using different variations of MinION library construction for our ability to detect and differentiate among ‘Ca. P. vitis’ species and other closely related strains associated with grapevine yellows diseases.