Project Number: 2092-21220-003-003-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2023
End Date: Jun 30, 2024
The objectives of this research are to 1) characterize Potato mop-top virus isolates collected in the U.S. and 2) directly visualize Potato mop-top virus and Tobacco rattle virus in the potato root system.
Objective 1: Develop accurate diagnostic assays for Potato mop-top virus (PMTV) detection, the complete genomes of PMTV isolates in the U.S. are essential. USDA-ARS Prosser, WA research team has access to plant tissue containing PMTV from the Washington State University Seed Lot Trial, which contains samples from 13 U.S. States and Canada spanning seven years. Total ribonucleic acid (RNA) will be extracted from 40 samples that previously tested positive for PMTV and used for next generation sequencing (NGS). Additionally, 15 soil samples will be collected from Spongospora subterranea (Sss)- and PMTV-affected fields (Five locations from CO, OR, and WA). Since RNA extracted from the soil is often poor quality and not amenable for NGS, we will use bait plants. Briefly, soil samples will be enriched for Sss spores by sieving and used in bait assays to infect tomato plants with PMTV. Total RNA will be extracted from the PMTV-positive plants and used for NGS. CLC workbench will be used for quality control, sequence assembly into contigs, elimination of tomato genes, contig annotation, and identification of variation between samples. Objective 2: Direct visualization of viruses in potato roots over time will be conducted using tissue-printing hybridization. Tissue culture plantlets will be grown in a specially designed petri dish growth chamber containing a nutrient solution. Tobacco rattle virus (TRV)-infected stubby root nematodes (SRNs) or PMTV-infected Sss sporosori will be transferred to the root to allow inoculation. Previous work with an SRN-related nematode has shown that it takes several hours for a nematode to feed at a single location, and it can take up to 4 hours for TRV to move to adjacent cells. Despite this data, the time required for SRN to transmit TRV or for TRV to begin moving within the root is unknown. Similarly, the timing of Sss infection and PMTV entry into plant roots are unknown. We propose visualizing the viruses at 4, 8, 16, 24, 48, and 72 hours after inoculation. This time frame may need to be adjusted as we begin our experiment. It is believed that SRNs seek root tips for feeding, but Sss is likely to infect along the root (based on root galling locations). We will allow feeding by SRNs and Sss at different locations on the roots to determine the likely point of initial infection and subsequent movement of the viruses. After feeding, roots will be "printed" onto a nylon membrane by pressing the tissue firmly on top of the membrane. The quality of the root press will be visualized under ultraviolet light. A digoxigenin-labeled nucleic acid probe will then enable visualization of TRV or PMTV within the root tissue using chemiluminescent detection. Localization of the viruses in roots will be compared between inoculation site and the time after inoculation.