Location: Temperate Tree Fruit and Vegetable Research
Project Number: 2092-21220-003-031-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2024
End Date: Aug 30, 2025
Objective:
The goal of our study is to identify viral pathogens in the U.S. populations of Scab Pathogen Spongospora subterranea (Sss) that could be developed or propagated as biological control agents for the powdery scab pathogen and the associated virus, Potato Mop Top Virus (PMTV), transmitted by Sss. Objective 1: Use a metagenomic (HTS) approach to examine the virome of Sss cultures carrying PMTV and those that are PMTV-free. Objective 2: Screen field and laboratory populations of Sss in the U.S. for the presence of SsHLV and SsALV1-3. Objective 3: Screen field and laboratory populations of Sss in the U.S. for the presence of other viral pathogens potentially infecting powdery scab pathogen.
Approach:
Objective 1: The two cultures of cab Pathogen Spongospora subterranea (Sss) will be subjected to the metagenomic (HTS) analysis, using two grams pooled spores per sample. Total RNA will be extracted from germinating spores of Sss using the Zymo Research Quick-RNA Fecal/Soil Microbe Microprep kit. DNAse-treated, purified RNA will then be depleted of ribosomal RNA using the RiboMinus kit for RNA-seq. Before proceeding further, quality of resulting ribo-depleted RNA will be checked by fragment analysis. The ribo-depleted RNA will then be submitted to the Genomics Resources Core at the University of Idaho or to a commercial vendor for library preparation, multiplexing, and sequencing on the Illumina HiSeq platform. Bioinformatics analysis of the high-throughput sequencing data will be performed as described previously. Any matches to the custom-prepared virus sequence database entries will be confirmed through the RT-PCR or PCR verification using specific primers, with subsequent Sanger-based sequencing of amplified PCR fragments.
Objective 2: We will collect or receive Sss samples from potato fields in the states of Washington and Oregon, either in person or through collaborators. The powdery scab-affected tubers will be examined for the presence of visual symptoms and will be tested for the presence of Sss and Potato Mop Top Virus (PMTV) using PCR and RT-PCR, respectively. All samples positive for Sss will be tested for the Sss viruses discovered through the bioinformatics analysis, SsHLV and SsALV1-3. Results from Objective 2 will be used to determine SsHLV and SsALV1-3 infection rates among field populations of Sss.
Objective 3. The data obtained from Objectives 1 and 2 will be analyzed for: 1) discovery of any new viruses besides SsHLV and SsALV1-3; and 2) any correlations between the presence/absence of Sss viruses and severity of Sss and PMTV symptoms in potato tubers.
