Location: Genetic Improvement for Fruits & Vegetables Laboratory
Project Number: 8042-21000-305-014-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2021
End Date: Jun 30, 2024
Objective:
Common scab is an economically costly disease of potato that is present in all potato growing regions. There is remarkable localized diversity in the distribution of pathogenic Streptomyces. While neighboring fields can radically differ in the severity of common scab pressure, disease pressure is generally stable in a field site over many years, likely due to the ability of Streptomyces to survive in soil for decades even when potatoes are not grown. Therefore, a diagnostic assay to detect and quantify the common scab pathogen in a field would significantly reduce potato grower losses to common scab and improve overall soil health. This project will validate a qPCR assay for detection of common scab pathogens in field settings.
1. Determine the diagnostic efficacy of the txtAB qPCR assay for the detection and quantification of pathogenic Streptomyces in the field through controlled sampling of field sites with a spectrum of disease pressure.
2. Assess the spatial variability of the common scab pathogen based on the txtAB qPCR assay and identify soil covariates that impact common scab symptoms and the txtAB qPCR assay.
3. Test the accuracy of the txtAB qPCR diagnostic assay in the greenhouse for the seven phytopathogenic Streptomyces thought to be the most prevalent in the United States.
4. Optimize a digital droplet PCR (ddPCR) assay from the txtAB qPCR assay to improve the sensitivity of detection of pathogenic Streptomyces.
Approach:
1. To test the validity of the txtAB qPCR assay in field settings, we will employ parallel field trials in the 2021 growing season at several commercial potato fields in North Dakota. Fields with common scab disease pressure varying from light to severe will be planted to locally grown potato cultivars with moderate to high susceptibility to common scab. Trials will include six planting blocks, with the block spaced to capture variable common scab pressure. Each block will contain eight 5-hill plots. At or near planting, eight soil samples will be collected from each block. For each sample, soil from 5 cores will be mixed and dried before DNA preparation. Quantitative real-time PCR using primer pair StrepF/R will be performed. At harvest, tubers from each 5-hill plot will be scored for expression of common scab symptoms. We will calculate the Pearson’s product-moment correlation to accurately determine whether the txtAB qPCR assay provides meaningful prediction of common scab pressure in the field.
2. We will perform a covariate spatial analysis to test whether there is a significant spatial component to the distribution of the detected txtAB levels across the 48 soil samples in three of the state trials. Additionally, we will analyze soil parameters and perform a covariate analysis on both the txtAB data and the disease data from each 5-hill plot to determine whether any of the measured soil parameters are significantly associated with the distribution of either dataset.
3. We will perform controlled greenhouse studies on the accuracy of the txtAB qPCR assay with the most prevalent species in the United States: S. scabiei, S. stelliscabiei, S. europascabiei, S. turgidiscabies, S. canutascabiei, S. acidscabies, and S. caviscabies. The same protocol will be used as for the field samples to test the accuracy of the assay against multiple species of the pathogen.
4. The previously developed qPCR primers and probe will be utilized for the development of the ddPCR assay. Cycling conditions and reaction reagents will be optimized as needed. Pearson’s product-moment correlation analysis will be conducted comparing serially diluted Streptomyces standard with known pathogen levels to expected values generated by ddPCR.
