Research Plan

The research objectives of this study are to:

1. Characterize the temporal dynamics of the potato rhizosphere microbiome through the growing season and in relation to powdery scab status
2. Assay soils for their ability to suppress powdery scab
   1. Identify abiotic and biotic factors associated with disease suppression
   2. Identify cropping systems and/or management practices associated with reduced disease  

Objective 1: 

Characterize the temporal dynamics of the potato rhizosphere microbiome through the growing season and in relation to powdery scab status.

Materials and Methods

Field selection and location. An observational field study was conducted in four commercial potato fields selected based on their powdery scab disease history. Within each field, four 0.1-acre grid areas were established for environmental sensor placement and soil and plant sampling. The sensor unit was installed in each plot after potato planting and hilling. Flags were used to mark the sensor location and plot corners. GPS coordinates and elevation for each location were recorded. Hourly soil water and temperature were collected by the sensor unit. The sensors were installed in April and bulk and rhizosphere soils were sampled in April, May, June, July, and August. Previous management history (i.e., previous crops, fumigation events and fungicide applications, soil amendments, etc.) and in-season management practices were documented for each field.

Bulk soil and rhizosphere sampling. We sampled the potato rhizospheres and bulk soils when plots are established and monthly thereafter until plant senescence. To sample bulk soils, 20 standard (1.87 cm diameter) soil cores to a depth of approximately 20 cm were collected within each field plot, 10 m of the sensor. Soil cores were pooled into a collection bag and mixed well by shaking and turning the bag upside-down 10 times. Ten grams of each soil were placed into a 15 ml centrifuge tube. Centrifuge tubes were placed on ice for transport to the laboratory and stored at -20°C until DNA extraction. Based on our experimental design, this resulted in 64 bulk soil samples per year (4 months x 4 fields x 4 locations within field = 64 bulk soil samples per year). At the first sample date, the bag of bulk soil from each plot was used for soil chemical and soil health analyses.

To sample potato rhizospheres, three plants from each location were carefully removed from the soil so that plant roots are left mostly intact. The plants were shaken to remove loose soil from the root system. A 1.0 g sample of root was collected for each plant, and placed in a 15 ml centrifuge tube with 10 ml of phosphate-buffered saline Tween 20 (PBST). Roots were subjected to 3 minutes of vigorous shaking in the field and the cleaned root were removed from the tube. The rhizosphere soil suspended in PBST was stored on ice for transport to the laboratory. In the laboratory, rhizosphere soils were thawed and centrifuged to pellet the soil. The resulting rhizosphere soil pellet was used for DNA extraction. Based on our experimental design, this resulted in 240 rhizosphere samples per year (5 months x 4 fields x 4 locations within field x 3 plants = 240 samples per year).

Objective 2: 

Assay soils for their ability to suppress powdery scab by identifying abiotic and biotic factors associated with disease suppression and by identifying systems and/or management practices associated with reduced disease.

Materials and Methods

Collection of soils, 2022. A preliminary greenhouse experiment was set up to compare disease suppression from different soils collected in Oregon. Soils with diverse management histories were collected from ten locations. Soils were collected from potato and non-potato agricultural systems in Oregon (e.g., forage crops, long-term pasture, etc.) (Figure 1). Approximately, 12 gallons of each field soil were obtained from each location. For each soil, the chemical and physical properties (i.e., fertility, soil particle sizes, etc.), microbial biomass, and basal respiration were determined.

Collection of soils, 2023. A greenhouse experiment was set up to compare disease suppression from different soils collected in Oregon. Soils with diverse management histories were collected from six locations that were informed by the preliminary work from 2022. Soils were collected from potato and non-potato agricultural systems in Oregon (e.g., forage crops, long-term pasture, etc.). Approximately, 20 gallons of each field soil were obtained from each location. For each soil, the chemical and physical properties (i.e., fertility, soil particle sizes, etc.), microbial biomass, and basal respiration were determined.

Greenhouse experiment, 2022. A greenhouse pot experiment was set up to examine disease suppressive activity of the collected field soils. Three different potting mixtures of soil were used in this experiment: potting mixture 1 (PM1) 95% field soil with 5% S. subterranea-infested potting soil, potting mixture 2 (PM2) 10% field soil, 85% potting soil, and 5% S. subterranea-infested potting soil and potting mixture 3 (PM3) 10% autoclaved field soil, 85% potting soil, and 5% S. subterranea-infested potting soil (Figure 2). The three potting mixtures are being used to determine the relative importance of soil edaphic and soil biological factors leading to powdery scab suppressive activity. One treatment consisting of potting soil alone (95% potting soil and 5% S. subterranea-infested potting soil) was included as a control. This experimental design results in a total of 31 treatments. Ten replicate pots, approximately 15 cm diameter, was prepared with each of the potting soil mixtures and one seed tuber of the susceptible cultivar ‘Shepody’ was planted in each of the pots. The experiment was conducted in the plant pathology greenhouse at the HAREC temperatures ranged from 15°C to 44°C, and pots were watered daily to promote S. subterranea tuber infection.

Greenhouse experiment, 2023. A greenhouse pot experiment was set up to examine disease suppressive activity of the collected field soils. Inoculum was applied to all pots at a rate of 5 spores per gram of soil. Three different potting mixtures of soil were used in this experiment: potting mixture 1 (PM1) 100% field soil, potting mixture 2 (PM2) 15% field soil and 85% potting soil, and potting mixture 3 (PM3) 15% autoclaved field soil and 85% potting soil. The three potting mixtures are being used to determine the relative importance of soil edaphic and soil biological factors leading to powdery scab suppressive activity. One treatment consisting of potting soil alone and one treatment consisting of autoclaved potting soil was included as a control. This experimental design results in a total of 20 treatments. Six replicate pots, approximately 35 cm diameter, were prepared with each of the potting soil mixtures and one seed tuber of the susceptible cultivar ‘Kennebec’ was planted in each of the pots. The experiment was conducted in the plant pathology greenhouse at the HAREC, temperatures ranged from 15°C to 32°C, and pots were watered daily to promote S. subterranea tuber infection.

Soil and rhizosphere sampling, 2022. At the start of the experiment, a sample of each potting mixture was collected and stored at -80°C. Bulk and rhizosphere soils were sampled three times throughout the course of the experiment by destructively sampling. Root galling (disease) was assessed on roots during destructive sampling. Bulk soils were sampled directly from each pot. To sample rhizosphere soils, the plant was removed from its pot, then shaken to remove all loose soil from the plant root system. A 0.8 to 1.0 g sample of root was collected for each plant, placed in a 15 ml centrifuge tube with 10 ml of phosphate buffered saline Tween 20 (PBST), and subjected to 3 minutes of vigorous shaking. The cleaned root was removed from the tube and the rhizosphere soil suspended in PBST was centrifuged to pellet the soil. The resulting rhizosphere soil pellet was used for DNA extraction.

Soil and rhizosphere sampling, 2023. At the start of the experiment, a sample of each potting mixture was collected and stored at -80°C. Bulk and rhizosphere soils were sampled once 95 days after planting by destructively sampling. Root galling (disease) was assessed on roots during destructive sampling. Bulk soils were sampled directly from each pot. To sample rhizosphere soils, the plant was removed from its pot, then shaken to remove all loose soil from the plant root system. A 0.8 to 1.0 g sample of root was collected for each plant, placed in a 15 ml centrifuge tube with 10 ml of phosphate buffered saline Tween 20 (PBST), and subjected to 3 minutes of vigorous shaking. The cleaned root was removed from the tube and the rhizosphere soil suspended in PBST was centrifuged to pellet the soil. The resulting rhizosphere soil pellet was used for DNA extraction.

Disease assessment, 2022 & 2023. Powdery scab was assessed for tubers generated in at the end of the experiment. For each pot, the number of tubers were counted, and powdery scab presence was recorded. Powdery scab incidence and severity were estimated for each pot and averaged for each soil treatment. Roots were surface sterilized and assessed for infection by S. subterranea with PCR. 2023, galls were counted on roots to determine severity.

DNA extraction, soil microbiome assay, and bioinformatics pipeline. DNA was extracted from all the soil samples (control, bulk soil, and rhizosphere). Bacterial and fungal community composition were assessed using amplicon sequencing. For library preparation, DNA samples were used as templates for PCR amplification of the 16S and ITS regions. Amplicon libraries were indexed and sequenced on the Illumina NextSeq 2000 platform (300 bp paired-end) at the Oregon State University (OSU) Center for Quantitative Life Sciences. Taxonomy was assigned from the UNITE and SILVA databases for fungi and bacteria, respectively. Sampling depth was normalized, to account for the difference in sequencing depth, and data may be transformed prior to statistical analysis.