Progress 07/01/18 to 08/31/19

Outputs
Target Audience:AmebaGone has been actively engaged with a number of stakeholders interested in our technology. This includes potato growers in the state of Wisconsin, the potato pathology extension program at the University of Wisconsin, industry partners that are assisting us with production, and investors interested in investing in our technology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The PI of this project, Ryan Kessens, has attended two conferences related to professionl development and training. These include the Wisconsin Potato and Vegetable Growers Association conference where he learned about prominent potato diseases and management practices. At the American Phytopathological Society conference, Ryan met with scientists from a diverse range of agricultural companies and spoke with extension leaders across the U.S. At this conference, emerging techniques and new discoveries in the field of plant pathology were presented. How have the results been disseminated to communities of interest?AmebaGone periodically updates growers, extension leaders, collaborators, and potential investment partners on progress being made with the potato soft rot treatment outlined in this proposal. This entails sending emails and making phone calls to our stakeholders. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1 - Task 1: The Phase I pipeline used to select the most promising Dicty strains for our potato soft rot treatment began with 119 strains from our collection. These Dicty strains were screened for their ability to feed on Dd and Pcc at 10° C. This assay was performed by inoculating Dd or Pcc on a low nutrient medium (SM2 agar) that supports both bacterial and Dicty growth. Dicty spores from individual strains were then inoculated on top of the bacterial growth and incubated at 10° C to mimic potato storage temperatures. Dicty strains that successfully fed on Dd or Pcc created visible clearings in the lawn of bacterial growth and ultimately produced sporangia (fruiting bodies) that rose from the agar surface.From this initial screen, 36 Dicty strains that are capable of feeding on both Dd and Pcc at 10° C were identified. Objective 1 - Task 2: Our next task was to identify Dicty species capable of feeding on biofilms of Dd and Pcc. Microporous polycarbonate membranes (MPMs) are widely reported to support biofilm formation of numerous Enterobacteriaceae species (2, 63, 70, 71). We wanted to see if Dd and Pcc would form biofilms on MPMs and determine if our Dicty strains were capable of feeding on these biofilms. Membranes were placed on top of SM2 agar to provide Dd and Pcc with nutrients for growth. Bacteria were then inoculated on the surface of the MPMs and growth was monitored over the course of 1 week by washing bacteria off the membranes and performing dilution plating for colony counting. Growth of both bacterial strains plateaued around 4 dpi.From these results, we determined that the best time to collect inoculated MPMs for biofilm analysis was at 2 dpi. Scanning electron microscopy (SEM) is commonly used to confirm biofilm formation by detecting extracellular polymeric substance (EPS) that forms the biofilm matrix (2). Samples of Dd and Pcc after 2 days of growth on MPMs in the presence and absence of Dicty have been submitted for SEM analysis, but we are still waiting on the results of this experiment.Of the 36 Dicty strains identified from our initial screen against Dd and Pcc, 17 strains were isolated from regions of Russia that were included in our screen because of their potential for cold-tolerance. However, we decided to discontinue efforts on these strains due to difficulties getting EPA approval for non-native species and international protocols (i.e. Nagoya Protocol) designed to prevent the exploitation of genetic resources. Our remaining 19 strains (U.S. isolates) were tested for their ability to feed on Dd and Pcc growing on MPMs. These experiments were performed by establishing Dd and Pcc growth on MPMs overlaid on SM2 agar at 37° C for 24 hr. Dicty spores were then applied to the center of bacterial growth in a 5 uL drop containing 1000 spores. Bacteria and Dicty were incubated at 10° C for 2 weeks before remaining bacteria were washed off and colonies were counted.While no Dicty strains produced a statistically significant reduction in Dd viability compared to the non-treated control, likely due to the high level of variability using the colony counting method, we did notice trends for some Dicty species between experiments. Treating Dd lawns with Cohen 36, Cohen 9, WS-15, WS-20, and WS-69 consistently reduced the number of viable bacteria byapproximately 100,000-fold compared to the non-treated control. Cohen 9 was the only Dicty strain that produced a statistically significant reduction in viability of Pcc compared to the non-treated control. Other Dicty strains capable of reducing the number of viable Pcc by at least 100,000-fold were Cohen 35, Cohen 36, WS-647, and WS-69. Objective 2 - Task 1: This objective was modified to take full advantage of the Storage Research Facility (SRF) in Hancock that AmebaGone now has access to. A full summary of this experiment is included in Objective 3 - Task 2 of the Phase II Work Plan since this experiment will also be performed during the Phase II project. Briefly, cut seed potatoes will be inoculated with Dd or Pcc and treated with three Dicty strains at three application rates. Cut seed potatoes will be stored at 12°C and 98% relative humidity (RH) for one week before being assessed for soft rot development. Surviving seed potatoes will be planted at the Hancock Research Station and plants will be monitored for blackleg through the summer. Potatoes will be harvested in the fall and have their quality assessed. Results from the short-term storage experiment will be included in the final Phase I report while field data will be included in the Phase II project. Objective 2 - Task 2: From the data generated in Objective 1, we noticed that Dicty strains Cohen 9, Cohen 36, and WS-69 were capable of feeding on both Dd and Pcc when these bacteria were cultured on SM2 agar and MPMs. These strains were also particularly effective feeders as all three reduced the number of viable Dd and Pcc on MPMs at 10° C by 100,000-fold compared to the non-treated control.To see if these strains could suppress soft rot development on seed potato tubers, we stab-inoculated tubers with Dd or Pcc and treated with spores from each Dicty strain. Seed potatoes were surface-sterilized and punctured using a sterile screw to a depth of 1.5 mm. Overnight cultures of Dd and Pcc were suspended in 10 mM potassium phosphate buffer, diluted to an OD600 of approximately 0.003, and administered as a 5 uL drop into the wound. Next, 5 uL of a Dictyspore suspension (100,000 spores) was added to the wound. Inoculated seed potatoes were placed in a plastic container with moist paper towels and were misted with water twice a day to maintain a high humidity. After 3 days at room temperature, seed potatoes were sliced in half and the area of macerated tissue was quantified using ImageJ. All three strains reduced the severity of soft rot caused by Dd and Pcc. Cohen 36 was the most effective strain on both Dd and Pcc: reducing the area of tissue maceration by 60% and 35%, respectively. Treating seed potatoes with WS-69 reduced the area of tissue maceration by 50% and 30% for Dd and Pcc, respectively. Finally, Cohen 9 was the least effective, but still able to reduce tissue maceration caused by Dd and Pcc by 25% and 20%, respectively. Objective 2 - Task 3: Dicty must be capable of sporulating at temperatures as cold as 10°C on a potato surface if our product is to be applied as a one-time pre-planting or post-harvest treatment. Sporulation was assessed by inoculating small potato discs (5x6 mm) with 10 uL of Dd or Pcc suspensions at an OD600 of 3 x 10-5 and Dicty spores at a concentration of 1 x 107 spores/mL. Potato discs were kept in a covered 96-well plate for two weeks at 10°C followed by visual inspection for sori using a dissecting microscope. Representative images of a strain producing many sori (WS-517) and a strain producing few sori (WS-69) can be seen in Figure 7. Of the 11 strains evaluated so far, only Cohen 9 and WS-20 are unable to sporulate in the presence of both pathogens. These strains will be evaluated again to determine the relative abundance of sori produced, which will be considered when determining the best strain(s) to use in our final product.

Publications