Source: AMEBAGONE, INC. submitted to NRP
COMBATING POTATO SOFT ROT WITH FREE-LIVING PHAGOCYTES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1020469
Grant No.
2019-33610-30186
Cumulative Award Amt.
$646,368.00
Proposal No.
2019-02681
Multistate No.
(N/A)
Project Start Date
Sep 1, 2019
Project End Date
Aug 31, 2022
Grant Year
2019
Program Code
[8.2]- Plant Production and Protection-Biology
Recipient Organization
AMEBAGONE, INC.
510 CHARMANY DR STE #58
MADISON,WI 53719
Performing Department
(N/A)
Non Technical Summary
AmebaGone, Inc is developing a novel microbial pesticide for the prevention of soft rot in potato tubers. Each year, an estimated 15-30% of the U.S. potato crop is lost to soft rot, representing a market loss of $0.57B-$1.13B. There is currently no effective treatment available for this disease. Our solution is a biological treatment that uses the predatory power of Dictyostelids ("Dicty" for short) instead of harsh chemicals. Dicty are microscopic organisms that live in the soil where they feed selectively on bacteria. AmebaGone has access to over 3,000 Dicty strains from diverse geographic and climatic regions. We have identified Dicty strains that feed on bacteria that cause potato soft rot: Dickeya and Pectobacterium. Strains capable of feeding on Dickeya and Pectobacterium on potatoes at cool temperatures relevant to storage conditions (~50°F) have also been discovered. Three strains identified during Phase I research are even capable of reducing soft rot severity in whole tubers. Conveniently, Dicty produce large numbers of semi-dormant spores that are highly resistant to environmental stress and long-term storage. Spore germination releases metabolically active Dicty cells that immediately begin feeding on surrounding bacteria. These spores will be the active ingredient in our future product and efforts to develop a large-scale fermentation method for spore cultivation are underway. AmebaGone is also focused on maximizing product shelf-life, spore germination, and compatibility with other potato treatments. Finally, we will conduct trials with product prototypes under conditions that mimic commercial potato storage facilities.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
100%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21513101103100%
Knowledge Area
215 - Biological Control of Pests Affecting Plants;

Subject Of Investigation
1310 - Potato;

Field Of Science
1103 - Other microbiology;
Goals / Objectives
Through NSF, NIH, and USDA-funded research, AmebaGone has demonstrated that Dicty are capable of feeding on diverse human and plant Enterobacteriaceae pathogens. Specifically, our USDA Phase I research demonstrates that multiple Dicty strains are capable of feeding on the soft rot pathogens Dickeya and Pectobacterium on potatoes at cold storage temperatures. Major technical questions we must now address for successful commercialization of our potato soft rot treatment and other products in our pipeline are outlined below.Can Dicty spores be produced at an industrial scale?Will Dicty spores remain efficacious after packaging and storage?What application rate and additives are necessary for optimal efficacy?Will our treatment work under commercial potato storage conditions?AmebaGone will address these questions by accomplishing the following Objectives in Phase II.Objective 1: Scale up production of Dicty spores from the most effective strainsTask 1: Use a high throughput screening assay to identify Dicty strains from Alaska that feed on Dd and Pcc at 10° C on potatoScreen is successful if 5 strains reduce growth of Dd and/or Pcc by >90%Task 2: Select 5 Dicty strains from Task 1 for large-scale spore production using solid-state fermentationProduction is successful if we can produce 10 trillion spores in one monthObjective 2: Optimize product formulation and application rateTask 1: Maximize Dicty spore viability following lyophilizationLyophilization is successful if >90% viability is achievedTask 2: Develop a method for rehydrating and activating lyophilized sporesSpore activation is successful if >50% germination is achievedTask 3: Determine product shelf lifeProduct should retain 90% efficacy after 1 year of storageObjective 3: Conduct seed treatment and storage trials to determine if Dicty can effectively reduce soft rot under commercial conditions and determine if our product can treat other common potato diseases and is compatible with treatments for these diseasesTask 1: Determine if Dicty are compatible with fungicides commonly applied to potatoesSuccessful if at least two fungicides reduce Dicty viability by <10%Task 2: Determine if Dicty can feed on Fusarium species, H. solani, and the causative agent of potato common scab (Streptomyces scabies)Not essential but helpful for product registration and increased marketTask 3: Conduct seed potato trials to determine which Dicty strains and application rates are most effective.Successful if seed potato survival is >80% of the non-treated controlTask 4: Conduct potato storage trials using most efficacious Dicty strains and application rates determined from Task 3Successful if Dicty treatment is more effective than current standard
Project Methods
In vitro bacterial viability assay -In summary, organic red potatoes are surface-sterilized and cut into 5 mm thick slices using a vegetable mandolin. Slices are then stacked and punched using a 6 mm stainless steel cork borer. This produces 5x6 mm potato discs that are loaded into each well of a 96-well plate to serve as a natural food source for inoculated Dd and Pcc. The Dd and Pcc strains used express the bacterial lux operon, which encodes a luciferin-luciferase system that produces light in metabolically active cells. After inoculating potato cores with bacteria (~100 CFUs per well), Dicty spores are administered as treatments (~100,000 spores per well) using 5-6 replicates per strain. Luminescence readings are then recorded using a plate reader to monitor the proliferation of bacterial cells.Dicty spore cultivation -Cultivation of Dicty spores on vermiculite is relatively simple. Coarse vermiculite (grade 4) is sterilized in a wire mesh cage by autoclaving. Sterile vermiculite is then saturated with an inorganic solution (10 mM potassium phosphate) containing high density E. coli seeded with Dicty spores. The use of an inorganic buffer further reduces the possibility of contamination as the only carbon sources present for microbial growth are E. coli and Dicty cells. Saturated vermiculite is then placed in a high humidity chamber (>90% RH) under constant light to induce sporulationDicty spore lyophilization - Dicty spores will be suspended in 0, 5, and 10% non-fat skim milk (NFSM buffer; 10 mM potassium phosphate) at concentrations of 1 x 10^8, 1 x 10^9, and 1 x 10^10 spores/mL. Spore viability will be determined by plating 100 spores suspended in 200 uL of E. coli on an SM2 agar plate. After 5 days, plaques in the bacterial lawn caused by Dicty feeding will be counted, and the percentage of viable spores will be determined. Viability will be assessed before freezing the spores at -20° C, after freezing, and after lyophilization. Efficacy of rehydrated spores will be assessed by treating Dd and Pcc in our luminescent assay. If we are unable to achieve at least 90% spore viability or treatment efficacy after lyophilization in NFSM, we will explore the use of additional stabilizers. Using the optimal spore and NSFM concentration identified in the previous experiment, we will include trehalose, lactose, and sucrose at concentrations of 5 and 10%. Viability will be assessed before freezing the spores at -20° C, after freezing, and after lyophilization. Efficacy of rehydrated spores will be assessed by treating Dd and Pcc in our luminescent assay. If we are still unable to achieve at least 90% spore viability or treatment efficacy, and our data comparing viability before and after freezing suggests most of our spores are dying before lyophilization, we will explore different rates of freezing.We will subject Dicty spores to both direct and progressive cooling regimens. Using the optimal spore and stabilizers identified in the previous experiments, Dicty spores will be frozen directly (-12° C and -20° C) and progressively (4° C for 2 hr, -12° C for 8 hr, and -20° C overnight). Viability will be assessed before freezing the spores at -20° C, after freezing, and after lyophilization. Efficacy of rehydrated spores will be assessed by treating Dd and Pcc in our luminescent assay.Dicty spore germination -Lyophilized spores will be reconstituted in peptone at concentrations of 1, 2, and 4% in the presence or absence of NFSM at a concentration of 10%. Spores will then be incubated at temperatures of 25, 35, or 45° C for 30 min. Percent germination will be assessed using the viability assay described previously and the efficacy of rehydrated spores will be assessed by treating Dd and Pcc in our luminescent assay.Assessing Dicty feeding habits on other potato pathogens -We will assess the ability of Dicty to suppress fungal growth by coculturing H. solani or Fusarium spores with Dicty spores on minimal media.If fungal sporulation is successful, radial growth of emergent hyphae will be recorded daily to determine if Dicty can reduce fungal growth on agar. To confirm that our Dicty strains are compatible with fungal growth media, we will grow Dicty in the presence of E. coli on this media to determine if Dicty can feed and sporulate. Experiments will be performed at 10°C and 25°C. Image analysis of growth area will be performed using ImageJ. In contrast to these fungal pathogens, potato common scab is caused by the gram-positive bacterium S. scabies. We will first attempt to find a growth medium that supports both S. scabies and Dicty by testing SM2, oatmeal, and yeast malt extract agar. If none of these media support growth of both S. scabies and Dicty, S. scabies will be cultured on an MPM on top of oatmeal agar and incubated at 28°C to first establish bacterial growth. The membrane will then be transferred to water agar and inoculated with Dicty spores. These assays will be performed in 96-well plates and luminescence will be recorded with a multimode plate reader to assess bacterial viability over time.Soft rot field trial -AmebaGone will treat cut seed potatoes with Dicty spores to determine if our product can provide protection against soft rot during this critical time. Seed potatoes will be cut and dip-inoculated with a suspension of Dd or Pcc. This will be followed by treatments with our three Dicty strains using three application rates: 1 x 10^6, 1 x 10^7, and 1 x 10^8 spores/mL. These rates include our current application rate for in vitro experiments (1 x 10^7 spores/mL) plus rates that are ten-fold lower and ten-fold higher. Controls will include non-inoculated potatoes, Dd and Pcc without Dicty, and Dicty treatments at 1 x 10^8 spores/mL without bacteria to ensure that Dicty are non-toxic to potatoes. Approximately 320 cut seed pieces will be used for each treatment condition. Potatoes will be allowed to dry before being stored for one week at 12°C and 98% relative humidity (RH). After 24 hr, and then one week post-treatment, seed potatoes will be visually inspected by an experienced plant pathologist at the SRF for symptoms of soft rot. Seed potatoes will then be planted in field research plots at the UW-Hancock ARS using a randomized complete block design with 4 replications. The percent of potato plants that emerge will be recorded for each treatment and symptoms of blacklegwill be monitored throughout the growing season. After harvest, potatoes will be counted, weighed, and inspected for signs of soft rot that had developed in the field.Soft rot storage trials -AmebaGone will conduct potato storage trials at the UW-Hancock SRF beginning in the fall of 2019.Our first trial will be conducted at the medium scale using naturally-inoculated tubers if the pathogens are confirmed to be present. Artificial inoculations, such as those used in our seed potato trials, can also be used if naturally-infected tubers cannot be sourced at the initiation of the trial in 2019. We will treat potatoes with three Dicty strains at two application rates, include a non-treated control, and include hydrogen peroxide as a standard tuber surface disinfectant. Dicty spores will be lyophilized, rehydrated, and activated according to the best methods identified in Objective 2. Fungicides will still be omitted for this trial until our compatibility tests have been performed. Potatoes will be stored at 10°C and 97% RH for up to three months. During this time, weekly assessments of soft rot development will be taken by a trained plant pathologist. At the end of the experiment, 100 randomly selected potato tubers per treatment will be removed from storage and evaluated for external and internal symptoms of soft rot or other disease. External disease ratings will be taken as percent tuber coverage; internal ratings will be taken as percentage of cut surface diseased.

Progress 09/01/19 to 08/31/22

Outputs
Target Audience: Nothing Reported Changes/Problems:Major Changes This project was approved for funding in September of 2019. Unfortunately, funds were not made available until March 20 of 2020. What opportunities for training and professional development has the project provided?The lead PI on this project, Dr. Ryan Kessens, worked with Anaga Gopalkrishnan during the fall of 2019. Anaga was a student in the Master of Science in Biotechnology program at the University of Wisconsin - Madison. The internship was part of a requirement for the program that Anaga needed for graduation. Ryan provided mentorship by training Anaga in basic microbiological techniques and assay development. Dr. Ryan Kessens was provided with several professional development activities stemming from this project. During the summer of 2019, Ryan attended the American Phytopathological Society conference in Cleveland, Ohio. This conference brings together academic, government, and industry research groups working on highly diverse plant pathogens for 5 days of seminars, networking, and committee meetings. In October of 2019, Ryan and Dr. Marcin Filutowicz, Amebagone's founder and CSO, attended the World Agri-tech Summit in London to pitch AmebaGone's technology to potential investors. This twoday conference included a short pitch of AmebaGone's technology presented by Ryan, a networking booth with visual aids describing our technology, and multiple meetings with potential investors and collaborators from other companies. This conference provided multiple collaborative leads that are still being pursued by AmebaGone. How have the results been disseminated to communities of interest?Ryan Kessens and Marcin Filutowicz coauthored an advertorial in the International Sugar Journal describing the work AmebaGone is doing to develop a treatment for potato soft rot. AmebaGone was invited to write this article after meeting an editor of the journal at the World Agri-tech Summit in London. Interestingly, soft rot of sugar beets is a major problem in the sugar industry, which is why there is interest in novel approaches to treating this devastating disease. The full article can be found here: https://internationalsugarjournal.com/paper/targeting-soft-rot-of-tuber-crops-with-soil-dwelling-phagocyticpredators- of-bacteria/. What do you plan to do during the next reporting period to accomplish the goals?N/A

Impacts
What was accomplished under these goals? Objective 2: Optimize product formulation and application rate Task 1: Maximize Dicty spore viability following lyophilization. Lyophilization is successful if >90% viability is achieved A novel liquid growth method was developed to produce Dicty aspidocytes that provide the necessary efficacy post lyophilization and can be economically scaled-up for mass production while maintaining sterility throughout the process. The aspidocyte phase of Dicty growth was discovered in 2007 in Dr. Kay's laboratory of molecular biology at the University of Cambridge. He discovered that the aspidocyte growth phase can be induced by starvation or toxic stress of liquid growing Dicty cultures and demonstrated that they are immobile but fully viable and are resistant to lysis by detergents. He also discovered that when favorable conditions for growth return, aspidocytes return to the amoebic phase within hours. A liquid growth development buffer containing 5mM Na2HPO4, 5mM KH2PO4, 1mM CaCl2 and 2mM MgCl2 and autoclaved E. Coli B/r as a growth bacteria is inoculated with spores at a concentration of 5 x 104 spores and grown for 72hrs at 25° C to reach a concentration of 1 x 107 live feeding amoeba/ml. After the 72 hour growth period the liquid development buffer is replaced with a 2% sodium alginate solution to induce amoeba starvation causing complete formation of aspidocytes after another 72 hour period. The resulting aspidocyte/2% sodium alginate solution was lyophilized with or without the presence of mannitol or glycine excipients to enhance the final material properties. Utilizing the final product lyophilized materials as an inoculum for liquid development buffer instead of spores resulted in live feeding amoeba growth, demonstrating that the final product can be used as starting inoculum for future Dicty production runs. Task 2: Develop a method for rehydrating and activating lyophilized spores. Spore activation is successful if >50% germination is achieved Lyophilized aspidocyte powders were rehydrated using sterile water at 2X the volume of the pre-lyophilized solution (i.e. 10 ml of solution was lyophilized then rehydrated into 20 ml of sterile water post lyophilization). The reconstituted aspidocyte solution was plated onto SM2D Broth agar plates with K. oxytoca or Dickeya as a growth bacteria and grown for 7 days at 25° C to determine efficacy of lyophilized materials. Resulting final spore concentrations on SM2D Broth agar plates ranged from 3.5-5 x 10 4 spores/mm2 representing a 70-95% successful germination rate. Task 3: Determine product shelf life. Product should retain 90% efficacy after 1 year of storage Freeze-dried aspidocyte samples were prepared in June of 2022 and will be retested for 1 year efficacy in June of 2023. Objective 3: Conduct potato seed treatment and storage trials to determine if Dicty can effectively reduce soft rot under commercial conditions and determine if our product can treat other common potato diseases and is compatible with treatments for these diseases Task 1: Determine if Dicty are compatible with fungicides commonly applied to potatoes. Successful if at least two fungicides reduce Dicty viability by <10% At field application relevant concentrations of Syngenta STADIUM®plaque clearing and mature sporangia formation for Dicty spores of AKKSA (D. sphaerocephalum), Maryland Sa (D. purpureum), and Maryland 18a (D. purpureum) and NW2 (D. sphaerocephalum) was observed, although there was an observed reduction in plaque formation and Dicty development. This demonstrates that Dicty are able to complete their life cycle in the presence of STADIUM®, a commonly used chemical pesticide used to treat soft potato rot. Due to Covid-19 related delays we have no further data to report for this task.? Task 3: Conduct seed potato trials to determine which Dicty strains and application rates are most effective. Successful if seed potato survival is >80% of the non-treated control This task was not completed due to delays and restrictions that occurred because of Covid-19 safety concerns for staff. Task 4: Conduct potato storage trials using most efficacious Dicty strains and application rates determined from Task 3. Successful if Dicty treatment is more effective than current standard Large scale potato seed treatments and storage trials were delayed due to the restrictions imposed by the Covid-19 pandemic. Instead in house experiments were pursued and conducted with the aid and supervision of our scientific advisor, Prof. Amanda Gavens, and other experts at the Hancock Agricultural Research Station. A small-scale in house potato storage trial was conducted utilizing spore application rate of 5x106 spores/ml, determined from spray trials with a 1L Solo sprayer (https://us.solo.global/handheld-and-backpack-sprayers/1077-418-farm-landscape-one-hand-sprayer-1-liter.html) to maximize the spores delivered to the potato surface while minimizing the amount of water sprayed per potato. Representative Snowden ( i.e. Solanum tuberosum ) variety potatoes provided by outreach specialist Dr. Stephen Jordan of the Hancock Research Station were tested for the efficacy of Dicty isolate AKK5A to prevent potato soft rot caused by either Pcc or Dd biofilm forming pathogen strains. Snowden potatoes were infected with Pcc or Dd by puncture wounds from metal tweezers that were dipped in live liquid growth cultures of Pcc or Dd biofilm forming bacteria. Each positive control and treated potato were punctured 60 times to deliver the desired pathogen to the interior starch mass where potato soft rot biofilms form. Spores were applied to the potato surface using a 1L Solo sprayer. Negative control potatoes were not inoculated with Dd or Pcc and did not receive any AKK5A spore treatment. All potatoes were stored at 25° C for 42 days and the percent of infection was determined by total surface area of potato damaged by biofilm forming pathogen for slices for triplicate potatoes of each condition and results are shown in figures 5 and 6. Treatment with AKK5A reduced Dd pathogen total surface area from 5% to 1%. Treatment with AKK5A reduced Pcc pathogen total surface area from 39% to 2%. The larger total surface area of pathogenetic bacteria for Pcc positive control was due to one whole potato being completely infected by Pcc biofilm forming bacteria.

Publications


    Progress 09/01/20 to 08/31/21

    Outputs
    Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Dr. Noah Stern participated in the Wisconsin Center for Technological Advancement Investor Readiness Workshop affiliated with University of Wisconsin Extension. Crafting a One-page teaser, Mock pitch slide deck, presenting to angel investor groups and preparing and implementing materials for website development. Dr. Noah Stern worked with Morgan Warner a high school student participant in the DWD Youth Apprenticeship Program. The internship was part of a requirement for the program that Morgan needed for graduation. Dr. Stern provided mentorship by training Morgan in basic microbiological techniques and assay development. How have the results been disseminated to communities of interest?Drs. Ryan Kessens and Marcin Filutowicz coauthored an advertorial in the International Sugar Journal describing the work AmebaGone is doing to develop a treatment for potato soft rot. AmebaGone was invited to write this article after meeting an editor of the journal at the World Agri-tech Summit in London. Interestingly, soft rot of sugar beets is a major problem in the sugar industry, which is why there is interest in novel approaches to treating this devastating disease. The full article can be found here: https://internationalsugarjournal.com/paper/targeting-soft-rot-of-tuber-crops-with-soil-dwelling-phagocyticpredators-of-bacteria/. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Scale up production of Dicty spores( aspidocytes and amoebae from the most effective strains. Further optimization of the novel liquid growth Dicty spore cultivation method will be performed to maximize the number of spores produced per ml of amoebae grown in liquid medium. Spore production will be scaled up to produce the billions of spores required for field and storage trials. Objective 2: Optimize product formulation and application rate. (I'm lost here) Task 1: Maximize Dicty spore/aspidocyte viability following lyophilization or sodium alginate preparation. Task 2: Test difference in germination rate and efficacy of treatment when comparing rehydration methods for lyophilized spores and sodium alginate beads. Task 3: Determine product shelf life, product should be stable for 1 year of storage. Objective 3: Conduct seed treatment and storage trials to determine if Dicty can effectively reduce soft rot under commercial conditions and determine if our product can treat other common potato diseases and is compatible with treatments for these diseases. Seed potato storage trials will be conducted at the Potato and Vegetable Storage Research Facility in Hancock, WI in the fall of 2020. These experiments will be conducted with the aid and supervision of our scientific advisor, Prof. Amanda Gevens, and other experts at the Hancock Agricultural Research Station. In the summer of 2021, field trials will also be conducted at the Hancock Agricultural Research Station. These two trials will be performed using a variety of Dicty strains and application rates. Based on promising in vitro results, Dicty strains AKK5A (D. sphaerocephalum), Maryland 5a (D. purpureum), and Maryland 18a (D. purpureum) will be included in these experiments. In addition to assessing potato soft rot severity, potatoes from the field trial will also be assessed for symptoms of potato common scab. Potato common scab rating is a routine practice conducted at the Hancock Agricultural Research Station. If our treatment shows a clear reduction in common scab symptoms, we will explore this feature in future field trials as it will add additional value to AG Dicty based technology/ Additional experiments will be conducted to determine if Dicty spore viability is inhibited by other commonly used fungicides chemical treatments for potato seed tuber treatment

    Impacts
    What was accomplished under these goals? Major activities conducted/experiments completed: A liquid growth spore production media was optimized to induce amoebic phase growth into spore germination in wild-type strains. Transitioning from solid to liquid phase fermentation allows AG to significantly scale up live amoebae production and spores under sterile batch conditions. Developed a novel method for inducing the formation of Dicty "aspidocytes", partially dormant organisms that when conditions are favorable quickly resume active feeding and are resistant to detergents (Serafimidis, Ioannis, et al. "A new environmentally resistant cell type from Dictyostelium."Microbiology153.Pt 2 (2007): 619.). Engineered a scalable pipeline for production of stable food-grade sodium alginate - calcium chloride beads infused with either Dicty aspidocytes or spores. To accomplish that, a batch reactor slurry of 2% sodium alginate and dicty spores or aspidocytes are dripped using a peristaltic pump or sprayed using aerosol into a 2% calcium chloride bath system. Produced Dicty "active beads" are washed dried and weighed at room temperature for storage. Our current experiments have demonstrated that aspidocyte and spore Dicty "active beads" are stable for a storage period of 70 days, although testing is still ongoing to determine how long they are viable for. Data collected: Germination of spores and growth in liquid batch reactors was assessed by quantifying the change in live amoebae over time using a hemocytometer counter. Mature spore production from liquid batch reactor amoebae was assessed quantifying spores/mm2 produced. The transition from amorphous amoeba growth phase to the spherical aspidocyte growth phase in batch reactors was quantified using a hemocytometer counter. Viability and bacterial clearing growth of the aspidocytes phase was assessed by inoculating agar plates with liquid batch reactor culture and quantifying total spore production. After a "shelf-life" storage period of 70 days stable sodium alginate - calcium chloride beads infused with either Dicty aspidocytes, amoebae or spores were rehydrated with sterile water and inoculated on lawns of bacteria grown on agar plates, viability was quantified by the total number of spores produced/mg alginate bead. Summary of statistics and discussion of results: Spores of wild-type strains AKK5A (D. sphaerocephalum), Maryland 5a (D. purpureum), and Maryland 18a (D. purpureum), DC-61 (unknown species), Cohen 9 (D. mucoroides), and NW2 (D. sphaerocephalum) were inoculated in liquid development media consisting of 5 mM Na2HPO4, 5 mM KH2PO4, 1mM CaCl2, 2mM MgCl2 and K. oxytoca as a prey bacteria. After 48 hours numbers of live amoebae in the liquid phase reach a maximum density ranging from 4x106-2x107 amoebae/ml. Liquid media from 48hr batch reactor cultures of live amoebae was inoculated on lawns of bacteria and sporangia formation resulted in spore concentrations ranging from 4.2-6x108 spores/ ml reactor media. After a 72hr incubation period in a 2% sodium alginate solution a 90% transition from live growing amoebae phase to the aspidocyte phase was observed for wild-type strains AKK5A (D. sphaerocephalum), Maryland 5a (D. purpureum), and Maryland 18a (D. purpureum), DC-61 (unknown species), Cohen 9 (D. mucoroides), and NW2 (D. sphaerocephalum). After a 70 day storage period rehydrated sodium alginate beads containing spores or aspidocytes were plated on media with lawns of K. oxytoca grown on agar plates, and spore production was quantified. It ranged between 2.3x107-3.7x108 spores/ mg for wild-type strains AKK5A (D. sphaerocephalum), Maryland 5a (D. purpureum), and Maryland 18a (D. purpureum), DC-61 (unknown species), Cohen 9 (D. mucoroides), and NW2 (D. sphaerocephalum). Key outcomes or other accomplishments achieved: The developed liquid growth media and combined stable sodium alginate - calcium chloride preservation of spores and aspidiocytesis promising development for production pipeline of AG products. That process will be scaled up to test Dicty activity after long storage. Objective 2: Optimize product formulation and application rate This objective will be addressed during our no cost extension now that we have developed a scalable pipeline for production of stable food-grade sodium alginate - calcium chloride beads infused with either Dicty aspidocytes or spores as mentioned in Objective 1. Objective 3: Conduct seed treatment and storage trials to determine if Dicty can effectively reduce soft rot under commercial conditions and determine if our product can treat other common potato diseases and whether it is compatible with treatments for these diseases Potato seed treatments and storage trials were delayed due to the restrictions imposed by the Covid-19 pandemic. If safety and health concerns are successfully mitigated in 2022, field and storage trials will be conducted at the Potato and Vegetable Storage Research Facility in Hancock, WI. These experiments will be conducted with the aid and supervision of our scientific advisor, Prof. Amanda Gavens, and other experts at the Hancock Agricultural Research Station. We also anticipate field trials conducted at the Hancock Agricultural Research Station. Based on promising in vitro results, Dicty strains AKK5A (D. sphaerocephalum), Maryland 5a (D. purpureum), and Maryland 18a (D. purpureum) will be included in these experiments. Major activities conducted/experiments completed: Initial experiments to determine if Dicty spore viability is inhibited by commonly used fungicides chemical treatments for potato seed tuber treatment. Spores were treated with Syngenta STADIUM®, containing Azoxystrobin, Difenoconazole and Fludioxonil and inoculated on agar plates. Data collected: Dicty feeding on bacterial lawns was assessed by photographing the stages of their development. Summary of statistics and discussion of results: At field application relevant concentrations of Syngenta STADIUM® plaque clearing and mature sporangia formation for Dicty spores of AKK5A (D. sphaerocephalum), Maryland 5a (D. purpureum), and Maryland 18a (D. purpureum) and NW2 (D. sphaerocephalum) was observed, although there was an observed reduction in plaque formation and Dicty development Key outcomes or other accomplishments achieved: This demonstrates that Dicty are able to complete their life cycle in the presence of STADIUM®, a commonly used chemical pesticide used to treat soft potato rot.

    Publications


      Progress 09/01/19 to 08/31/20

      Outputs
      Target Audience: Nothing Reported Changes/Problems:This project was approved for funding that was supposed to administered to AmebaGone in September of 2019. Unfortunately, funds were not made available until March 20 of 2020. What opportunities for training and professional development has the project provided?The lead PI on this project, Dr. Ryan Kessens, worked with Anaga Gopalkrishnan during the fall of 2019. Anaga was a student in the Master of Science in Biotechnology program at the University of Wisconsin - Madison. The internship was part of a requirement for the program that Anaga needed for graduation. Ryan provided mentorship by training Anaga in basic microbiological techniques and assay development. Dr. Ryan Kessens was provided with several professional development activities stemming from this project. During the summer of 2019, Ryan attended the American Phytopathological Society conference in Cleveland, Ohio. This conference brings together academic, government, and industry research groups working on highly diverse plant pathogens for 5 days of seminars, networking, and committee meetings. In October of 2019, Ryan and Dr. Marcin Filutowicz, Amebagone's founder and CSO, attended the World Agri-tech Summit in London to pitch AmebaGone's technology to potential investors. This two-day conference included a short pitch of AmebaGone's technology presented by Ryan, a networking booth with visual aids describing our technology, and multiple meetings with potential investors and collaborators from other companies. This conference provided multiple collaborative leads that are still being pursued by AmebaGone. How have the results been disseminated to communities of interest?Ryan Kessens and Marcin Filutowicz coauthored an advertorial in the International Sugar Journal describing the work AmebaGone is doing to develop a treatment for potato soft rot. AmebaGone was invited to write this article after meeting an editor of the journal at the World Agri-tech Summit in London. Interestingly, soft rot of sugar beets is a major problem in the sugar industry, which is why there is interest in novel approaches to treating this devastating disease. The full article can be found here: https://internationalsugarjournal.com/paper/targeting-soft-rot-of-tuber-crops-with-soil-dwelling-phagocytic-predators-of-bacteria/. What do you plan to do during the next reporting period to accomplish the goals? Objective 1: Scale up production of Dicty spores from the most effective strains Further optimization of the novel Dicty spore cultivation method will be performed. This includes testing and optimizing the following parameters: Humidity, light intensity, and temperature Spore seeding concentration E. coli concentration Faster spore harvesting methods AmebaGone is currently exploring the possibility of using electrostatic attraction to harvest spores. Cellulose is a major component of the spore cell wall and has an attraction to electrostatic charge. Experiments will be performed to determine if an electrostatic charge can be generated that is strong enough to remove spores from sporangia without reducing the viability of the spores. Objective 2: Optimize product formulation and application rate Task 1: Maximize Dicty spore viability following lyophilization Lyophilization is successful if >90% viability is achieved Task 2: Develop a method for rehydrating and activating lyophilized spores Spore activation is successful if >50% germination is achieved Task 3: Determine product shelf life Product should retain 90% efficacy after 1 year of storage Objective 3: Conduct seed treatment and storage trials to determine if Dicty can effectively reduce soft rot under commercial conditions and determine if our product can treat other common potato diseases and is compatible with treatments for these diseases Seed potato storage trials will be conducted at the Potato and Vegetable Storage Research Facility in Hancock, WI in the fall of 2020. These experiments will be conducted with the aid and supervision of our scientific advisor, Prof. Amanda Gevens, and other experts at the Hancock Agricultural Research Station. In the summer of 2021, field trials will also be conducted at the Hancock Agricultural Research Station. These two trials will be performed using a variety of Dicty strains and application rates. Based on promising in vitro results, Dicty strains AKK5A (D. sphaerocephalum), Maryland 5a (D. purpureum), and Maryland 18a (D. purpureum) will be included in these experiments. In addition to assessing potato soft rot severity, potatoes from the field trial will also be assessed for symptoms of potato common scab. Potato common scab rating is a routine practice conducted at the Hancock Agricultural Research Station. If our treatment shows a clear reduction in common scab symptoms, we will explore this feature in future field trials as it will add additional value to our potato soft rot treatment.

      Impacts
      What was accomplished under these goals? Every year, an estimated 15-30% of the total potato crop in the U.S. is lost topotato soft rot. The economic impactis believed to be well over $510 million annually, and there is no effective treatment available to U.S. growers. AmebaGone is developing novel biocontrol agents to fight the bacteria that cause this devastating disease. Our approach utilizes natural, soil-dwelling organisms called Dictyostelids ("Dicty") that feed on bacteria as their primary food source. Dicty are an all-natural alternative to the chemical pesticides and disinfectants that are currently used to limit disease on potatoes. Dicty are hardy, environmentally tolerant, and self-propagating, making them an excellent choice for sustainable agriculture. Through the first year of this Phase 1 supported research, AmebaGone has identified three strains of Dicty capable of feeding on and limiting the growth of the bacteria that cause potato soft rot. In small-scale lab experiments, we have shown that these three strains, when used alone and/or in combination, can reduce bacterial growth by more than 10-fold. Interestingly, we have also discovered that these Dicty strains can feed on the bacterium that causes another major potato disease:potato common scab. In the second year, we will scale our trials to more closely mimic real-world applications of a future product. The development of a novel fermentation method for scaling up Dicty production is being pursued at AmebaGone and by our partners at BioPharm Laboratories. Production of Dicty for trials in year two will rely on one of these methods or a combination of both approaches. Objective 1: Scale up production of Dicty spores from the most effective strains Major activities conducted/experiments completed An in vitro assay was developed to quickly screen Dicty strains for antibacterial activity against the soft rot pathogens Dickeya dianthicola (Dd) and Pectobacterium carotovorum subsp. carotovorum (Pcc). The Dd and Pcc strains expressed a luminescent reporterthat could be detected and quantified using a plate reader. In-total, 33 Dicty strains collected from Alaska were screened using this method. A novel Dicty spore cultivation method using solid-state fermentation is being developed. Terry cloth fabric is being used as a solid-state to support Dicty and the E.coli substrate that they feed on in an alginate matrix.Terry cloth fabric is cheap, reusable, and can be tightly packed for efficient use of space. Data collected Bacterial viability was assessed by quantifying luminescence from metabolically active bacterial cells using a plate reader with a luminometer.Non-treated bacteria were compared to Dicty-treated bacteria to assess viability.Experiments were conducted in a 96-well all-white plate. Using the novel spore cultivation method being developed at AmebaGone, Dicty strains AKK5A (Dictyostelium sphaerocephalum), Maryland 5a (D. purpureum), and Maryland 18a (D. purpureum) were seeded on Terry cloth fabric with E.coli in an alginate matrix. Spore viability was determined by performing a plaque assay in which Dicty spores were culture with a thin layer of E.coli growth on an agar plate. Summary statistics and discussion of results Of the 33 Dicty strains screened, 3 strains provided the most robust and reproducible reduction in bacterial growth: AKK5A, Maryland 5a, and Maryland 18a. Strain AKK5A reduced Dd viability by >10-fold and Pcc viability by >50-fold compared to the non-treated control. Dicty strain Maryland 18a reduced viability of both bacteria by >100-fold. Interestingly, Dicty strain Maryland 5a only reduced bacterial viability when combined with AKK5A.The AKK5A/Maryland 5a combination reduced the viability of both bacteria by >50-fold. Attempts to cultivate Dicty strain AKK5A on Terry cloth fabric were unsuccessful. Eachstrain was also cultivated on agar since this is the current standard method of cultivating spores. Maryland 5a produced ~51,000 spores/mm2 on fabric and ~40,000 spores/ mm2 on agar. Maryland 18a produced ~49,000 spores/mm2 on fabric and ~48,000 spores/ mm2 on agar. Maryland 5a spores had a viability of 26% on agar while viability dropped to 16% on cloth. Maryland 18a spores had a viability of 35% on agar while viability dropped to 14% on cloth. Key outcomes or other accomplishments achieved In Objective 1, we describe the development of an in vitro assay for rapidly screening Dicty strains against bacterial pathogens of interest growing in solid medium. This assay is ~10x faster than the previous method used at AmebaGone and will allow AmebaGone to rapidly screen in a solid medium its collection of over 3,000 Dicty strains against otherbacterial pathogens. In Objective 1, we also describe the optimization of a novel Dicty spore cultivation method utilizing cheap and reusable Terry cloth fabric. This is a completely novel method of Dicty spore production that AmebaGone will file a provisional patent on in year 2 of this grant. Objective 2: Optimize product formulation and application rate This objective will be addressed in year 2 as we move forward with 1 or 2 strains of Dicty. Objective 3: Conduct seed treatment and storage trials to determine if Dicty can effectively reduce soft rot under commercial conditions and determine if our product can treat other common potato diseases and is compatible with treatments for these diseases Major activities conducted/experiments completed Potato seed treatments and storage trials will be conducted in year 2. Once these results indicate the best strains for commercial use, experiments will be conducted to determine compatibility of these strains with commonly used potato fungicides. Potato common scab is a disease that causes blemishes on the tuber surface that significantly reduces fresh market value and can promote secondary infections and shrinkage. This disease is caused by the gram-positive bacterial pathogen Streptomyces scabies, with no treatments available to growers. We have investigated the ability of ten Dicty strains to feed on S. scabies at 10°C and 28°C on two different types of media: SM2D agar and oatmeal agar. These experiments were performed by first establishing a lawn of S. scabies growth on polycarbonate filters overlaying SM2D or oatmeal agar at 10°C or 28°C. Once a dense lawn of bacterial growth had been established, spores from Dicty strains AKK5A, Cohen 9 (D. mucoroides) , DC-7 (unknown species), DC-61 (unknown species), HP4 (Polysphondylium pallidum), HP8 (P. candidum), Maryland 5a, Maryland 18a, NW2 (D. sphaerocephalum), or NW16B (D. mucoroides) were spotted in the center of the lawn and placed back at 10°C or 28°C.. Data collected Images were taken of bacterial lawns and Dicty feeding was assessed by looking for signs of clearing and sporangia development. SM2D agar at 10°C - Dicty strains AKK5A, Cohen 9, DC-61, and NW2 showed clear signs of feeding on S. scabies. Oatmeal agar at 10°C - Dicty strains AKK5A, Cohen 9, DC-61, HP8, and NW2 showed clear signs of feeding on S. scabies. SM2D agar at 28°C - Dicty strains AKK5A, Cohen 9, DC-7, HP4, Maryland 5a, Maryland 18a, and NW2 showed clear signs of feeding on S. scabies. Oatmeal agar at 28°C - Dicty strains Cohen 9, DC-7, DC-61, HP4, HP8, Maryland 5a, Maryland 18a, NW2, and NW16B showed clear signs of feeding on S. scabies. Summary statistics and discussion of results The only two Dicty strains that were capable of feeding on S. scabies under all conditions were Cohen 9 and NW2. While we saw no signs of feeding on oatmeal agar at 28°C, Dicty strain AKK5A was able to feed on S. scabies under the other three conditions. Key outcomes or other accomplishments achieved This is the first report of Dicty strains capable of feeding on the potato common scab pathogen, Streptomyces scabies.

      Publications

      • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Presentation to investors at the 2019 World Agri-Tech Summit in London
      • Type: Journal Articles Status: Published Year Published: 2020 Citation: Kessens, R. and Filutowicz, M. Targeting soft rot of tuber crops with soil-dwelling phagocytic predators of bacteria. International Sugar Journal. 2020, 122 (1454): 142-144