Source: MONTANA BIOAGRICULTURE INC. submitted to
CURVULARIA: FUNGAL SYMBIOSIS FOR DROUGHT PROTECTION IN WHEAT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1024569
Grant No.
2020-39410-33217
Cumulative Award Amt.
$600,000.00
Proposal No.
2020-06715
Multistate No.
(N/A)
Project Start Date
Sep 30, 2020
Project End Date
Sep 29, 2023
Grant Year
2020
Program Code
[8.2]- Plant Production and Protection-Biology
Project Director
Bradley, C.
Recipient Organization
MONTANA BIOAGRICULTURE INC.
510 EAST KENT AVE
MISSOULA,MT 59801
Performing Department
(N/A)
Non Technical Summary
There is a critical need for innovative technology to reduce drought induced crop losses on more than 45 million acres of US dryland grown small grains. Montana BioAgriculture, MBAI, licensed patents for a plant symbiotic fungus, Curvularia protuberata, which confers tolerance to drought in wheat and barley. MBAI sees opportunity to bring C. protuberata to commercial use. Phase 1 demonstrated proof of concept: Low cost spore production using commercially scalable culture conditions; Efficient delivery by spore coating seeds; Up to 10 days of increased tolerance to water stress in wheat grown from nonsterile seed, planted in nonsterile soil. Phase II will advance C. protuberata to a commercial product with objectives and work plan to:Develop methods to product ensure efficacy over long term culture maintenance, production scale up and deliveryConfirm efficacy with different wheat varieties and integration with agronomic practicesDemonstrate efficacy in field trialsGenerate the economic information needed to raise capital for commercial production and marketing.MBAI will raise capital for commercial production and marketing to help grain farmers respond to climate change and unpredictable rainfall. Based on phase 1 results, MBAI can sell C. protuberata as low cost "drought insurance" while providing attractive return to investors.As a commercial product C. protuberata will contribute to food security and advance development and delivery of science for agricultural to mitigate climate impacts
Animal Health Component
40%
Research Effort Categories
Basic
(N/A)
Applied
40%
Developmental
60%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20315491102100%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1549 - Wheat, general/other;

Field Of Science
1102 - Mycology;
Goals / Objectives
The goal of phase II is to develop C. protuberata to the point where MBAI can raise private capital to finance commercial production and marketing. Specific objectives are to address the principal challenges in advancing from proof of concept to a commercial product:1. Develop methods to ensure efficacy is maintained through culture storage, spore production and delivery in commercial channels2. Confirm efficacy across multiple wheat varieties, refine delivery methods for spring and winter wheat3. Determine feasibility of colonizing wheat with both C. protuberata and endophytic fungal insect pathogens4. Develop strategies to integrate C. protuberata with existing agronomic practices5. Estimate capital and operating cost for commercial production of C. protuberata spores6. Define regulatory requirements and cost7. Demonstrate efficacy in field trials
Project Methods
MBAI will conduct the project in a series of 7 tasksTask 1 Ensure commercial efficacyTask 2 Confirm efficacy across multiple spring and winter wheat varieties.Task 3 Interactions between beneficial endophytesTask 4. Agronomic practicesTask 5. C. protuberata Spore production.Task 6. Regulatory ComplianceTask 7. Field trialsIn laboratory work MBAI will use standard methods for:Quantifying spore preparations, serial dilutions and plating for colony forming unitsLaboratory wheat propagation: Winter and spring wheat cultivars grown in standard container, soil, fertilizer and photoperiod conditions in laboratory growth chambers. Winter wheat will be germinated and grown to early tillering, typical for fall emergence, then placed in cold storage and returned to growth chambers to resume growth to stage 39-45 (Zadocks et al. 1974) before assessment of colonization or foliar spore applications.Confirming Curvularia colonization of plant tissues Root colonization will be determined by surface sterilizing root sections in a sequence of ethanol, sodium hypochlorite and sterile water and plating for outgrowth of C. protuberata from root. The effectiveness of surface sterilization is verified by imprinting sections of un-dissected plants on agar plates and observing for growth of bacteria and fungi.Laboratory water deficit stress Water stress is initiated when watering is stopped. After control plants show wilt symptoms, typically 5 to 7 days without water, plants are rehydrated at varying intervals to assess recovery from stress. An alternative method is to measure effect of the interval between water additions. Inoculated wheat and un-inoculated controls are grown with regular watering then switched to watering with defined water volume at different intervals, for example 6, 8, 10, 12 and 14 days. Effects of water deficit is assessed by observation and photographing plants and by measuring biomass at sample intervals or at the end of experiments.Assay for presence/absence of the Curvularia thermotolerance virus. We will use simple sequence repeat (SSR) analysis with two single-primer PCR reactions and by sequence analysis of the rDNA ITS1-5.8S-ITS2 region as outlined previously by Marques et al. 2007. We will also use Northern blot analysis for testing RNA1 and RNA2 of the virus and RT-PCR using transcripts of the virus as probes, and RT-PCR using primers specific for a section of the RNA 2.Field TrialsThe project will include spring wheat field trial in Montana and North Dakota in year 1 with addition of trials one or more states with large wheat areas with predominantly winter wheat, Kansas, Oklahoma, Colorado and or Washington in year 2:Spring wheat 2021: Spring wheat seed treatment and or foliar spray applied spring 2021 results assessed Spring Summer 2021.Winter wheat Fall 2021: Seed treatment Fall planting 2021, follow up foliar spray as needed spring growth 2022. Assessing results summer 2022Spring wheat 2022: Spring wheat seed treatment and or foliar spray applied spring 2022, planted wheat, results assessed Summer 2022Trial design assumes four to six treatment variables in comparison with untreated controls in a minimum of 3 replicate plots per treatment and controls with typical plot size for research trials. Wheat will be grown without irrigation or cover and subject to natural weather conditions. In year two we will coordinate with collaborators at one or more locations for treatments including rain covers. The Montana site has research plots placed with local growers at two locations in addition to the research center each with different soil types and rainfall patterns. Variables to be evaluated in trials will include:Wheat varietySpore coated seed versus foliar spray, especially for winter wheatSpore doseApplication timing for foliar sprayFungicide seed treatment and foliar sprayCombination of C. protuberata with endophytic wheat stem sawfly pathogens year 2.Efficacy will be assessed by:Observation and photographs at regular intervalsAssays for C. protuberata colonization, plots sampled at approximate midpoint of growing season and assayed for C. protuberata colonization and drought response compounds as developed in task 1.Yield.Weather conditions including temperature and rainfall during trials will be obtained for each site.

Progress 09/30/20 to 09/29/23

Outputs
Target Audience:The project has multiple target audiences, university extension, grain growers in key wheat growing states, potential investors, potential partners for marketing and distribution. During the phase II we worked with university extension researches in Montana and North Dakota, and discussed the project with extension researchers in Kansas and Nebraska. We initiated discussions for venture financing and with agriculture chemical companies for financing commercial development and marketing as one product in a portfolio of biological products under development. Changes/Problems:Phase II met objectives except for objective 7 field trials where we have not generated sufficient performance data to support commercialization. Trial locations were limited to Montana and North Dakota; planned trials in Nebraska and Kansas were not conducted due to covid restrictions. Extreme drought during trials limited useful data. Assays of root samples taken from field plots showed inconsistent colonization by Cp. MBA is working to define conditions to improve root colonization and plans additional field trials with provate support. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Discussions and review of field trials with individual university extension researchers in Montana, and North Dakota . Disseminated project information to potential venture investors and to interested agriculture chemical companies. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During the phase II project MBA: • Developed a streamlined genetic assay method for quality control in long term culture storage and spore production. Evaluated efficacy of Curvularia protuberata, Cp, spore preparations from different original sources and differing storage conditions. • Confirmed Cp root colonization in multiple varieties of spring and winter wheat • Demonstrated that endophytic colonization of wheat by Cp did not inhibit disease resistance induced by fungal insect pathogens (development of endophytic fungal insect pathogens in related work) • Demonstrated Cp resistance to commercial fungicide seed treatments. Continued work with both powder and liquid formulations suitable for application as commercial seed treatments. • Confirmed economics in spore production for field trials. Spore production to treat 5 million acres of wheat seed would have a cost for major equipment of less than $2 million and cost of product of about $0.50/acre. • Determined that in Cp will not require EPA pesticide registration • Conducted two tears of research field trials in both spring and winter wheat at university experiment stations in Montana and North Dakota. Sampled and assayed for Cp colonization of root samples from all different treatments of Cp spore preparations and wheat varieties in spring and winter wheat field trials.

Publications


    Progress 09/30/21 to 09/29/22

    Outputs
    Target Audience:The project has multiple target audiences, university extension, grain growers in key wheat growing states, potential investors, potential partners for marketing and distribution. During the first year of phase II we worked with university extension researches in Montana and North Dakota, and discussed the project with extension researchers in Kansas and Nebraska. We initiated discussions with two companies regarding potential finance for further commercial development and marketing. Changes/Problems:The first two seasons of field trials did not generate sufficent efficacy data. Montana BioAgricuture obtained a no cost extension for the project primarily to enable an additional season of field tests. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Discussions and review of field trials with individual university extension researchers in Montana, and North Dakota. What do you plan to do during the next reporting period to accomplish the goals?The principal activity will be to conduct additional spring wheat trials in 2023. With continuing field trials, we will also continue work in to meet objectives 1, 2, 4 and 5. Continued field trials will require continued production of spore formulations generating data for objectives 1, and 5. We will continue scaled up spore production and monitor spore production for quality control and maintenance of efficacy through production pitch up. We will also continue to assess spore yields and refine capital and operating cost estimates for commercial scale spore production. For objective 2 field trials lab work will continue with tests in different wheat varieties in parallel with different varieties in field trials. For objective 4 we will include comparisons of seed with standard fungicide treatments and untreated seed in fungal seed treatments. In laboratory experiments, C. protuberata growth and root colonization was not inhibited with fungicide treated seed. However, with limited field trials to date we have not been able to assess interactions with fungicide treated seed under field conditions.

    Impacts
    What was accomplished under these goals? During the second year of phase II MBAI: • Developed a streamlined genetic assay method for quality control in long term culture storage and spore production. Evaluated efficacy of Curvularia protuberata Cp spore preparations from different original sources and differing storage conditions. • Confirmed Cp root colonization in multiple varieties of spring and winter wheat • Demonstrated that endophytic colonization of wheat by Cp did not inhibit disease resistance induced by fungal insect pathogens (development of endophytic fungal insect pathogens in related work) • Demonstrated Cp resistance to commercial fungicide seed treatments. Continued work with both powder and liquid formulations suitable for application as commercial seed treatments. • Confirmed economics in spore production for field trials • Determined that in Cp will not require EPA pesticide registration • Conducted a second year of research field trials in spring wheat at university experiment stations in Montana and North Dakota, completed winter wheat field trials at both locations. Sampled and assayed for Cp colonization of root samples from all different treatments of Cp spore preparations and wheat varieties in spring and winter wheat field trials.

    Publications


      Progress 09/30/20 to 09/29/21

      Outputs
      Target Audience:The project has multiple target audiences, university extension, grain growers in key wheat growing states, potential investors, potential partners for marketing and distribution. During the first year of phase II we worked with university extension researches in Montana and North Dakota, and discussed the project with extensionn researhers in Kansas and Nebraska. We initiated discussions with two compnaies regarding potential finnce for further commercial development and marketing. Changes/Problems:Phase II is on track for meeting objectives except for objective 7 field trials where severe drought limited useful performance data. Project may require an additional year of research scale field trials to demonstrate efficacy. Slippage Phase II performance period is 9/30/2020 - 9/29/2022. The original work plan proposed an initial winter wheat trial with fall planting in 2020. However, with the September 30 start date and covid restrictions this trial could not be carried out. Covid restrictions and staff limits in Kansas and Nebraska are also limiting 2021 winter wheat trial at those locations. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Discussions and review of field trials with individual university extension researchers in Montana, and North Dakota . What do you plan to do during the next reporting period to accomplish the goals?Continue work in each of the project objectives 1. Continued evaluation of maintaining Curvularia thermo tolerance virus in long term culture storage and in spore production scale up 2. Continue evaluation of Cp colonization of different wheat varieties, especially colonization in through winter wheat vernalization 3. Additional experiments testing colonization by both C. protuberata and endophytic fungal insect pathogens, especially from seed treatment by both fungi. 4. Test efficacy of Cp applied to commercial seed treated with fungicides, laboratory experiments with soil planted seeds and in small plot field trials 5. Refine estimates for Cp spore production capital and operating cost. Prepare preliminary design for production plant including types and cost for major equipment 6. Continue to monitor US EPA rules for plant biostimulants. Review Canadian regulations and prepare necessary applications 7. Continue field trials: 2021 fall planted winter wheat in Montana and North Dakota, 2022 spring wheat trials Montana North Dakota, fall planted winter wheat in additional states. Commercialization: Continue discussions with two companies identified in year 1, if necessary, identify and initiate discussions with addition potential investors and marketing partners.

      Impacts
      What was accomplished under these goals? During the first year of phase II MBAI: Developed assay methods for quality control in long term culture storage and spore production Confirmed Cp root colonization in multiple varieties of spring and winter wheat Demonstrated feasibility for endophytic colonization of wheat by both Cp and fungal insect pathogens Developed spore formulations suitable for application as commercial seed treatments and demonstrated Cp resistance to commercial fungicide seed treatments Confirmed economics in spore production for field trials Determined that in Cp will not require EPA pesticide registration Conducted research field trials in spring wheat at university experiment stations in Montana and North Dakota and initiated winter wheat trials at both locations.

      Publications