Progress 03/01/19 to 10/31/19

Outputs
Target Audience:C. protuberata drought tolerance is one component of an overall MBAI program to develop plant symbiotic microbes as solutions for the principal issues of wheat production in North America: drought, wheat stem sawfly and Fusarium head blight. Principal target audience for this program has been potential investors. MBAI prepared and presented program summaries, power point presentations and preliminary financial forecasts to multiple venture capital companies and other sources of private capital. MBAII was successful in securing a term sheet for a $3 million equity investment to support this program. Changes/Problems:Timeline for phase 1 was to short to complete winter wheat experiments. Equipment and set up for winter wheat vernalization took longer than expected. MBAI applied for but did not receive a no cost extension. Experiments with winter wheat are continuing. Results will be submitted with phase II proposal. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Results provided to a venture capital company were important in obtaining equity investment for commercially developing C. protuberata as one of several fungal based products for solving issues of small grain production. What do you plan to do during the next reporting period to accomplish the goals?The phase 1 project is complete. MBAI will submit a SBIR phase II proposal to continue developing C. protuberata as a commercial product. Venture capital investment will also support continued development. Next steps in development are: Scale up spore production to 20kg substrate to support field trials Multi state research field trials in collaboration with university cooperative extension Continued work with winter wheat and alternative inoculation methods Research to assay for and methods to ensure Curvularia thermal tolerance virus is maintained in culture storage, production scale up and commercial application Compatibility for application with commercial fungicide treated seed Economic analysis Design for pilot plant to support commercial introduction on at least 100,000 acres. Investigate regulatory requirements for marketing in Canada.

Impacts
What was accomplished under these goals? What was accomplished under these goals? MBAI met the phase 1 goal; research met each of the objectives, demonstrating feasibility of C. protuberata as a commercial product for increasing tolerance of wheat to water stress. Objective 1. Spore production Phase 1 demonstrated that solid substrate culture is necessary for spore production by C. protuberata. MBAI developed bench scale spore production using low cost, grain based substrates, nutrient additions and culture conditions which produced up to 2 X1010 spores per Kg of substrate in a 7 day culture time. At spore application rates found effective in colonizing roots of wheat (discussed on objective 2 and 3 below), one Kg of culture substrate would produce sufficient spores to inoculate 10 acres of wheat. Preliminary economic analysis based on MBAI experience with commercial scale solid substrate fungal culture showed that cost of product for C. protuberata spores would be less than $1.00/acre. C. protuberata would be would be very profitable sold in the range of 4$ to $6 per acre as "insurance" against yield and quality loss due to extended periods between rainfall. Capital cost for commercial spore production to treat 8 million acres of wheat would be no more than $10 million, with revenue of $40 million if sold at $5.00/acre treated. Objective 2 Commercially feasible inoculation methods In phase 1 MBAI developed spore formulations and seed coating as a cost effective means of inoculating wheat to establish C. protuberata as a root endophyte. Concentrating spores on to seeds reduces the number of spores required to inoculate and acre of wheat compared to foliar sprays or soil application. Seed coating as a delivery method also aids marketing; supplying spore formulations to seed coating operations rather than through distribution to individual farmers. Results showed that application rates of about 1,000 spores per seed provided consistent inoculation and endophytic colonization. In some experiments as few as 100 spores per seed were effective. At 1,000 spores per seed and typical dryland seeding rates of 1,000,000 seeds per acre, effective seed treatment will require about 1 X 109 spores/acre. Experiments tested a number of different carriers for spores. Two formulations were found cost effective: as diluents for spores, adhering spores to seed and suitable for commercial seed coating systems. One was a clay based carrier and the second a water based formulation using a selected molecular weight of methyl cellulose. One issue with seed coating is application to commercial seed treated with fungicides and insecticides. C. protuberata spores were coated onto commercial seeds fungicide treated seeds which were then plated on agar. C. protuberata hyphae grew from seed indicating tolerance to the fungicide. Objective 3 Water stress tolerance Experiments using spring wheat varieties grown from spore treated seed showed colonization by C. protuberata and tolerance to up to 10 days more water stress than uninoculated controls. Roots assays confirmed colonization with outgrowth of C. protuberata on nutrient agar from surface sterilized roots. Tolerance to water stress was shown in both experiments with nonsterile seed in sterile soil and in representative nonsterile soil. Experiments with winter wheat testing endophytic colonization and water stress tolerance after cold period vernalization are in progress. Conclusion C. protuberata spores produced in solid substrate culture using commercially feasible substrates and culture conditions, applied to seed using commercially feasible formulations and application provided up to 10 days of tolerance to water deficit compared to controls. Analysis of regulatory requirements indicates that C. protuberata can be field tested with permits from USDA APHIS PPQ and marketed as a microbial "plant biostimulant" to improve stress tolerance in plants without the need for EPA registration.

Publications