Desiccation resistant pseudomonad strains and treatment of agricultural maladies therewith
Inventors
Slininger, Patricia J. • Schisler, David A. • Shea Andersh, Maureen A.
Assignees
US Department of Agriculture USDA
Publication Number
US-10982185-B2
Publication Date
2021-04-20
Expiration Date
2038-09-26
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Abstract
Novel strains of Pseudomonas fluorescens are disclosed. Several novel mutated strains of Pseudomonads are engineered by repetitive culturing of a parent strain under stressed conditions. Various enriched populations are screened and ranked based on relative performance indices including viable cell yield during growth, efficacy in suppression of dry rot disease, cell growth recovery after dry storage and resistance to switchgrass hydrolysate.
Core Innovation
The invention provides novel strains of Pseudomonas fluorescens that are tolerant of long-term storage under desiccated conditions and maintain their viability and biological control efficacy when rehydrated. These novel strains are developed through engineered mutagenesis, involving selective pressure environments that induce mutations and select for enhanced desiccation tolerance, growth yield, and efficacy in suppressing fungal agricultural diseases such as Fusarium dry rot. The strains are produced by repetitively culturing parent strains under stressed conditions including cycles of growth, desiccation without osmoprotectants, and dry storage, followed by screening for superior performance indices.
The problem addressed is the difficulty in producing and delivering Pseudomonads on a commercial scale due to their low resilience under desiccated storage conditions as they are Gram-negative, non-spore-forming bacteria. Existing chemical fungicides, particularly azole-based compounds, are less effective due to widespread resistance in fungal pathogens and pose human health concerns. There is an industrial need for stable biological control agents (BCAs) that remain viable after long periods of dry storage and are effective in controlling fungal diseases in agricultural products, especially potatoes affected by Fusarium species.
The invention also provides methods for producing these novel microbial strains via engineered mutagenesis and methods of using the strains as BCAs applied to agricultural products. The process includes culturing parent strains, passaging, subjecting to stress conditions such as desiccation and dry storage at varying temperatures, measuring characteristics such as viable cell yield and disease suppression efficacy, and selecting strains outperforming parent strains. Additionally, the invention covers compositions comprising the novel strains, their dried formulations suitable for long-term storage, and kits for applying the strains in agricultural contexts.
Claims Coverage
The patent includes several inventive features directed to novel strains of Pseudomonas fluorescens, methods of generating such strains through engineered mutagenesis, compositions comprising these strains, and methods and kits for treating agricultural products with these strains.
Novel desiccation resistant Pseudomonas fluorescens strains
The invention provides novel strains capable of surviving rapid air drying in droplets of spent broth without osmoprotection and capable of surviving non-refrigerated storage conditions, produced from parent strains of Pseudomonas fluorescens, including strains deposited under specific ARS Culture Collection accession numbers.
Identifying characteristics defining novel strains
The novel strains have identifying characteristics including viable cell yield during growth, efficacy in suppression of dry rot disease, cell growth recovery after dry storage, tolerance to switchgrass hydrolysate, enhanced capability and tolerance to withstand long-term partially or fully desiccated storage, and growth and bioefficacy phenotypes more active after rehydration compared to parent strains.
Method of generating novel microbial strains by engineered mutagenesis
A method comprising culturing a parent strain, passaging samples, optionally pelletizing and suspending samples, subjecting samples to stress conditions to create stressed samples, measuring characteristics, and selecting novel mutant strains outperforming the parent strain, optionally repeating the process with selected mutants for further improvement.
Compositions comprising novel strains
Compositions comprising the novel strains, at least partially dehydrated, with water content less than about 30% or less than about 15%.
Methods of treating agricultural products with novel strains
Methods of treating agricultural products, specifically potatoes, by applying the novel strains, optionally rehydrating dehydrated compositions before application to control dry rot-inducing fungi.
Kits for applying novel strains in agriculture
Kits comprising containers holding the novel strains, optional solutions, diluents or applicators, and instructions for applying the novel strains to agricultural products.
The claims collectively cover novel desiccation-resistant Pseudomonas fluorescens strains, their identifying characteristics, methods for creating and using them as BCAs for fungal disease suppression in agriculture, compositions and formulations containing these strains, and kits facilitating their application.
Stated Advantages
Provides novel strains of Pseudomonads that are resilient to long-term dry storage.
Enables production of novel microbial strains with engineered mutagenesis to achieve user-defined desirable characteristics.
Offers methods of preventing and treating various fungal infections in agricultural environments.
Provides methods for preventing and treating potato maladies including Fusarium dry rot, late blight, pink rot, and sprouting.
Delivers broad spectrum microbial control agents as alternatives to antifungal chemical compounds.
Supplies kits for inoculating agricultural areas with novel BCA strains to control fungal populations.
Enables production of broad spectrum antifungal microbial alternatives as coproducts of renewable lignocellulosic biomass biorefineries.
Documented Applications
Use of novel strains as biological control agents for fungal agricultural diseases, including postharvest and field diseases in potatoes such as Fusarium dry rot, late blight, pink rot, and sprouting.
Application of strains to agricultural products, particularly various types of potatoes, to suppress dry rot-inducing fungi.
Production and use of broad spectrum antifungal microbial alternatives derived from switchgrass hydrolysate in lignocellulosic biorefineries.
Integration of the novel strains in integrated pest management systems, optionally combined with chemical treatments to reduce chemical dosages or to comply with organic certification requirements.
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