Desiccation resistant pseudomonad strains and treatment of agricultural maladies therewith

Patent metadata

Field	Value
Publication number	US-10982185-B2
Application number	US-201816142745-A
Country	US
Kind code	B2
Filing date	Sep 26, 2018
Priority date	Sep 26, 2018
Publication date	Apr 20, 2021
Grant date	Apr 20, 2021

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.

First claim

The claimed invention is:

1. A novel strain having identifying characteristics of being capable of surviving rapid air drying in droplets of spent broth without osmoprotection, and being capable of surviving non-refrigerated storage conditions, wherein the novel strain is produced from a parent strain of Pseudomonas fluorescens , and the novel strain is selected from the group consisting of: Agricultural Research Service (ARS) Culture Collection Patent Deposit Accession Numbers NRRL B-67667; NRRL B-67668; NRRL B-67669; NRRL B-67670; NRRL B-67671; NRRL B-67672; NRRL B-67673; NRRL B-67674; NRRL B-67675; and combinations or cocultures thereof.

2. The novel strain of claim 1 , wherein the identifying characteristics further include viable cell yield during growth, efficacy in suppression of dry rot disease, and cell growth recovery after dry storage.

3. The novel strain of claim 1 , wherein the identifying characteristics further include tolerance to switchgrass hydrolysate.

4. The novel strain of claim 1 , wherein the identifying characteristics further include growth and bioefficacy phenotypes that are at least about 1.6 times more active after rehydration from partially or fully desiccated conditions as compared to the parent strain of Pseudomonas fluorescens.

5. The novel strain of claim 1 , wherein the identifying characteristics further include growth and bioefficacy phenotypes that are from about 1.6 to about 6 times more active after rehydration from partially or fully desiccated conditions as compared to the parent strain of Pseudomonas fluorescens.

6. The novel strain of claim 1 , wherein the identifying characteristics further include enhanced capability and tolerance to withstand long-term storage; enhanced capability and tolerance to withstand partially or fully desiccated storage, while maintaining growth and bioefficacy phenotypes when rehydrated; enhanced growth and bioefficacy phenotypes; and combinations thereof, wherein the enhancements are when compared to the parent strain.

7. A composition comprising the novel strain of claim 1 .

8. The composition of claim 7 , which is at least partially dehydrated and has less than about 30% water content.

9. The composition of claim 7 , which is at least partially dehydrated and has less than about 15% water content.

10. A method of generating a novel microbial strain of claim 1 having identifying characteristics of being capable of surviving rapid air drying in droplets of spent broth without osmoprotection, and being capable of surviving non-refrigerated storage conditions, the method comprising: (a) culturing a parent strain of the novel microbial strain in a first medium for a time sufficient to achieve a predetermined culture density and create a base culture; (b) passaging the base culture by drawing one or more samples from the base culture to create at least one passaged sample; (c) optionally pelletizing the at least one passaged sample and suspending each passaged sample separately in a second medium to create a series of passaged samples; (d) subjecting the series of passaged samples to at least one stress condition to create a series of stressed samples; (e) measuring at least one characteristic of the series of stressed samples to create at least one measured characteristic; and (f) determining whether the measured characteristic is superior to the same characteristic of the parent strain to select at least one novel mutant strain from the series of stressed samples; further comprising replacing the parent strain in step (a) with one or more of the selected mutant strains from step (f) and repeating steps (a) to (f).

11. The method of claim 10 , wherein the measured characteristic is selected from the group consisting of: enhanced capability and tolerance to withstand long-term storage; enhanced capability to tolerate at least partially desiccated storage, while maintaining growth and bioefficacy phenotypes when rehydrated; enhanced growth and bioefficacy phenotypes; and combinations thereof.

12. The method of claim 10 , wherein the measured characteristic includes enhanced capability to tolerate at least partially desiccated storage, while maintaining growth and bioefficacy phenotypes when rehydrated.

13. The method of claim 10 , wherein the parent strain is a Pseudomonas fluorescens strain and selected from the group consisting of: strain P22:Y:05, having ARS Culture Collection Patent Deposit Accession Number NRRL B-21053; strain S11:P:12, having ARS Culture Collection Patent Deposit Accession Number NRRL B-21133; strain S22:T:04 having ARS Culture Collection Patent Deposit Accession Number NRRL B-21102; and combinations or cocultures thereof.

14. The method of claim 10 , further comprising preparing a dried culture from the novel mutant strain.

15. A method of treating an agricultural product with a novel strain having identifying characteristics of being capable of surviving rapid air drying in droplets of spent broth without osmoprotection, and being capable of surviving non-refrigerated storage conditions, wherein the novel strain is selected from the group consisting of: Agricultural Research Service (ARS) Culture Collection Patent Deposit Accession Numbers NRRL B-67667; NRRL B-67668; NRRL B-67669; NRRL B-67670; NRRL B-67671; NRRL B-67672; NRRL B-67673; NRRL B-67674; NRRL B-67675; and combinations or cocultures thereof, the method comprising applying the novel strain to the agricultural product; wherein the agricultural product is any type of potato.

16. The method of claim 15 , further comprising: (a) rehydrating a dehydrated composition comprising the novel strain to produce a rehydrated novel strain and (b) applying the rehydrated novel strain to the agricultural product.

17. The method of claim 16 , wherein the dehydrated composition contains no more than about 30% water based on weight.

18. The method of claim 16 , further comprising applying the rehydrated novel strain to the agricultural product to control dry rot-inducing fungi.

19. A kit for carrying out the method of claim 15 , the kit comprising a first container means containing the novel strain; optionally other container means comprising a solution, diluent, or applicator for the novel strain; and written information including procedures for applying the novel strain to the agricultural product.

Assignees

• Us Agriculture

Inventors

• Slininger Patricia J
• Schisler David A
• Shea Andersh Maureen A

Classifications

• A01N63/27

  Pseudomonas · CPC title

• C12R2001/39

  Pseudomonas fluorescens · CPC title

• C12N1/205

  Bacterial isolates · CPC title

• C12N1/20 Primary

  Bacteria; Culture media therefor · CPC title

• A01N63/10

  Animals; Substances produced thereby or obtained therefrom · CPC title