DESIGNED COMPLEX ENDOPHYTE COMPOSITIONS AND METHODS FOR IMPROVED PLANT TRAITS

• US 20160366892A1
• Filed: 04/29/2016
• Published: 12/22/2016
• Est. Priority Date: 05/01/2015
• Status: Active Grant

• Current Assignee: Indigo Agriculture, Inc.
• Original Assignee: Indigo Agriculture, Inc.
• Inventors: Pelaez, Julianne Naomi, Ambrose, Karen V., Boghigian, Brett A., Djonovic, Slavica, Gray, Paul Andrew, Toledo, Gerardo V., Marquez, Luis Miguel, von Maltzahn, Geoffrey
• Granted Patent: US 10,212,944 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A01G 22/00 Cultivation of specific crops or plants not otherwise provided for
• CPC Class Codes: A01G 22/05 Fruit crops, e.g. strawberries, tomatoes or cucumbers
• CPC Class Codes: A01G 22/15 Leaf crops, e.g. lettuce or spinach
• CPC Class Codes: A01G 22/20 Cereals
• CPC Class Codes: A01G 22/22 Rice
• CPC Class Codes: A01G 22/25 Root crops, e.g. potatoes, yams, beet or wasabi
• CPC Class Codes: A01G 22/35 Bulbs; Alliums, e.g. onions or leeks
• CPC Class Codes: A01G 22/40 Fabaceae, e.g. beans or peas
• CPC Class Codes: A01G 22/50 Cotton
• CPC Class Codes: A01G 22/55 Sugar cane
• CPC Class Codes: A01G 22/60 Flowers; Ornamental plants
• CPC Class Codes: A01H 17/00 Symbiotic or parasitic combinations including one or more new plants, e.g. mycorrhiza lichens A01H15/00
• CPC Class Codes: A01N 43/16 with oxygen as the ring hetero atom
• CPC Class Codes: A01N 63/20 Bacteria; Substances produced thereby or obtained therefrom
• CPC Class Codes: A01N 63/22 Bacillus
• CPC Class Codes: A01N 63/25 Paenibacillus
• CPC Class Codes: A01N 63/30 Microbial fungi; Substances produced thereby or obtained therefrom
• CPC Class Codes: C12N 1/14 Fungi culture of mushrooms A01G18/00; as new plants A01H15/00; Culture media therefor
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor

Abstract

This invention relates to methods and materials for providing a benefit to a plant by associating the plant with a complex endophyte comprising a host fungus further comprising a component bacterium, including benefits to a plant derived from a seed or other plant element treated with a complex endophyte. For example, this invention provides purified complex endophytes, purified complex endophyte components such as bacteria or fungi, synthetic combinations comprising said complex endophytes and/or components, and methods of making and using the same.

Claims

1. A method of creating a designed complex endophyte comprising at least one bacterium within a heterologous fungus, comprising:

a. isolating and purifying a bacterium;

b. isolating and purifying a fungus;

c. co-culturing said bacterium with said fungus in a liquid medium; and

d. incubating the co-cultured bacterium and fungus at a temperature not exceeding 35.5 degrees Celsius.

2. The method of claim 1, wherein step a) is conducted at a temperature range selected from the group consisting of:

between 18°

C. and 36°

C., between 20°

C. and 30°

C., between 20°

C. and 26°

C., between 20.0°

C. and 25.5°

C., between 22.0°

C. and 25.0°

C., and between 23.0°

C. and 24.0°

C.

3. The method of claim 1, further comprising, after step b):

treating said fungus with one or more antibiotics to cure the fungus of any component bacteria.

4. The method of claim 1, further comprising plating the co-cultures on 2% water agar for a minimum of 1 week, then transferring said co-culture to full-strength PDA/LB agar.

5. The method of claim 1, wherein the co-cultured bacterium and fungus are incubated at a temperature range selected from the group consisting of:

between 18°

C. and 36°

C., between 20°

C. and 30°

C., between 20°

C. and 26°

C., between 20.0°

C. and 25.5°

C., between 22.0°

C. and 25.0°

C., and between 23.0°

C. and 24.0°

C.

6. The method of claim 1, wherein the incubated co-cultured bacterium and fungus are further treated with gentamicin.

7. The method of claim 1, wherein the co-cultured bacterium and fungus are further plated onto broth and/or agar, with gentamicin.

8. The method of claim 6, wherein the cultures are washed with magnesium chloride no fewer than three times.

9. The method of claim 6, wherein the concentration of gentamicin is selected from the group consisting of:

5 micrograms per mL, 20 micrograms per mL, 50 micrograms per mL, and 75 micrograms per mL.

10. A synthetic composition comprising a designed complex endophyte further comprising a fungal endophyte encapsulating a heterologous bacterial endophyte, further comprising a plant element, wherein the synthetic composition is capable of providing a trait of agronomic importance to said plant element or plant with which said plant element is associated or grown from.

15. The synthetic composition of claim 10, wherein said trait of agronomic importance is selected from the group consisting of:

germination rate, emergence rate, shoot biomass, root biomass, seedling root length, seedling shoot length, and yield.

16. The synthetic composition of claim 10, wherein the trait of agronomic importance is improved under normal watering conditions.

17. The synthetic composition of claim 10, wherein the trait of agronomic importance is improved under conditions of water limitation.

19. The synthetic composition of claim 10, wherein said synthetic composition further comprises an agronomic formulation that further comprises one or more of the following:

a stabilizer, or a preservative, or a carrier, or a surfactant, or an anticomplex agent, fungicide, nematicide, bactericide, insecticide, and herbicide, or any combination thereof.

20. The synthetic composition of claim 10, wherein said designed complex endophyte is present in an amount of at least about 10̂

2 CFU per plant element.

21. The synthetic composition of claim 10, wherein said designed complex endophyte comprises a host fungus from a class selected from the group consisting of:

Dothideomycetes, Sordariomycetes, Mucoromycotina, Pezizomycotina, or any of the corresponding anamorph or teleomorph taxonomy of the preceding.

22. The synthetic composition of claim 10, wherein said designed complex endophyte comprises a bacterium from a class selected from the group consisting of:

Bacilli, Gammaproteobacteria, Actinobacteria.

29. The synthetic composition of claim 10, comprising a fungus that further comprises a nucleic acid sequence at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:

561 through SEQ ID NO;

758.

30. The synthetic composition of claim 10, comprising a bacterium that further comprises a nucleic acid sequence at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:

1 through SEQ ID NO;

560.

31. The synthetic composition of claim 10, wherein said designed complex endophyte is selected from the compositions listed in Table 3.

49. A plurality of the synthetic composition of claim 10, placed in a medium that promotes plant growth, wherein said medium is soil, wherein said synthetic compositions are placed in the soil with substantially equal spacing between each.

53. The plant, plant element, or progeny of the plant grown from the synthetic composition of claim 10, wherein said plant or progeny of the plant comprises in at least one of its plant elements said fungal endophyte, said bacterial endophyte, designed complex endophyte, fungal host, or bacterial component.

11-14. -14. (canceled)

18. (canceled)

23-28. -28. (canceled)

32-48. -48. (canceled)

50-52. -52. (canceled)

54. A method of inoculating a plant with a designed complex endophyte, comprising contacting a plant element of said plant with a formulation comprising said designed complex endophyte.

55-57. -57. (canceled)

58. A method of improving a trait of agronomic importance in a plant, comprising contacting a plant element with a formulation comprising a designed complex endophyte further comprising a fungus further comprising a bacterium;

as compared to an isoline plant not contacted with said designed complex endophyte.

59. A method of improving a trait of agronomic importance in a plant, comprising growing said plant from a plant reproductive element that has been contacted with a formulation comprising a designed complex endophyte;

as compared to an isoline plant grown from a plant reproductive element not contacted with said designed complex endophyte.

60-88. -88. (canceled)

89. A method for preparing a synthetic composition, comprising associating the surface of a plurality of plant elements with a formulation comprising a designed complex endophyte, wherein the designed complex endophyte comprises a bacterium within a heterologous fungus and is present in the formulation in an amount capable of modulating at least one of:

a trait of agronomic importance, the transcription of a gene, the expression of a protein, the level of a hormone, the level of a metabolite, and the population of endogenous microbes in plants grown from said seeds, as compared to isoline plants not associated with, or grown from plant elements associated with, said formulation.

90-109. -109. (canceled)

110. A method of improving the efficacy of a bacterial endophyte in an application, comprising utilizing a designed complex endophyte, wherein said designed complex endophyte comprises said bacterial endophyte.

111. A method of improving the efficacy of a fungal endophyte in an application, comprising utilizing a designed complex endophyte, wherein said designed complex endophyte comprises said fungal endophyte.

112-118. -118. (canceled)