AGRICULTURAL ENDOPHYTE-PLANT COMPOSITIONS, AND METHODS OF USE

• US 20170020138A1
• Filed: 09/04/2014
• Published: 01/26/2017
• Est. Priority Date: 06/26/2013
• Status: Active Grant

• Current Assignee: Indigo Ag, Inc.
• Original Assignee: Symbiota, Inc.
• Inventors: VON MALTZAHN, Geoffrey, JACK, Allison, NAYDICH, Alexander, FLAVELL, Richard Bailey, TOLEDO, Gerardo V., JOHNSTON, David Morris, DJONOVIC, Slavica, MARQUEZ, Luis Miguel, MILLET, Yves Alain, SADOWSKI, Craig, LYFORD, Jeffrey
• Granted Patent: US 10,136,646 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A01C 1/00 Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
• CPC Class Codes: A01C 1/06 Coating or dressing seed
• CPC Class Codes: A01N 25/00 Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application , e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
• CPC Class Codes: A01N 63/20 Bacteria; Substances produced thereby or obtained therefrom
• CPC Class Codes: G01N 33/5097 involving plant cells immunoassays of plant cells G01N33/56961

Abstract

This application relates to methods and materials for providing a benefit to a seed or seedling of an agricultural plant (e.g., an agricultural grass plant or a Gycine plant), or the agricultural plant derived from the seed or seedling. For example, this application provides purified bacterial populations that include novel seed bacterial endophytes, and synthetic combinations of seeds and/or seedlings (e.g., cereal or soy seeds and/or cereal or soy seedlings) with heterologous seed-derived bacterial endophytes.

Claims

1. A method for preparing an agricultural seed preparation, comprising:

a) contacting the surface of a plurality of Glycine agricultural plant seeds with a formulation comprising a purified bacterial population at a concentration of at least about 10² CFU/ml in a liquid formulation or about 10² CFU/gm in a non-liquid formulation, the bacterial population comprising a seed bacterial endophyte exhibiting the ability to produce or induce in a Glycine agricultural plant grown from the Glycine agricultural plant seed an activity selected from production of an auxin, production of an antimicrobial, production of a siderophore, mineral phosphate solubilization, production of a cellulase, production of a chitinase, production of a xylanase, and production of acetoin, wherein the seed bacterial endophyte is present in the formulation in an amount capable of providing a benefit to the plant seeds or to agricultural plants derived from the plant seeds, wherein the formulation further comprises a stabilizer, a fungicide, a preservative, or a combination thereof; and

b) packaging the contacted seeds in a container.

2. The method of claim 1, wherein the bacterial endophyte is amplified within a tissue of the Glycine agricultural plant.

3. The method of claim 1, wherein the contacted seeds are packaged in the container such that they are substantially stable at about 25 degrees C. for at least 7 days.

4. The method of claim 1, wherein the bacterial endophyte comprises the ability to induce in the Glycine agricultural plant at least two activities selected from production of an auxin, production of an antimicrobial, production of a siderophore, mineral phosphate solubilization, production of a cellulase, production of a chitinase, production of a xylanase, and production of acetoin.

5. The method of claim 1, wherein the contacted seeds and/or the Glycine agricultural plant grown therefrom comprises an increased production of a polypeptide selected from LOC100796978 or gi|356536151/Alpha-L-fucosidase 2-like and gi|571436840/Pectinesterase/pectinesterase inhibitor 18-like or gi|358248512/Uncharacterized protein LOC100796978 and gi|356508869/Lysosomal alpha-mannosidase-like or gi|358248512/Uncharacterized protein LOC100796978 and gi|571436840/Pectinesterase/pectinesterase inhibitor 18-like or gi|358249064/Uncharacterized protein LOC100795412 and gi|351723615/Aspartate aminotransferase glyoxysomal isozyme AAT1 precursor or gi|358249064/Uncharacterized protein LOC100795412 and gi|356508869/Lysosomal alpha-mannosidase-like or gi|358249064/Uncharacterized protein LOC100795412 and gi|358248512/Uncharacterized protein LOC100796978 or gi|358249064/Uncharacterized protein LOC100795412 and gi|571436840/Pectinesterase/pectinesterase inhibitor 18-like, and/or ii) a reduced production of a polypeptide selected from gi|356506190/Transketolase, chloroplastic and gi|356509275/Glutamate synthase [NADH], amyloplastic-like or gi|356506190/Transketolase, chloroplastic and gi|356516458/Staphylococcal nuclease domain-containing or gi|356506190/Transketolase, chloroplastic and gi|356533407/Embryonic protein DC-8-like or gi|356506190/Transketolase, chloroplastic and gi|356535993/Beta-conglycinin, alpha chain or gi|356506190/Transketolase, chloroplastic and gi|356575855/Beta-conglycinin, beta chain-like or gi|356506190/Transketolase, chloroplastic and gi|571477629/Low-temperature-induced 65 kDa prot. or gi|356509275/Glutamate synthase [NADH], amyloplastic-like and gi|356516458/Staphylococcal nuclease domain-containing or gi|356509275/Glutamate synthase [NADH], amyloplastic-like and gi|356533407/Embryonic protein DC-8-like or gi|356509275/Glutamate synthase [NADH], amyloplastic-like and gi|356535993/Beta-conglycinin, alpha chain or gi|356509275/Glutamate synthase [NADH], amyloplastic-like and gi|356575855/Beta-conglycinin, beta chain-like or gi|356509275/Glutamate synthase [NADH], amyloplastic-like and gi|571477629/Low-temperature-induced 65 kDa prot. or gi|356533407/Embryonic protein DC-8-like and gi|356535993/Beta-conglycinin, alpha chain or gi|356533407/Embryonic protein DC-8-like and gi|356575855/Beta-conglycinin, beta chain-like or gi|356533407/Embryonic protein DC-8-like and gi|571477629/Low-temperature-induced 65 kDa prot. or gi|356575855/Beta-conglycinin, beta chain-like and gi|356535993/Beta-conglycinin, alpha chain or gi|571477629/Low-temperature-induced 65 kDa prot. and gi|356535993/Beta-conglycinin, alpha chain or gi|571477629/Low-temperature-induced 65 kDa prot. and gi|356575855/Beta-conglycinin, beta chain-like as compared to a reference Glycine agricultural plant.

6. The method of claim 1, wherein the contacting in (a) comprises spraying, immersing, coating, encapsulating, injecting or dusting the seeds with the formulation.

7. The method of claim 1, wherein the purified bacterial population comprises a plurality of seed bacterial endophyte entities.

8. The method of claim 1, wherein the purified bacterial population comprises a plurality of taxonomically diverse seed bacterial endophyte entities.

9. The method of claim 1, wherein the bacterial population comprises a bacterial endophyte comprising a 16S nucleic acid sequence at least 97% identical to a bacterial endophyte selected from Table 1.

10. The method of claim 1, wherein the purified bacterial population comprises i) a first bacterial endophyte comprising a first 16S nucleic acid sequence at least 97% identical to a bacterial endophyte selected from Table 1, and ii) a second bacterial endophyte comprising a second 16S nucleic acid sequence at least 97% identical to a bacterial endophyte selected from Table 1, wherein the first and second 16S nucleic acid sequence are not 100% identical.

11. The method of claim 1, wherein the contacting results in a concentration of at least 10² CFU of the bacterial endophyte on the surface of the seed.

12. The method of claim 1, wherein the bacterial population is obtained from an interior seed compartment and/or wherein the bacterial population comprises a seed bacterial endophyte.

13. The method of claim 1, wherein the bacterial population is obtained from a plant species other than the seeds to which the formulation is contacted.

14. The method of claim 1, wherein the bacterial population is obtained from a plant species other than the seeds to which the formulation is contacted, and wherein the bacterial population is propagated in culture.

15. The method of claim 1, wherein the bacterial population is obtained from a plant cultivar or a seed thereof different from the cultivar of the seeds to which the formulation is contacted.

16. The method of claim 1, wherein the bacterial population is obtained from a surface sterilized seed.

23. The method of claim 1, wherein the benefit is selected from the group consisting of:

increased root biomass, increased root length, increased height, increased shoot length, increased leaf number, increased water use efficiency, increased overall biomass, increase yield, increased photosynthesis rate, increased tolerance to drought, increased heat tolerance, increased salt tolerance, increased resistance to nematode stress, increased resistance to a fungal pathogen, increased resistance to a bacterial pathogen, increased resistance to a viral pathogen, a detectable modulation in the level of a metabolite, and a detectable modulation in the proteome, relative to reference seeds or agricultural plants derived from the reference seeds.

24. The agricultural seed preparation prepared by the method of claim 1.

17-22. -22:

(canceled)

25-46. -46. (canceled)

47. A method of making a seed treatment, the method comprising:

(a) inoculating an isolated cultured bacterial endophyte selected from Table 1 into a liquid medium;

(b) culturing the bacterial endophyte in the medium at a temperature of about 4 to about 37 degrees C. until the cell or spore density in the medium is at least about 10² CFU/mL; and

(c) collecting the vegetative cells or spores under conditions suitable to produce a seed treatment.

48-57. -57. (canceled)

58. A method of screening a modulator of a Glycine trait, comprising:

(a) applying a library of bacterial entities to a population of seeds of a Glycine agricultural plant;

(b) measuring a trait in seedlings or plants derived from the seeds, the trait selected from the group consisting of root biomass, root length, height, shoot length, leaf number, water use efficiency, overall biomass, yield, photosynthesis rate, tolerance to drought, heat tolerance, salt tolerance, resistance to nematode stress, resistance to a fungal pathogen, resistance to a bacterial pathogen, resistance to a viral pathogen, the level of a metabolite, and proteome expression; and

(c) identifying at least one of the bacterial entities present in the library capable of modulating the trait, relative to reference seedlings or plants.

59-150. -150. (canceled)