PLANT-ENDOPHYTE COMBINATIONS AND USES THEREFOR

• US 20170223967A1
• Filed: 06/26/2015
• Published: 08/10/2017
• Est. Priority Date: 06/26/2014
• Status: Abandoned Application

• Current Assignee: Indigo Ag, Inc.
• Original Assignee: AIT Austrian Institute of Technology GmbH (Government of Austria), Indigo Agriculture, Inc.
• Inventors: PASTAR, Milica, AMBROSE, Karen, ZHANG, Xuecheng, MITTER, Birgit, SESSITSCH, Angela, REICHENBERGER, Guenther, PFAFFENBICHLER, Nikolaus
• Application Number:
  US15/321,595
• Publication Number:
  US 20170223967A1
• Time in Patent Office: Days
• CPC Class Codes: A01N 63/25 Paenibacillus
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: G01N 33/0098 Plants or trees wood G01N33/46

Abstract

The disclosure provides materials and methods for conferring improved plant traits or benefits on plants. The materials can include a formulation comprising an exogenous endophytic bacterial population, which can be disposed on an exterior surface of a seed or seedling, typically in an amount effective to colonize the plant. The formulations can include at least one member selected from the group consisting of an agriculturally compatible carrier, a tackifier, a microbial stabilizer, a fungicide, an antibacterial agent, an herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, and a nutrient.

Claims

1. A seed or seedling of an agricultural plant having disposed on an exterior surface of the seed or seedling a formulation comprising an exogenous endophytic bacterial population consisting essentially of an endophytic bacterium comprising a 16S rRNA nucleic acid sequence at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:

1-10, wherein the exogenous endophytic bacterial population is disposed on an exterior surface of the seed or seedling in an amount effective to colonize the plant, the formulation further comprising at least one member selected from the group consisting of an agriculturally compatible carrier, a tackifier, a microbial stabilizer, a fungicide, an antibacterial agent, an herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, and a nutrient.

2. The seed or seedling of claim 1, wherein the exogenous endophytic bacterial population is disposed in an amount effective to be detectable within a target tissue of the mature agricultural plant selected from a fruit, a seed, a leaf, a stem, a shoot, a flower, or a root, or portion thereof.

3. The seed or seedling of claim 2, wherein the exogenous endophytic bacterial population is disposed in an amount effective to be detectable in an amount of at least about 100 CFU of the endophytic bacterial population per gram fresh weight of the target tissue.

4. The seed or seedling of claim 1, comprising at least about 100 CFU per gram fresh weight of the exogenous endophytic bacterial population on its exterior surface.

5. The seed or seedling of claim 1, wherein the agricultural plant is a monocot.

6. The seed or seedling of claim 1, wherein the agricultural plant is a corn plant.

7. The seed or seedling of claim 1, wherein the agricultural plant is a dicot.

8. The seed or seedling of claim 1, wherein the exogenous endophytic bacterial population is disposed on an exterior surface in an amount effective to be detectable in an amount of at least about 100 CFU in the mature agricultural plant.

9. The seed or seedling of claim 1, wherein the exogenous endophytic bacterial population is disposed in an amount effective to increase fruit or grain biomass or yield from the resulting plant by at least 5% when compared with a reference agricultural plant.

10. The seed or seedling of claim 1, wherein the exogenous endophytic bacterial population is disposed in an amount effective to increase the biomass of the agricultural plant or portion thereof by at least 5% when compared with a reference agricultural plant.

11. The seed or seedling of claim 1, wherein the exogenous endophytic bacterial population is disposed in an amount effective to increase the rate of seed germination when compared with a reference agricultural plant.

12. The seed or seedling of claim 1, wherein the exogenous endophytic bacterial population is disposed in an amount effective to detectably induce production of auxin in the seed or seedling.

13. The seed or seedling of claim 1, wherein the seed is a corn seed, and wherein the formulation further comprises at least about 10,000 CFU of the exogenous endophytic bacterial population consisting essentially of an endophytic bacterium comprising a 16S rRNA nucleic acid sequence at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:

1-10 disposed on the exterior surface of the seed, the formulation comprising a microbial stabilizer.

14. The seed or seedling of claim 1, wherein the seed or seedling is packaged in a container, wherein the container further comprises a label describing said seeds or seedlings and/or said exogenous endophytic bacterial population.

15. The seed or seedling of claim 14, wherein the container comprises at least 1000 of said seeds or seedlings.

16. A method for preparing a seed or seedling comprising an endophytic bacterial population, said method comprising applying to a seed or seedling a formulation comprising an endophytic bacterial population consisting essentially of an endophytic bacterium comprising a 16S rRNA nucleic acid sequence at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:

1-10.

17. The method of claim 16, wherein the formulation further comprises at least one member selected from the group consisting of an agriculturally compatible carrier, a tackifier, a microbial stabilizer, a fungicide, an antibacterial agent, an herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, and a nutrient.

18. The method of claim 16, wherein the endophytic bacterial population is applied in an amount effective to be detectable within a target tissue of the mature agricultural plant selected from a fruit, a seed, a leaf, a stem, a shoot, a flower, or a root, or portion thereof.

19. The method of claim 18, wherein the endophytic bacterial population is applied in an amount effective to be detectable in an amount of at least about 100 CFU of the endophytic bacterial population per gram fresh weight of the target tissue.

20. The method of claim 16, wherein the seed is a monocot seed.

21. The method of claim 16, wherein the monocot seed is a corn seed.

22. The method of claim 16, wherein the endophytic bacterial population is applied in an amount effective to increase water use efficiency of a plant grown from the seed by at least 5% when compared with a reference agricultural plant grown under the same conditions.

23. The method of claim 16, wherein the endophytic bacterial population is applied in an amount effective to increase photosynthetic rates of a plant grown from the seed by at least 8% when compared with a reference agricultural plant grown under the same conditions.

24. The method of claim 16, wherein the endophytic bacterial population is applied in an amount effective to increase the biomass of a plant, or plant part, grown from the seed under abiotic-stress free conditions by at least 5% when compared with a reference agricultural plant grown under the same conditions.

25. The method of claim 16, wherein the endophytic bacterial population is applied in an amount effective to increase fruit or grain biomass or yield from the resulting plant by at least 5% when compared with a reference agricultural plant that was grown under stress-free conditions.

26. The method of claim 16, wherein the endophytic bacterial population is applied in an amount effective to increase the rate of seed germination by at least 5% when compared with a reference agricultural plant grown under the same conditions.

27. The method of claim 16, wherein the endophytic bacterial population is applied in an amount effective to increase plant height by at least 5% when compared with a reference agricultural plant grown under the same conditions.

45. The method of any of claim 16, 31, 33, 38, 39, 41, or 44, wherein the endophytic bacterium is capable of exhibiting production of an auxin, nitrogen fixation, production of an antimicrobial, production of a siderophore, mineral phosphate solubilization, production of a cellulase, production of a chitinase, production of a xylanase, or production of acetoin.

47. The method of any of claim 16, 31, 33, 38, 39, 41, or 44, wherein the endophytic bacterium is capable of utilizing arabinose as a substrate.

48. The method of any one of claim 16, 31, 33, 38, 39, 41, or 44, wherein the bacterial endophyte is present at a concentration of at least 10̂

2 CFU/seed on the surface of the seeds after contacting.

49. The method of any one of claim 16, 31, 33, 38, 39, 41, or 44, 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 tolerance to low nitrogen stress, increased nitrogen use efficiency, increased overall biomass, increase grain 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 reference seeds.

50. The method of claim 49, wherein the benefit comprises at least two benefits 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 tolerance to low nitrogen stress, increased nitrogen use efficiency, increased overall biomass, increase grain 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 plants derived from reference seeds.

51. The method of claim 50, wherein the benefit is increased tolerance to low nitrogen stress or increased nitrogen use efficiency, and the endophytic bacterium is non-diazotrophic.

52. The method of claim 50, wherein the benefit is increased tolerance to low nitrogen stress or increased nitrogen use efficiency, and the endophytic bacterium is diazotrophic.

28. The method of 16, wherein the seed is a corn seed, and wherein the formulation comprises at least about 1,000 CFU of the endophytic bacterium applied on the exterior surface of the seed, the formulation comprising a microbial stabilizer.

29. The method of claim 28, further comprising packaging the seeds in a container, wherein the container further comprises a label describing said seeds and/or said exogenous endophytic bacterial population.

30. The method of claim 29, wherein the container comprises at least 1000 of said seeds.

31. A method for conferring one or more fitness benefits to an agricultural plant comprising:

a. Providing a seed or seedling of the plant;

b. Contacting said seed or seedling with a formulation comprising an exogenous endophytic bacterial population consisting essentially of an endophytic bacterium comprising a 16S rRNA nucleic acid sequence at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs;

1-10, wherein the exogenous endophytic bacterial population is disposed on an exterior surface of the seed or seedling in an amount effective to colonize the mature plant, wherein the formulation further comprises at least one member selected from the group consisting of an agriculturally compatible carrier, a tackifier, a microbial stabilizer, a fungicide, an antibacterial agent, an herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, and a nutrient; and

c. Allowing the seed or seedling to grow under conditions that permit the endophytic bacterium to colonize the plant.

32. The method of claim 31, wherein the one or more of the fitness benefits are selected from the group consisting of increased germination, increased biomass, increased flowering time, increased plant biomass, increased fruit or grain yield, increased biomass of the fruit or cob, and increased drought tolerance.

33. A method for conferring one or more fitness benefits to a monocot agricultural plant comprising:

a. Providing a monocot seed of the plant;

b. Applying to an exterior surface of the monocot seed a formulation comprising an exogenous endophytic bacterial population consisting essentially of an endophytic bacterium comprising a 16S rRNA nucleic acid sequence at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs;

1-10, wherein the exogenous endophytic bacterial population is applied to an exterior surface of the seed in an amount effective to colonize the mature plant, wherein the formulation further comprises at least one member selected from the group consisting of an agriculturally compatible carrier, a tackifier, a microbial stabilizer, a fungicide, an antibacterial agent, an herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, and a nutrient; and

c. Allowing the seed to grow under conditions that permit the endophytic bacterium to colonize the plant.

34. The method of claim 33, wherein the one or more of the fitness benefits is selected from the group consisting of increased germination rate, shortened time before onset of flowering, increased stomatal conductance, increased photosynthetic rates, increased plant height, and increased drought tolerance as compared to an agricultural plant grown under the same conditions.

35. The method of claim 33, wherein the one or more of the fitness benefits is selected from the group consisting of increased biomass, increased root biomass, increased leaf biomass, increased fruit or grain yield, and increased biomass of the fruit or cob as compared to an agricultural plant that was grown under stress-free conditions.

36. The method of claim 33, wherein the seed is allowed to grow under abiotic-stress free conditions and the one or more of the fitness benefits is selected from the group consisting of increased germination rate, increased biomass, shortened time before onset of flowering, increased plant biomass, increased fruit or grain yield, increased biomass of the fruit or cob, increased stomatal conductance, increased photosynthetic rates, increased plant height, and increased drought tolerance compared to a reference agricultural plant grown under the same conditions.

37. A method of obtaining greater flowering uniformity in a population of agricultural monocot plants comprising:

a. Providing a population of monocot seeds;

b. Applying to an exterior surfaces of the population of seeds a formulation comprising an exogenous endophytic bacterial population consisting essentially of an endophytic bacterium comprising a 16S rRNA nucleic acid sequence at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs;

1-10, wherein the exogenous endophytic bacterial population is applied on an exterior surface of the seed in an amount effective to colonize the mature plant, wherein the formulation further comprises at least one member selected from the group consisting of an agriculturally compatible carrier, a tackifier, a microbial stabilizer, a fungicide, an antibacterial agent, an herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, and a nutrient; and

c. Allowing the population of seeds to grow at least to the flowering stage, wherein the population of plants has a reduction in the standard deviation in the flowering time of at least 5% when compared with a population of reference agricultural plants grown under the same conditions.

38. A method for preparing a monocot seed comprising an endophytic bacterial population, said method comprising applying to an exterior surface of a monocot seed a formulation comprising an endophytic bacterial population consisting essentially of an endophytic bacterium comprising a 16S rRNA nucleic acid sequence at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:

1-10.

40. The method of claim 38 or 39, wherein the contacting comprises spraying, immersing, coating, encapsulating, or dusting the seeds or seedlings with the formulation.

39. A method for treating seedlings, the method comprising:

a) contacting foliage or the rhizosphere of a plurality of agricultural plant seedlings with a seed a formulation comprising an endophytic bacterial population consisting essentially of an endophytic bacterium comprising a 16S rRNA nucleic acid sequence at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs;

1-10; and

b) growing the contacted seedlings.

41. A method for modulating a plant trait comprising applying to vegetation or an area adjacent the vegetation, a seed a formulation comprising an endophytic bacterial population consisting essentially of an endophytic bacterium comprising a 16S rRNA nucleic acid sequence at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:

1-10, wherein the formulation is capable of providing a benefit to the vegetation, or to a crop produced from the vegetation.

42. The method of claim 41, wherein the vegetation is dicot seedlings.

43. The method of claim 41, wherein the vegetation is monocot seedlings.

44. A method for modulating a plant trait comprising applying a formulation to soil, the seed a formulation comprising an endophytic bacterial population consisting essentially of an endophytic bacterium comprising a 16S rRNA nucleic acid sequence at least 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs:

1-10, wherein the formulation is capable of providing a benefit to seeds planted within the soil, or to a crop produced from plants grown in the soil.

46. The method of claim 46, wherein the bacterial endophyte exhibits at least two of:

production of an auxin, nitrogen fixation, 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.

53. A synthetic combination comprising a purified bacterial population in association with a plurality of seeds or seedlings of an agricultural plant, wherein the purified bacterial population comprises a first bacterial endophyte capable of at least one of:

production of an auxin, nitrogen fixation, production of an antimicrobial, production of a siderophore, mineral phosphate solubilization, production of a cellulase, production of a chitinase, production of a xylanase, utilization of arabinose as a carbon source, and production of acetoin, or a combination of two or more thereof, wherein the first bacterial endophyte comprises a 16S rRNA nucleic acid sequence at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs;

1-10, and wherein the bacterial endophyte is present in the synthetic combination in an amount effective to provide a benefit to the seeds or seedlings or the plants derived from the seeds or seedlings.

54. The synthetic combination of claim 53, wherein the synthetic combination is disposed within a packaging material selected from a bag, box, bin, envelope, carton, or container.

55. The synthetic combination of claim 54, comprising 1000 seed weight amount of seeds, wherein the packaging material optionally comprises a dessicant, and wherein the synthetic combination optionally comprises an anti-fungal agent.

56. The synthetic combination of claim 53, wherein the purified bacterial population is localized on the surface of the seeds or seedlings.

57. The synthetic combination of claim 53, wherein the first bacterial endophyte is obtained from a plant species other than the seeds or seedlings of the synthetic combination.

58. The synthetic combination of claim 53 wherein the first bacterial endophyte is obtained from a plant cultivar different from the cultivar of the seeds or seedlings of the synthetic combination.

59. The synthetic combination of claim 53, wherein the first bacterial endophyte is obtained from a plant cultivar that is the same as the cultivar of the seeds or seedlings of the synthetic combination.

60. The synthetic combination of claim 53, wherein the bacterial population comprises two or more families of bacterial endophytes.

61. The synthetic combination of claim 53, wherein the bacterial population further comprises a second bacterial endophyte having an 16S nucleic acid sequence less than 95% identical to that of the first bacterial endophyte.

62. The synthetic combination of claim 53, wherein the bacterial population further comprises a second bacterial endophyte, wherein the first and second bacterial endophytes are independently capable of at least one of production of an auxin, nitrogen fixation, production of an antimicrobial, production of a siderophore, mineral phosphate solubilization, production of a cellulase, production of a chitinase, production of a xylanase, utilization of arabinose as a carbon source, or production of acetoin, or a combination of two or more thereof.

63. The synthetic combination of claim 53, wherein the bacterial population further comprises a second bacterial endophyte, wherein the first and second bacterial endophytes are capable of synergistically increasing at least one of:

production of an auxin, nitrogen fixation, production of an antimicrobial, production of a siderophore, mineral phosphate solubilization, production of a cellulase, production of a chitinase, production of a xylanase, utilization of arabinose as a carbon source, or production of acetoin, or a combination of two or more thereof, in an amount effective to increase tolerance to drought relative to a reference plant.

64. The synthetic combination of claim 53, wherein the first bacterial endophyte is capable of at least two of:

production of an auxin, nitrogen fixation, production of an antimicrobial, production of a siderophore, mineral phosphate solubilization, production of a cellulase, production of a chitinase, production of a xylanase, utilization of arabinose as a carbon source, and production of acetoin.

65. The synthetic combination of claim 53, 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 grain 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 a reference plant.

66. The synthetic combination of claim 53, wherein the benefit comprises at least two benefits 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 tolerance to low nitrogen stress, increased nitrogen use efficiency, increased overall biomass, increase grain 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 a reference plant.

67. The synthetic combination of claim 53, wherein the combination comprises seeds and the first bacterial endophyte is associated with the seeds as a coating on the surface of the seeds.

68. The synthetic combination of claim 53, wherein the combination comprises seedlings and the first bacterial endophyte is contacted with the seedlings as a spray applied to one or more leaves and/or one or more roots of the seedlings.

69. The synthetic combination of claim 53, wherein the synthetic combination further comprises one or more additional bacterial endophyte species.

70. The synthetic combination of claim 53, wherein the effective amount is at least 1×

10̂

3 CFU/per seed.

71. The synthetic combination of claim 53, wherein the combination comprises seeds and the effective amount is from about 1×

10̂

2 CFU/per seed to about 1×

10̂

8 CFU/per seed.

72. An agricultural product comprising a 1000 seed weight amount of a synthetic combination produced by the step of contacting a plurality of agricultural plant seeds with a liquid formulation comprising a bacterial population at a concentration of at least 1 CFU per agricultural plant seed, wherein at least 10% of the CFUs present in the formulation are one or more bacterial endophytes, wherein the purified bacterial population comprises a first bacterial endophyte capable of at least one of:

production of an auxin, nitrogen fixation, production of an antimicrobial, production of a siderophore, mineral phosphate solubilization, production of a cellulase, production of a chitinase, production of a xylanase, utilization of arabinose as a carbon source, and production of acetoin, or a combination of two or more thereof, wherein the first bacterial endophyte comprises a 16S rRNA nucleic acid sequence at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs;

1-10, under conditions such that the formulation is associated with the surface of the seeds in a manner effective for the bacterial endophytes to confer a benefit to the seeds or to a crop comprising a plurality of agricultural plants produced from the seeds.

73. The agricultural product of claim 72, wherein the bacterial endophytes are present in a concentration of from about 10̂

2 to about 10̂

5 CFU/ml.

74. The agricultural product of claim 73, wherein the bacterial endophytes are present in a concentration is from about 10̂

5 to about 10̂

8 CFU/seed.

79. The agricultural product or formulation of claim 72 or 75, 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 tolerance to low nitrogen stress, increased nitrogen use efficiency, increased overall biomass, increase grain 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 a reference plant, or a combination thereof.

80. The agricultural product or formulation of claim 72 or 75, wherein the bacterial population comprises two or more families of bacterial endophytes.

81. The agricultural product or formulation of claim 72 or 75, wherein the formulation further comprises a second bacterial endophyte having a 16S nucleic acid sequence less than 95% identical to that of the first bacterial endophyte.

82. The agricultural product or formulation of claim 72 or 75, wherein the bacterial endophyte exhibits:

production of auxin, production of a siderophore, mineral phosphate solubilization, production of a cellulase, production of a chitinase, production of a xylanase, and production of acetoin, but not increase nitrogen fixation relative to a reference plant.

83. The agricultural product or formulation of claim 72 or 75, wherein the product or formulation comprises two or more bacterial endophyte species.

75. An agricultural formulation comprising a purified bacterial population and an agriculturally acceptable carrier, the bacterial population comprising a first bacterial endophyte capable of at least one of:

production of an auxin, nitrogen fixation, production of an antimicrobial, production of a siderophore, mineral phosphate solubilization, production of a cellulase, production of a chitinase, production of a xylanase, utilization of arabinose as a carbon source, and production of acetoin, or a combination of two or more thereof, wherein the first bacterial endophyte comprises a 16S rRNA nucleic acid sequence at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs;

1-10, and wherein the first bacterial endophyte is present in an amount effective to confer a benefit to an agricultural plant seed to which the formulation is applied or to an agricultural plant seedling to which the formulation is applied.

76. The agricultural formulation of claim 75, wherein the purified bacterial population consists essentially of two or more species of bacterial endophytes.

77. The agricultural formulation of claim 75, wherein the formulation is a liquid and the bacterial concentration is from about 10̂

3 to about 10̂

11 CFU/ml.

78. The agricultural formulation of claim 75, wherein the formulation is a gel or powder and the bacterial concentration is from about 10̂

3 to about 10̂

11 CFU/gm.