PLANTS CONTAINING BENEFICIAL ENDOPHYTES

• US 20160338360A1
• Filed: 12/24/2014
• Published: 11/24/2016
• Est. Priority Date: 12/24/2013
• Status: Active Grant

• Current Assignee: AIT Austrian Institute of Technology GmbH (Government of Austria)
• Original Assignee: AIT Austrian Institute of Technology GmbH (Government of Austria), Indigo Ag, Inc.
• Inventors: MITTER, Birgit, NAVEED, Muhammad, BERNINGER, Teresa, COMPANT, Stephane, SESSITSCH, Angela, VON MALTZAHN, Geoffrey, FLAVELL, Richard Bailey, TOLEDO, Gerardo V., DJONOVIC, Slavica, MARQUEZ, Luis Miguel, JOHNSTON, David Morris, MILLET, Yves Alain, LYFORD, Jeffrey, LEFF, Jonathan W., SAMAYOA, Phillip, SADOWSKI, Craig
• Granted Patent: US 10,271,554 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: G01N 33/0098 Plants or trees wood G01N33/46

Abstract

This application relates to methods and materials for providing a benefit to a seed, seedling or plant by producing seeds that are internally colonized with endophytes. Beneficial endophytes with particular characteristics are provided. These may be used in the methods described to provide a benefit to a seed, seedling or plant.

Claims

47-1. The method of claim 46, wherein the protein involved in the establishment of symbiosis is a protein selected from the group consisting of ribosomal proteins, proteins homologous to those involved in nodule formation in legumes, and proteins associated with micorrhiza.

48-1. The method of any one of claims 1-47, 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, increased yield, increased germination rate, decreased time to start germination, decreased mean germination time, decreased time to 50% germination, decreased time to final germination, increased germination index, increased germination energy, increased coefficient of uniform germination, earlier flowering, 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, altered metabolite expression, and altered protein expression, relative to reference seeds or agricultural plants derived from the reference seeds.

49-1. The method of any one of claims 1-48, 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 overall biomass, increased yield, increased germination rate, decreased time to start germination, decreased mean germination time, decreased time to 50% germination, decreased time to final germination, increased germination index, increased germination energy, increased coefficient of uniform germination, earlier flowering, 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, altered metabolite expression, and altered protein expression, relative to reference seeds or agricultural plants derived from reference seeds.

53. A method for generating a synthetic agricultural preparation, comprising:

a) contacting at least one flower of a plurality of agricultural plants with a formulation comprising a purified bacterial population, the bacterial population comprising a bacterial endophyte exhibiting the ability to produce an auxin, an antimicrobial, a siderophore, a cellulase, a chitinase, a xylanase, HCN, NH₃, AHL, PHB or acetoin, in an agricultural plant grown from plant seeds produced by the flower.

54. A method of screening a modulator of a plant trait, comprising:

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

(b) measuring a trait in seedlings or plants derived from the seeds produced by the flowers, wherein the trait is selected from the group consisting of root biomass, root length, height, shoot length, leaf number, water use efficiency, overall biomass, yield, increased germination rate, decreased time to start germination, decreased mean germination time, decreased time to 50% germination, decreased time to final germination, increased germination index, increased germination energy, increased coefficient of uniform germination, earlier flowering, 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, altered levels of a metabolite, and altered protein 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.

55. The method of claim 54, wherein the library comprises at least about 10-100 bacteria of one or more species of bacterial endophytes.

56. A method of identifying a modulator of a plant trait, comprising:

(a) applying a bacterial population to flowers of a agricultural plant, the population comprising bacteria of one or more species of bacterial endophytes;

(b) measuring a trait in seedlings or plants derived from the seeds made by the flowers, the trait selected from the group consisting of root biomass, root length, height, shoot length, leaf number, water use efficiency, overall biomass, yield, increased germination rate, decreased time to start germination, decreased mean germination time, decreased time to 50% germination, decreased time to final germination, increased germination index, increased germination energy, increased coefficient of uniform germination, earlier flowering, 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 traits for which the bacterial population results in a modulation of the trait, relative to reference seedlings or plants.

57. The method of claim 56, wherein the bacterial endophytes comprise a plurality of bacterial endophyte entities.

58. A method of screening for a modulator of a plant trait, the method comprising:

(a) applying a single entity from a library of bacterial endophyte entities to at least one flower of a population of agricultural plants;

(b) measuring a trait in plants grown 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, increased germination rate, decreased time to start germination, decreased mean germination time, decreased time to 50% germination, decreased time to final germination, increased germination index, increased germination energy, increased coefficient of uniform germination, earlier flowering, 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;

(c) repeating steps (a) and (b) with one or more additional bacterial endophyte entities from the library; and

(d) identifying at least one of the bacterial endophyte entities as modulating at least one of the traits, relative to a reference seedling or plant.

59. The method of claim 58, wherein the library comprises at least 100 species of bacterial endophytes.

60. The method of claim 58, wherein one or more of the bacterial endophytes are obtained from a plant cultivar different from the cultivar of the population of seeds in step (a).

61. The method of claim 58, wherein the bacterial endophytes are bacterial endophytes are obtained from one or more surface sterilized seeds.

62. A method of identifying a modulator of a plant trait, the method comprising:

a) applying a purified bacterial population to flowers of an agricultural plant, the bacterial population comprising bacteria of one or more species of bacterial endophytes;

(b) measuring a trait in plants grown from the seeds produced by the flowers, the trait selected from the group consisting of root biomass, root length, height, shoot length, leaf number, water use efficiency, overall biomass, yield, increased germination rate, decreased time to start germination, decreased mean germination time, decreased time to 50% germination, decreased time to final germination, increased germination index, increased germination energy, increased coefficient of uniform germination, earlier flowering, 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 a modulation of at least one of the traits in the plants, relative to reference plants.

63. The method of claim 62, wherein the purified bacterial population comprises 2-6 different bacterial endophytes.

64. The method of claim 62, wherein the one or more bacterial endophytes are obtained from a plant cultivar different from the cultivar of the seeds in (a).

65. The method of claim 62, wherein the bacterial endophytes are obtained from a surface sterilized seed.

66. A method for preparing an agricultural seed preparation comprising a microbe localized in the seed, comprising:

a) contacting at least one flower of a plurality of agricultural plants with a formulation comprising a purified bacterial population, the bacterial population comprising a bacterial endophyte exhibiting the ability to induce in a seed made by the flower, a seedling grown from the seed or an agricultural plant grown from the seed an alteration in the population of microbes present in the seed or plant, as compared to a reference seed, seedling or plant.

67. The method of claim 66, wherein the alteration in the population of microbes is an increase in the number of bacteria of the Comamonadaceae or Xanthomonadaceae families.

68. The method of claim 66, wherein the alteration in the population of microbes is a decrease in the number of bacteria of the Actinomycetaceae, Chitinophagaceae, Phyllobacteriaceae, Microbacteriaceae, Exiguobacteraceae, Sphingomonadaceae, Phyllobacteriaceae, or Enterobacteriaceae families.

69. The method of claim 66, wherein the alteration in the population of microbes is the appearance of at least one microbe that is not present within the reference seed, seedling or plant, in addition to the bacterial endophyte.

70. The method of claim 69, wherein the at least one microbe that is not present within the reference seed, seedling or plant is of the Kineosporiaceae, Weeksellaceae, Geodermatophilaceae, Bacillaceae, Thermicanus, Weeksellaceae, or Geodermatophilaceae families.

71. The method of claim 66, wherein the alteration in the population of microbes is the disappearance of at least one microbe that is present within the reference seed, seedling or plant.

72. The method of claim 71, wherein the at least one microbe that is present within the reference seed, seedling or plant is of the Chitinophagaceae or Alcaligenaceae families.

74. The method of claim 66, wherein the bacterial endophyte is present in the formulation in an amount capable of providing a benefit to a seed derived from the flower, to a seedling derived from the seed, or to agricultural plants derived from the seed, as compared to a reference seed, seedling or plant.

75. The method of claim 74, 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, increased yield, increased germination rate, decreased time to start germination, decreased mean germination time, decreased time to 50% germination, decreased time to final germination, increased germination index, increased germination energy, increased coefficient of uniform germination, earlier flowering, 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, altered metabolite expression, and altered protein expression, relative to reference seeds or agricultural plants derived from the reference seeds.

76. The method of claim 75, 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 overall biomass, increased yield, increased germination rate, decreased time to start germination, decreased mean germination time, decreased time to 50% germination, decreased time to final germination, increased germination index, increased germination energy, increased coefficient of uniform germination, earlier flowering, 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, altered metabolite expression, and altered protein expression, relative to reference seeds or agricultural plants derived from reference seeds.

77. A method for preparing an agricultural seed preparation comprising a microbe localized in the seed, comprising:

a) contacting at least one flower of a plurality of agricultural plants with a formulation comprising a purified bacterial population, the bacterial population comprising a bacterial endophyte exhibiting the ability to induce in a seed made by the flower, a seedling grown from the seed or an agricultural plant grown from the seed an alteration in the level of expression of at least one protein, as compared to a reference seed, seedling or plant.

78. The method of claim 77, wherein the bacterial endophyte is present in the synthetic combination in an amount effective to increase the level of at least one protein involved in resistance against abiotic stress within the seed, a seedling derived from the seed or a cereal agricultural plant derived from the seed, as compared to a reference seed, seedling or plant.

79. The method of claim 78, wherein the protein involved in resistance against abiotic stress is a protein selected from those involved in response to heavy metals, proteins associated with ion transport and proteins involved in salt stress and water stress tolerance.

80. The method of claim 79, wherein the protein is a ribosomal protein.

81. The method of claim 77, wherein the bacterial endophyte is present in the synthetic combination in an amount effective to increase the level of at least one protein involved in symbiosis within the seed, a seedling derived from the seed or a cereal agricultural plant derived from the seed, as compared to a reference seed, seedling or plant.

82. The method of claim 81, wherein the protein involved in symbiosis is involved in a protein involved in the defense or establishment of symbiosis with microbes.

83. The method of claim 82, wherein the protein is involved in pathogen response, ribosomal protein, protein homologous to those involved in nodule formation in legumes, and protein associated with micorrhiza.

84. The method of claim 77, wherein the bacterial endophyte is present in the synthetic combination in an amount effective to increase the level of at least one protein involved in growth promotion within the seed, a seedling derived from the seed or a cereal agricultural plant derived from the seed, as compared to a reference seed, seedling or plant.

85. The method of claim 84, wherein the protein involved in growth promotion is selected from the group consisting of elongation factors, proteins related to nutrient acquisition and proteins involved in chromosomal segregation during mitosis.

89. A synthetic combination of a purified bacterial population in association with a seed of an agricultural plant, wherein the synthetic combination is produced by the steps of

(a) contacting at least one flower of a plurality of agricultural plants with a formulation comprising a purified bacterial population, the bacterial population comprising a bacterial endophyte exhibiting the ability to produce or induce in a seed made by the flower, a seedling grown from the seed or an agricultural plant grown from the seed an activity selected from catalase activity, oxidase activity, casein hydrolysis activity, gelatin hydrolysis activity, ACC-deaminase activity, exopolysaccharide activity, amylase activity, cellulase activity, chitinase activity, hemolytic activity, lipase activity, pectinase activity, phosphatase activity, protease activity, xylanase activity, production of an auxin, production of an antimicrobial, production of HCN, production of NH₃, production of AHL, production of PHB, production of a siderophore, mineral phosphate solubilization, and production of acetoin, wherein the bacterial endophyte is present in the formulation in an amount capable of providing a benefit to the seeds or to agricultural plants derived from the seeds, as compared to a reference seed, seedling or plant

(b) harvesting the seed.

90. The synthetic combination of claim 89, wherein the bacterial endophytes comprise a plurality of bacterial endophyte entities.

91. The synthetic combination of claim 89, wherein the synthetic combination is disposed within a packaging material selected from a bag, box, bin, envelope, carton, or container.

92. The synthetic combination of claim 89, 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.

93. The synthetic combination of claim 89, wherein the bacterial population comprises a first bacterial endophyte having a first 16S nucleic acid sequence and a second bacterial endophyte having a second 16S nucleic acid sequence, wherein the first and the second 16S nucleic acid sequences are less than 97% identical.

94. An agricultural product comprising a 1000 seed weight amount of a synthetic combination produced by the step of contacting at least one flower from a plurality of agricultural plants with a liquid formulation comprising a bacterial population, under conditions such that the formulation is associated with the flowers in a manner effective for the bacterial endophytes to confer a benefit to the seeds produced from the flowers or to a crop comprising a plurality of agricultural plants produced from the seeds.

95. The synthetic combination of claim 94 wherein the bacterial endophyte is a bacterial endophyte.

96. The synthetic combination of claim 94, wherein the synthetic combination is disposed within a packaging material selected from a bag, box, bin, envelope, carton, or container.

97. The synthetic combination of claim 94, comprising 1000 seed weight amount of seeds, wherein the packaging material optionally further comprises a dessicant, and wherein the synthetic combination optionally further comprises an anti-fungal agent.

98. The synthetic combination of claim 94, wherein the purified bacterial population comprises a first bacterial endophyte having a first 16S nucleic acid sequence and a second bacterial endophyte having a second 16S nucleic acid sequence, wherein the first and the second 16S nucleic acid sequences are less than 97% identical.

104. A method for generating an agricultural seed preparation, comprising:

a) isolating a bacterial endophyte derived from a plant living in a habitat of biotic or abiotic stress;

b) contacting at least one flower of a plurality of agricultural plants from a different habitat with a synthetic formulation comprising a purified bacterial population, the bacterial population comprising the isolated bacterial endophyte in an amount effective to colonize the at least one seed made by the flower, wherein the bacterial endophyte exhibits the ability to produce or induce in the at least one seed made by the flower, or in a seedling or an agricultural plant grown from the at least one seed, a functional activity selected from at least two of;

catalase activity, oxidase activity, casein hydrolysis activity, gelatin hydrolysis activity, ACC-deaminase activity, exopolysaccharide activity, amylase activity, cellulase activity, chitinase activity, hemolytic activity, lipase activity, pectinase activity, phosphatase activity, protease activity, xylanase activity, production of an auxin, production of an antimicrobial, production of HCN, production of NH₃, production of AHL, production of PHB, production of a siderophore, mineral phosphate solubilization, and production of acetoin.