Synergistic microbial strains for increasing the activity of nitrogen fixing microorganisms.

Abstract

Embodiments of the present disclosure provide methods and compositions for increasing nitrogen (N) fixation of nitrogen fixing bacteria or increasing N acquisition for plants in need of N acquisition. The method and composition embodiments include at least one live endophytic strain, which is isolated from one or more plants grown in a nutrient-limited and / or water-stressed environment. In some embodiments, the endophytic strain can be administered to a plant, where the endophytic strain synergistically increases the nitrogen fixation of the nitrogen fixing strain associated with the plant. In other embodiments, the nitrogen fixing strain is not associated with a plant.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 213,517, filed June 22, 2021.

[0002] (Sequence Listing Statement) The sequence listing associated with this application is provided in text format in lieu of hardcopy and is hereby incorporated by reference. The name of the text file containing the sequence listing is 3915-P1118WO2UW_Seq_list_Final_20220616_ST25.txt. The text file is 13KB. The text file was created on June 16, 2022. The text file will be submitted via EFS-Web with the filing of this application. [Background technology]

[0003] (background) Nitrogen (N) fixation in nature involves the use of nitrogen derived from the air. 2 It is an exclusively bacterial process that provides the essential N required for life by converting gases into usable metabolic products. Nitrogen can be shuttled between members of the microbial community, but diazotrophs (N fixing bacterium) are also commonly found in soils, which are associated with plants. Some plants house N fixing bacteria in specialized structures called root nodules, but bacteria can also survive within plant tissues as endophytes without causing disease. The endophytes provide the fixed N to the plant, presumably in exchange for receiving sugars and other specialized molecules provided by the plant.

[0004] Thus, a suitable plant microbiome can significantly improve plant growth and health. In addition to N, endophytic bacteria can also provide phosphorus, which has been shown to increase plant resistance to abiotic and biotic stresses.

[0005] Only in the past few years has the idea of ​​using nitrogen-fixing endophytes to produce N gained acceptance (Sharon L. Doty, 2017, Chapter 2: Endophytic Nitrogen Fixation: Controversy and a Path Forward, Functional Importance of the Plant Endophytic Microbiome: Implications for Agriculture, Forestry and Bioenergy, edited by Sharon L. Doty, Springer doi:10.1007 / 978-3-319-65897-1). It is now widely recognized that many non-legume species have symbiotic nitrogen-fixing endophytes, and that free-living nitrogen-fixing bacteria are often present in the soil. This has made harnessing the nitrogen-fixing capacity of nitrogen-fixing bacteria an area of ​​interest. Although some agricultural companies have developed nitrogen-fixing bioinoculants, the simple application of a single nitrogen-fixing strain has not resulted in the expected increase in crop yields. Artificial nitrogen fertilizer can also be produced through energy-requiring chemical processes. However, due to its high energy input, this is expensive and the cost is passed on to the consumer or farmer. Chemical fertilizers also have a negative impact on the environment due to the use of fossil fuels in their production and soil bacteria that convert excess fertilizer into nitrous oxide (a potent greenhouse gas), as well as by destroying aquatic ecosystems through leaching into waterways. In tropical agriculture, this pollution endangers sensitive coral reef ecosystems. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Sharon L. Doty, 2017, Chapter 2: Endophytic Nitrogen Fixation: Controversy and a Path Forward, Functional Importance of the Plant Endophytic Microbiome: Implications for Agriculture, Forestry and Bioenergy, edited by Sharon L. Doty, Springer Summary of the Invention [Means for solving the problem]

[0007] Thus, there remains a need to provide techniques for increasing the amount of fixed N produced by microorganisms to inexpensively produce nitrogen products that are not toxic to the environment. The methods should be broadly applicable to improve nitrogen availability to a variety of plants in a range of environments, as well as to any industrial process that requires nitrogen. The present disclosure addresses this and related needs.

[0008] (overview) This Summary is provided to introduce selected concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features of the present subject matter, nor is it intended to be used as an aid in determining the scope of the present subject matter.

[0009] In accordance with the above, in one aspect of the present invention, the present disclosure provides a method for synergistically increasing nitrogen acquisition in a plant in need thereof. The method may include the step of producing an inoculant for field treatment of a plant in need thereof. The inoculant may include a solution containing an effective amount of at least one live endophytic strain, the live endophytic strain being isolated from one or more plants grown in a nutrient-limited and / or water-stressed environment. The method may further include the step of applying the inoculant to a plant in need thereof, the live endophytic strain contacting at least one nitrogen-fixing bacterial strain associated with the plant and causing the nitrogen-fixing bacterial strain to fix nitrogen at a rate higher than the nitrogen fixation rate of the nitrogen-fixing bacterial strain in the absence of the live endophytic strain.

[0010] In another aspect of the present invention, the present disclosure provides a method for synergistically increasing the nitrogen fixation of at least one live nitrogen fixing bacterial strain, which may include contacting at least one live nitrogen fixing bacterial strain with an effective amount of a solution containing an effective amount of at least one live endophytic bacterial strain, the live endophytic bacterial strain being isolated from one or more plants grown in a nutrient-limited and / or water-stressed environment; contacting the live nitrogen fixing bacterial strain with the live endophytic bacterial strain causes the live nitrogen fixing bacterial strain to fix nitrogen at a rate that is higher than the rate of nitrogen fixation of the live nitrogen fixing bacterial strain in the absence of the live endophytic bacterial strain.

[0011] In another aspect of the present invention, the present disclosure provides an inoculant for synergistically increasing nitrogen acquisition in a plant in need thereof, which may comprise an effective amount of a solution derived from a lyophilized formulation comprising an effective amount of at least one live isolated endophytic strain, the live isolated endophytic strain being isolated from one or more plants grown in a nutrient-limited and / or water-stressed environment.

[0012] In some embodiments, the at least one live isolated endophytic strain comprises a 16S nucleic acid sequence set forth in SEQ ID NO:1, 5, and 10. In some embodiments, the at least one live isolated endophytic strain comprises a 16S nucleic acid sequence set forth in SEQ ID NO:1. In some embodiments, the at least one live isolated endophytic strain comprises a 16S nucleic acid sequence set forth in SEQ ID NO:5. In some embodiments, the at least one live isolated endophytic strain comprises a 16S nucleic acid sequence set forth in SEQ ID NO:10.

[0013] In some embodiments, the at least one live isolated endophytic strain comprises one or more markers selected from the sequences set forth in SEQ ID NOs: 2-4, 6-9, and 11-14. In some embodiments, the at least one live isolated endophytic strain comprises three markers selected from the sequences set forth in SEQ ID NOs: 2-4. In other embodiments, the at least one live isolated endophytic strain comprises four markers selected from the sequences set forth in SEQ ID NOs: 6-9. In yet other embodiments, the at least one live isolated endophytic strain comprises four markers selected from the sequences set forth in SEQ ID NOs: 11-14.

[0014] In some embodiments, the at least one live isolated endophytic strain is of a Sphingobium species. In other embodiments, the at least one live isolated endophytic strain is of a Herbiconiux species.

[0015] In some embodiments, the nutrient-limited and / or water-stressed environment is a primary substrate. In some embodiments, the primary substrate is cobble or sand. In other embodiments, the nutrient-limited and / or water-stressed environment is one of a lava field, a desert, an arid environment, a semi-arid environment, and / or a charred environment.

[0016] In some embodiments, the plant in need thereof is selected from the group of crop plants, bioenergy crop plants, forestry trees, horticultural plants, spice or medicinal plants, and turfgrass.

[0017] In some embodiments, the inoculum comprises a solution containing an effective amount of two or more live isolated endophytic strains.

[0018] In some embodiments, an effective amount of at least one live isolated endophytic strain is an amount that increases nitrogen fixation by a nitrogen fixing bacterial strain associated with the plant by at least 5% compared to the nitrogen fixation rate of the nitrogen fixing bacterial strain associated with the plant in the absence of the at least one live isolated endophytic strain.

[0019] In some embodiments, the inoculant may further comprise at least one live isolated strain of nitrogen fixing bacteria.

[0020] In some embodiments, the ratio of the at least one live isolated synergistic endophytic strain to the at least one live isolated nitrogen fixing bacterial strain is 1+n:1, where n is an integer from 0 to 20. In other embodiments, the ratio of the at least one live isolated endophytic strain to the at least one live isolated nitrogen fixing bacterial strain is 1:1+n, where n is an integer from 0 to 20.

[0021] In some embodiments, the inoculant is administered to a plant in need thereof and the at least one live isolated endophytic strain contacts the at least one nitrogen fixing bacterial strain associated with the plant, causing the nitrogen fixing bacterial strain associated with the plant to fix nitrogen at a rate that is higher compared to the nitrogen fixation rate of the nitrogen fixing bacterial strain associated with the plant in the absence of the at least one isolated endophytic strain.

[0022] Description of the drawings The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same becomes better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0023] [Figure 1] Figure 1. Acetylene reduction assay of diluted cultures showing that synergistic partners can increase the nitrogen fixation activity of different nitrogen fixers. Although the effect varied from strain to strain, all synergistic partners increased the activity of at least two nitrogen fixers. Open bars represent the nitrogen fixers tested alone, while striped bars represent mixtures with synergistic partners.

[0024] [Diagram 2] Figure 2. Acetylene reduction assay of diluted suspensions showing that a mixture of synergistic strains increases the activity of various nitrogen fixing bacteria. The synergistic mixture was processed as a single suspension containing each strain at OD600 0.2. The open bars represent the tested nitrogen fixing organisms alone, while the striped bars represent the mixture with the synergistic strains.

[0025] [Figure 3-1] Figures 3A-3B. Acetylene reduction assay of diluted cultures in nitrogen-free medium (NFM) (A) or diluted suspensions in NFM (B). Both A and B show that as the ratio of synergistic strains to nitrogen fixers increases, the nitrogen fixation activity also increases. White bars indicate the tested nitrogen fixing organisms alone, while striped bars indicate mixtures with synergistic strains. [Figure 3-2] Same as above.

[0026] [Figure 4]Figure 4. Acetylene reduction assay performed with a mixture of nitrogen fixing bacteria, where each nitrogen fixing organism was treated as a single cell suspension with an OD600 of 0.2, and mixed with various strains related to WW5. The results indicate that the synergistic activity observed in the partner strains is not a common trait of bacteria in general. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] (Detailed Description) The present disclosure is based on the surprising and novel discovery that plant-associated synergistic bacterial strains isolated from nutrient-limited and / or water-stressed environments (which may include, but are not limited to, Hawaiian lava-embedded plants or boulder-dominated riparian areas) can synergistically increase the nitrogen fixation of any nitrogen-fixing bacterial strain, regardless of whether the nitrogen-fixing bacterial strain is free-living, associated with the plant, or added as part of a combination with the endophytic bacterial strain (e.g., as an inoculant to the plant, or any other means known to those skilled in the art). The disclosed endophytic bacterial strains are synergistic partners that, when combined with a nitrogen-fixing bacterial strain, produce a total nitrogen fixation capacity that is greater than the sum of their individual nitrogen fixation capacities. Thus, a combination of one or more live synergistic bacterial strains can be used as a field treatment to increase nitrogen acquisition in plants. In some embodiments, one or more live endophytic bacterial strains are added to the soil surrounding the plant to synergistically increase the nitrogen fixation of existing nitrogen-fixing bacterial strains associated with the plant. In other embodiments, one or more live endophytic strains are added in combination with one or more live nitrogen fixing bacterial strains, and the combination is added to the soil surrounding the plant to synergistically increase the nitrogen fixation of existing nitrogen fixing bacterial strains associated with the plant. In other embodiments, the combination (e.g., endophytic strain and nitrogen fixing bacterial strain) may be used as part of a seed treatment / coating or other application to optimize plant growth or seed development by means well known to those skilled in the art.

[0028] As described in more detail in the Examples, these endophytic strains were isolated, characterized, and formulated in specific combinations to prepare as plant inoculants. Using acetylene reduction assay, these endophytic strains showed nitrogen fixation and synergistic effects when combined with nitrogen fixing strains. The observed effects with the diversity of nitrogen fixing strains suggested to those skilled in the art that the disclosed endophytic strains could function as synergistic partners with any nitrogen fixing strain to synergistically increase its nitrogen fixing ability. Thus, these data demonstrate the usefulness of using one or more live endophytic strains as synergistic partners to increase nitrogen fixation in host plants and reduce the need for external chemical fertilizers, providing an environmentally friendly and economically sustainable alternative to chemical fertilizers.

[0029] In accordance with the above, in one aspect of the present invention, the present disclosure provides a method for synergistically increasing nitrogen acquisition in a plant in need thereof. The method may include the step of producing an inoculant for field treatment of a plant in need thereof. The inoculant may include a solution containing an effective amount of at least one live endophytic strain, the live endophytic strain being isolated from one or more plants grown in a nutrient-limited and / or water-stressed environment. The method may further include the step of applying the inoculant to a plant in need thereof, the live endophytic strain contacting at least one nitrogen-fixing bacterial strain associated with the plant and causing the nitrogen-fixing bacterial strain to fix nitrogen at a rate higher than the nitrogen fixation rate of the nitrogen-fixing bacterial strain in the absence of the live endophytic strain.

[0030] In another aspect of the present invention, the present disclosure provides a method for synergistically increasing the nitrogen fixation of at least one live nitrogen fixing bacterial strain, which may include contacting at least one live nitrogen fixing bacterial strain with an effective amount of a solution containing an effective amount of at least one live endophytic bacterial strain, the live endophytic bacterial strain being isolated from one or more plants grown in a nutrient-limited and / or water-stressed environment; contacting the live nitrogen fixing bacterial strain with the live endophytic bacterial strain causes the live nitrogen fixing bacterial strain to fix nitrogen at a rate that is higher than the rate of nitrogen fixation of the live nitrogen fixing bacterial strain in the absence of the live endophytic bacterial strain.

[0031] In another aspect of the present invention, the present disclosure provides an inoculant for synergistically increasing nitrogen acquisition in a plant in need thereof, which may comprise an effective amount of a solution derived from a lyophilized formulation comprising an effective amount of at least one live isolated endophytic strain, the live isolated endophytic strain being isolated from one or more plants grown in a nutrient-limited and / or water-stressed environment.

[0032] As used herein, "nitrogen fixation," "nitrogen acquisition," and other grammatical variations of these phrases describe the chemical process by which diatomic nitrogen is converted into nitrogen-containing organic or inorganic molecules to provide nitrogen in a form that can be used for metabolism by living organisms.

[0033] The one or more live isolated endophytic strains are optionally isolated from one, two, three, four, five, six, seven, eight, nine, ten, or more than ten genera of plants. In another embodiment, the live isolated endophytic strains are optionally isolated from one, two, three, four, five, six, seven, eight, nine, ten, or more than ten species of plants.

[0034] The plant genera and species from which the one or more live endophytic strains are isolated include, but are not limited to, plants that survive in nutrient-limited and / or water-stressed conditions. In some embodiments, nutrient-limited and / or water-stressed conditions include lava, sand, desert, rock, semi-arid and arid climates, tropical, highly polluted, highly saline, highly mineralized, carbonized, exposed to radiation, low oxygen, marine, and soil or regolith lacking any one of the essential or preferred nutrients.

[0035] In some embodiments, the plant genus and species from which the one or more live endophytic strains are isolated includes a nutrient-limited and / or water-stressed environment that is the initial substrate. As used herein, the term "initial substrate" refers to the surface on which the plant grows being newly formed land. In some embodiments, the initial substrate is boulders or sand. In some embodiments, the initial substrate is lava. The lava can be a lava bed, lava field, or lava plain. Furthermore, the initial substrate can be in a highly polluted, highly saline, highly mineralized, carbonized, irradiated, low oxygen, low water, marine, arid, semi-arid, or tropical environment. Typically, such initial substrates have nutrient deficiencies, and thus plants that can establish initial growth have evolved to be able to compensate for such deficiencies in available nutrients. Such compensation can include the presence of an elaborate microbiome that facilitates the processing of nutrients (e.g., fixed nitrogen).

[0036] As used herein, the term "strain" refers to a genetic variant or subtype of a microorganism (e.g., a bacterium).

[0037] The one or more live endophytic strains include bacteria isolated and selected from one genus of bacteria, two genera of bacteria, three genera of bacteria, four genera of bacteria, five genera of bacteria, six genera of bacteria, seven genera of bacteria, eight genera of bacteria, nine genera of bacteria, ten genera of bacteria, or more than ten genera of bacteria. In one embodiment, the plurality of live endophytic strains includes bacteria from between six and eight genera. The one or more live endophytic strains include bacteria isolated and selected from one species of bacteria, two species of bacteria, three species of bacteria, four species of bacteria, five species of bacteria, six species of bacteria, or more than six species of bacteria. In one embodiment, the plurality of live endophytic strains are isolated and selected from between one and six species of a particular genus.

[0038] In yet another embodiment, the one or more live endophytic strains are helper strains that synergistically increase the nitrogen fixation rate of any nitrogen fixing strain. As used herein, "synergistically," "synergistic," or any grammatical variation of these terms refers to an interaction between at least one live endophytic strain (i.e., a helper strain) and any nitrogen fixing strain that results in a total increase in nitrogen fixation that is greater than the sum of their individual effects (i.e., a nitrogen fixing endophyte) or that results in a greater effect compared to the nitrogen fixation rate of the nitrogen fixing strain in the absence of the endophytic strain (i.e., a non-nitrogen fixing endophyte).

[0039] In some embodiments, the one or more live endophytic strains comprise the 16S rRNA sequence set forth in SEQ ID NO:1, 5, and 10. In some embodiments, the one or more live endophytic strains comprise the 16S rRNA sequence set forth in SEQ ID NO:1. In some embodiments, the live endophytic strains comprise the 16S rRNA sequence set forth in SEQ ID NO:5. In some embodiments, the live endophytic bacteria comprises the 16S rRNA sequence set forth in SEQ ID NO:10.

[0040] In yet other embodiments, the one or more live endophytic strains comprise at least one marker comprising a sequence as set forth in SEQ ID NOs: 2-4, 6-9, and 11-14. In some embodiments, the live endophytic strain comprises all three markers selected from SEQ ID NOs: 2-4. In some embodiments, the live endophytic strain comprises at least two markers selected from SEQ ID NOs: 2-4. In still other embodiments, the live endophytic strain comprises at least one marker selected from SEQ ID NOs: 2-4. In still other embodiments, the live endophytic strain comprises all four markers selected from SEQ ID NOs: 6-9. In some embodiments, the live endophytic strain comprises at least three markers selected from SEQ ID NOs: 6-9. In some embodiments, the live endophytic strain comprises at least two markers selected from SEQ ID NOs: 6-9. In still other embodiments, the live endophytic strain comprises at least one marker selected from SEQ ID NOs: 6-9. In still other embodiments, the live endophytic strain comprises all four markers selected from SEQ ID NOs: 11-14. In some embodiments, the live endophytic strain comprises at least three markers selected from SEQ ID NOs: 11-14. In some embodiments, the live endophytic strain comprises at least two markers selected from SEQ ID NOs: 11-14. In yet other embodiments, the live endophytic strain comprises at least one marker selected from SEQ ID NOs: 11-14.

[0041] In yet another embodiment, the one or more live isolated endophytic strains include strains derived from at least one Sphingobium species and at least one Herbiconiux species. In yet another embodiment, the live isolated endophytic strain comprises a 16S rRNA sequence set forth in SEQ ID NO:1 and includes all three markers selected from SEQ ID NOs:2-4 and is derived from a Sphingobium species (i.e., helper strain 1, WW5). In another embodiment, the live isolated endophytic strain comprises a 16S rRNA sequence set forth in SEQ ID NO:5 and includes all four markers selected from SEQ ID NOs:6-9 and is derived from a Herbiconiux species (i.e., helper strain 2, 11R-B). In yet another embodiment, the live isolated endophytic strain comprises a 16S rRNA sequence set forth in SEQ ID NO:10 and includes all four markers selected from SEQ ID NOs:11-14 and is derived from a Sphingobium species (i.e., helper strain 3, HT1-2). In some embodiments, the one or more live isolated endophytic strains comprise at least one strain selected from WW5, 11R-B, and HT1-2. In other embodiments, the live isolated endophytic strains comprise at least two strains selected from WW5, 11R-B, and HT1-2. In yet other embodiments, the live isolated endophytic strains comprise three strains selected from WW5, 11R-B, and HT1-2.

[0042] As used herein, the term "marker" refers to a nucleotide sequence that is unique to each endophytic strain. For example, WW5 strain (SEQ ID NO: 1) contains marker contig_60_9 (SEQ ID NO: 2), marker contig_68_34 (SEQ ID NO: 3), and marker contig_89_19 (SEQ ID NO: 4). 11R-B strain (SEQ ID NO: 5) contains marker contig_2_456500 (SEQ ID NO: 6), marker contig_3_405000 (SEQ ID NO: 7), marker contig_4_300500 (SEQ ID NO: 8), and marker contig_5_325500 (SEQ ID NO: 9). The HT1-2 strain (SEQ ID NO: 10) contains marker contig_3_1377 (SEQ ID NO: 11), marker contig_1_601 (SEQ ID NO: 12), marker contig_5_262 (SEQ ID NO: 13), and marker contig_1_592 (SEQ ID NO: 14).

[0043] In some embodiments, the at least one bacterial strain that fixes nitrogen is an endophytic strain or comprises an endophytic strain. It can be the same strain that is included in the one or more live endophytic strains isolated from one or more plants grown in nutrient-limited and / or water-stressed environments. In other embodiments, the nitrogen-fixing endophytic strain is a different strain from the one or more live endophytic strains isolated from one or more plants grown in nutrient-limited and / or water-stressed environments. In other embodiments, the at least one bacterial strain that fixes nitrogen is a non-endophytic bacterial strain or comprises a non-endophytic bacterial strain. In yet other embodiments, the at least one bacterial strain that fixes nitrogen is a nitrogen-fixing strain.

[0044] Those skilled in the art understand that nitrogen is a macronutrient required by all plants. Therefore, the microbial strains (e.g., one or more live endophytic strains) disclosed herein can provide additional nitrogen in any plant through synergistic activity with any nitrogen fixing strain. In some embodiments, the plant can include, but is not limited to, crop plants. In some embodiments, the crop plants can include, but are not limited to, corn, wheat, barley, rice, rapeseed, potato, and soybean. In still other embodiments, the crop plants can include, but are not limited to, fruit, nut, and vegetable crops, including, but are not limited to, tomatoes, strawberries, bananas, kale, spinach, lettuce, pumpkin, celery, broccoli, citrus, almonds, hazelnuts, walnuts, cherries, apples, pears, and peach trees. In some embodiments, the crop plants can include, but are not limited to, bioenergy crops. In some embodiments, the bioenergy crops may include, but are not limited to, poplar, eucalyptus, miscanthus, switchgrass, and willow.

[0045] In yet other embodiments, the plants may include forest trees, which may include, but are not limited to, Douglas fir, Western hemlock, Western red cedar, Loblolly pine, Ponderosa pine, oak, maple, ash, spruce, and sequoia.

[0046] In yet other embodiments, the plants may include horticultural plants, hi some embodiments, the horticultural plants may include, but are not limited to, azaleas, rhododendrons, roses, and hydrangeas.

[0047] In yet other embodiments, the plants may include spices or medicinal plants, hi some embodiments, the spices or medicinal plants may include, but are not limited to, ginseng, cumin, coriander, and turmeric.

[0048] In yet other embodiments, the plants may include turfgrass. In some embodiments, the turfgrass may include, but is not limited to, Kentucky bluegrass, fescue, and perennial ryegrass.

[0049] In some embodiments, one or more of the microbial strains described herein, together with the disclosed synergistic strains, may be added directly to soil to increase the activity of the diastrophic strain. In other embodiments, one or more of the microbial strains described herein, together with the disclosed synergistic strains, may be added directly to a plant containing at least one nitrogen fixing strain to increase the activity of the diastrophic strain. The synergistic strains (e.g., endophytic strains) may be added to the plant in many ways known to those of skill in the art. For example, in some embodiments, the synergistic strains are applied to the plant through foliar sprays, applied as a solution (e.g., an inoculant) to the spikes of root-containing or root-free plants, or applied to tissue culture plants. In some embodiments, the synergistic strains may be added in furrows or in irrigation solutions for irrigating plants and / or crops. In still other embodiments, the synergistic strains may be added to soil as dry powders or in any combination of methods known to those of skill in the art. Once a synergistic strain is incorporated into a plant, the scions of these plants may also continue to contain the synergistic strain and may transmit the cooperation between the plant and the microorganism indefinitely. Thus, any part of the plant or planting medium that contains the synergistic strain may be a continuous source of the synergistic strain.

[0050] In some embodiments, the isolated endophytic strain may be freeze-dried after the isolation process. In other embodiments, the isolated nitrogen-fixing strain may be freeze-dried after the isolation process. In yet other embodiments, one or more of the microbial strains disclosed herein may be freeze-dried after the isolation process. The microbial strains (e.g., endophytic strains, nitrogen-fixing strains and / or other disclosed microbial strains) may be freeze-dried according to any technique known to those skilled in the art.

[0051] As used herein, the term "inoculate" and grammatical variations thereof refer to contacting a plant with an inoculant composition. In some embodiments, the inoculant is applied by spraying, dipping, dusting, gassing, and other techniques known in the art. The inoculant composition may also be mixed into the soil or other substrate in which the plant seed is planted (before or after). In some embodiments, the inoculant may comprise a solution containing an effective amount of at least one live endophytic strain. In some embodiments, the inoculant may comprise a solution containing an effective amount of at least two live endophytic strains. In yet other embodiments, the inoculant may comprise a solution containing an effective amount of three or more live endophytic strains. In some embodiments, the live endophytic strain is a live isolated strain. As used herein, "isolated strain" refers to a 100% pure strain, which is free of any contaminating strains. For example, the live isolated endophytic strain WW5 is a 100% pure WW5 strain, which is free of any contaminating strains.

[0052] In some embodiments, the inoculant comprises a ratio of at least one live isolated endophytic strain to at least one live isolated nitrogen fixing bacterial strain. In some embodiments, the ratio of the at least one live isolated endophytic strain to at least one live isolated nitrogen fixing bacterial strain can be 1+n:1, where n is an integer between 0 and 20. In some embodiments, the ratio of the at least one live isolated endophytic strain to at least one live isolated nitrogen fixing bacterial strain can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1. In other embodiments, the ratio of the at least one live isolated endophytic strain to the at least one live isolated nitrogen fixing bacterial strain can be 1:1+n, where n is an integer between 0 and 20. In some embodiments, the ratio of the at least one live isolated endophytic strain to the at least one live isolated nitrogen fixing bacterial strain can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, and 1:20. In yet other embodiments, the ratio of two live isolated endophytic strains to the at least one live isolated nitrogen fixing bacterial strain can be 1+n:1+n:1, where n is an integer between 0 and 20. In some embodiments, n may be the same for the first and second endophytic strains. For example, the ratio may be 1:1:1, 2:2:1, 3:3:1, 4:4:1, 5:5:1, 6:6:1, 7:7:1, 8:8:1, 9:9:1, 10:10:1, 11:11:1, 12:12:1, 13:13:1, 14:14:1, 15:15:1, 16:16:1, 17:17:1, 18:18:1, 19:19:1, and 20:20:1. In other embodiments, n may be different for the first and second endophytic strains. For example, the ratio may be 1:2:1, 2:3:1, 3:4:1, 4:5:1, and any variation that may be determined by one of skill in the art.

[0053] In yet other embodiments, the inoculant may further comprise a solution containing at least one strain of live nitrogen fixing bacteria. In some embodiments, the inoculant may further comprise a solution containing at least two, three, four, five, six, seven or more strains of live nitrogen fixing bacteria. In some embodiments, the live nitrogen fixing strain is a live isolated nitrogen fixing strain. In some embodiments, the nitrogen fixing strain is HT1-9, the species is Azorhizobium sp., and the phylogenetic group is Alphaproteobacteria. In some embodiments, the nitrogen fixing strain is SherDot2 (SD2), the species is Azospirillum sp., and the phylogenetic group is Alphaproteobacteria. In some embodiments, the nitrogen fixing strain is WP4-2-2, the species is Burkholderia sp., and the phylogenetic group is Betaproteobacteria. In some embodiments, the nitrogen fixing strain is WPB, the species is Burkholderia vietnamiensis, and the phylogenetic group is Betaproteobacteria. In some embodiments, the nitrogen fixing strain is WP5, the species is Rahnella aceris, and the phylogenetic group is Gammaproteobacteria. In some embodiments, the nitrogen fixing strain is R10, the species is Rahnella aceris, and the phylogenetic group is Gammaproteobacteria. In yet other embodiments, the nitrogen fixing strain is SherDot1 (SD1), the species is Azotobacter beijerinckii, and the phylogenetic group is Gammaproteobacteria.

[0054] As used herein, "effective amount" and grammatical variations thereof refer to an amount of at least one live endophytic strain that increases nitrogen fixation in the nitrogen fixing strain, either isolated in culture or associated with the plant, by at least 5% compared to the nitrogen fixation rate of the nitrogen fixing strain, either isolated in culture or associated with the plant, in the absence of the endophytic strain. The increase in nitrogen fixation of the selected nitrogen fixing strain can be improved by at least 5%. For example, the increase in nitrogen fixation can be at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, or at least more than 30%. In some embodiments, the increase in nitrogen fixation can be at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%. In some embodiments, the nitrogen fixation of the selected nitrogen fixers can be improved by more than about 2-fold, e.g., about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, or more (e.g., greater than 100% nitrogen fixation, even up to and beyond 1000% nitrogen fixation).

[0055] Nitrogen fixation in the selected nitrogen fixing strains may be improved by between about 5% and about 2000%, between about 10% and about 1500%, between about 15% and about 1000%, between about 15% and about 800%, between about 20% and about 800%, between about 25% and about 800%, and between about 30% and about 750%. In some embodiments, nitrogen fixation may be improved by between about 50% and about 500%, between about 50% and about 400%, between about 50% and about 200%, and between about 75% and about 100%.

[0056] Unless specifically defined herein, all terms used herein have the same meaning as those terms would have to one of ordinary skill in the art in the field of this disclosure. For convenience, certain terms used in the specification, examples, and appended claims are provided here. These definitions are provided to assist in describing certain embodiments. As the scope of the present invention is limited only by the claims, these definitions are provided below. It is not intended to limit the invention.

[0057] Use of the term "or" in the claims and specification is used to mean "and / or" unless it is expressly stated that it refers to alternatives only or that the alternatives are mutually exclusive. However, the present disclosure supports a definition that refers to alternatives only and to "and / or."

[0058] The words "a" and "an", when used in conjunction with the word "comprising" in a claim or the specification, refer to one or more, unless specifically stated otherwise.

[0059] Unless the context clearly requires otherwise, throughout the specification and claims, words such as "comprise", "comprising" and the like should be interpreted in an open and inclusive sense, as opposed to a restrictive, exclusive or exhaustive sense. For example, the term "comprising" may be read to indicate "including, but not limited to". The term "consisting essentially of" or grammatical variations thereof indicate that the described subject matter may include additional elements not recited in the claims, but that the additional elements do not materially affect the basic and novel characteristics of the subject matter disclosed herein. Furthermore, the words "herein", "above", and "below" and words of similar import, when used herein, refer to this application as a whole and not to any particular portion of this application. Words using the singular or plural number also include the plural and singular number, respectively. The word "about" indicates a number that is within a small variation above or below the recited reference number. For example, "about" can refer to a number that is within a 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% greater or less than the indicated reference number.

[0060] Disclosed are materials, compositions, and components that can be used for the disclosed methods and compositions, can be used in combination with the disclosed methods and compositions, can be used in the preparation of the disclosed methods and compositions, or are products of the disclosed methods and compositions. When combinations of these materials, subsets of these materials, interactants of these materials, groups of these materials, and the like are disclosed, it is understood that each of the various individual combinations and collective combinations is specifically contemplated, although specific references to each and every combination and permutation of these compounds may not be expressly disclosed. This concept applies to all aspects of the disclosure, including but not limited to the steps in the described methods. Thus, specific elements of any of the above embodiments can be combined with elements in other embodiments, or substituted for elements in other embodiments. For example, when there are various additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method step or combination of method steps of the disclosed methods, and each of such combinations or subsets of combinations is specifically contemplated and should be considered as disclosed. Further, it will be understood that the embodiments described herein can be implemented using any suitable materials, such as those described elsewhere herein or known in the art.

[0061] Publications cited herein and the subject matter for which they are cited are hereby specifically incorporated by reference in their entireties. EXAMPLES

[0062] (Example) The present disclosure describes the isolation, purification, inoculant preparation, and demonstrated activity of multiple live endophytic strains isolated from plants for use in inoculating plants to provide nutrients to the plants without the need for excessive chemical fertilizers, and a method for synergistically increasing nitrogen fixation in multiple nitrogen fixing strains.

[0063] Example 1 (Isolation of synergistic strains) Plants growing in nutrient-poor or water-limited environments were sampled and surface sterilized. Approximately 10 g of tissue was then ground into 15 ml of sterile nitrogen-limited combined carbon medium (NLCCM) broth using a mortar and pestle. The resulting slurry was then centrifuged at low speed to sediment the plant debris. Serial dilutions were then made from the supernatant in NLCCM. To select for nitrogen fixers, the dilutions were plated on NLCCM agar plates and nitrogen-free NFCCM agar plates. The isolated colonies were then restreaked on new NLCCM or NFCCM agar plates. When the isolated colonies from the streaked plates were then restreaked on mannitol-glutamate-Luria broth (MG / L), the rich medium allowed the growth and isolation of multiple non-nitrogen fixing strains that clustered with the nitrogen fixers within what appeared as a single colony on the nitrogen-limited or nitrogen-free plates. Those strains that appeared on rich medium were candidates for synergy and were tested for their ability to increase nitrogen fixation in azotoxins by acetylene reduction.

[0064] Example 2 (Acetylene Reduction Assay) (Bacteria dilution suspension): Bacteria were grown on nutrient rich MG / L agar plates at 30°C. Nitrogen fixing strains were also grown on nitrogen limiting NLCCM agar plates. Bacteria were then suspended in liquid NLCCM or, where indicated, in nitrogen free medium (NFM) (Doty, S.L., Oakley, B., Xin, G., et al., Diazotrophic endophytes of native black cottonwood and willow, Symbiosis, 47, 23-33 (2009)). Cells of nitrogen fixing organisms grown on NL-CCM were given priority. Cells of each strain were then analyzed by optical density at 600 nm (OD ) unless otherwise stated in the figure legends. 600 ) and diluted to 0.4.

[0065] (bacterial culture): Bacteria were grown on MG / L agar plates at 30°C. Nitrogen fixing strains were also grown on NL-CCM agar plates. Isolated colonies were selected and prioritised for cells of nitrogen fixing organisms grown on NL-CCM to grow in 50 ml MG / L without mannitol at 30°C for 36 hours. Cultures were centrifuged at 5000 x g and washed in NFM, and this process was repeated twice. Cultures of each strain were grown to OD in NFM. 600 Diluted to 0.4.

[0066] (Stock ratio): When comparing ratios, nitrogen fixing organisms are 600 Dilute to 0.5, meanwhile, OD 600 The synergistic strains were diluted to create a series of dilutions with a NA of 0.05, 0.1, 0.5, 2.5, and 5.0, which when mixed together produced a series of ratios of nitrogen fixing organisms to synergistic partners.

[0067] (Acetylene reduction): The reduction of acetylene to ethylene was used as a proxy for nitrogen fixation activity because the nitrogenase enzyme performs both chemical reactions. Cell mixtures were made by combining diluted suspensions or diluted cultures in equal parts using a total volume of 150 μl in 17 ml amber septum vials prepared with 6 ml of NLCCM agar. The resulting 11 ml headspace was then dosed with 0.1 ml of 98.6% acetylene. After 2 days of incubation at 30°C, unless otherwise stated, the headspace was sampled by removing 5 ml of air from a 22 ml gas chromatography vial and replacing it with 5 ml of headspace from the experimental vial. Samples were analyzed by gas chromatograph with flame ionization detector (GC-FID, TRACE GC ULTRA, Thermo Scientific) using a HayeSep R column. High purity N2(g) was used as the carrier, H2(g) was used as the fuel, and synthetic air was used as the oxidizing gas. Peak areas were then converted to parts per million (ppm) using a standard curve of ethylene concentration.

[0068] Example 3 Sequencing of synergistic strains Whole genome sequencing of the above three synergistic strains indicates that all three strains are unique and novel species.

[0069] Table 1. Strains were identified using the Type (Strain) Genome Server (TYGS) protocol. The TYGS database consists of over 15,000 type strain species / subspecies genomes. 4 A score ≦70.0% is the threshold for potentially novel species (1-2). [Table 1]

[0070] Strain-specific regions of the genome for these three strains (eg, WW5, 11R-B1, and HT1-2).

[0071] In addition to using the 16s ribosomal gene (SEQ ID NOs: 1, 5, and 10) for strain identification, strain-specific primers for Sphingobium sp. WW5, Herbiconiux sp. 11R-B1, and Sphingobium sp. HT1-2 were designed using protocols adapted from Stets et al. (Stets MI et al., Quantification of Azospirillum brasilense FP2 bacteria in wheat roots by strain-specific quantitative PCR, Appl Environ Microbiol. 2015;81(19):6700-6709. doi:10.1128 / AEM.01351-15) and Jo et al. (Jo, J. et al., Microbial community analysis using high-throughput sequencing technology: a beginner's guide for microbiologists, J Microbiol., 58, 176-192(2020)). For each strain, the FASTA genome sequence was split into 500bp non-overlapping segments using the shred.sh (v.2.3.7) program from BBMap (v38.96). For each genera Sphingobium and Herbiconiux, a local database was constructed from seven complete genomes downloaded from the NCBI Reference Sequence database. The following steps were completed in Geneious Prime (v2022.1.1 Build 2022-03-15 11:43). The segmented FASTA file of candidate sequences for each strain was subjected to a BLASTn search against the local database, and segments with no hits were retained. The filtered list of candidate sequences was then subjected to a BLASTn search against a second local database of complete genomes constructed from our internal laboratory strains, again retaining only segments with no hits.Finally, the remaining candidate sequences were submitted online as queries against the complete NCBI nucleotide database, and segments for which no matches existed were designated as unique sequences and used to design strain-specific primers.

[0072] One primer set was designed for each unique sequence. The primers were designed in Geneious Prime using the Primer3 plug-in (v2.3.7) with the following settings: i) optimal amplicon length 400nt, range 300nt-500nt, ii) primer length 22nt-25nt, iii) Tm range 57°C-63°C, maximum Tm difference between primers 2°C, and iv) optimal GC% 50%, range 40%-60%. The resulting products (primer sets and amplicon sequences together) were mapped to the genome assemblies of their respective strains, and products that fell completely within the CDS were used as candidate primer sets.

[0073] A total of 47 strain-specific primer sets (SSPs) were identified for WW5. Of them, 18 SSPs targeted coding sequences (CDSs). Three of them are in known genes. Their primer sets and predicted products are included in Table 2. A total of 29 strain-specific primer sets (SSPs) were identified for 11R-B. Of them, 10 SSPs targeted annotated coding sequences (CDSs), only one of them targeted a known gene, and the other nine SSPs targeted CDSs annotated as hypothetical proteins. The four primer sets and predicted products for the one identified gene hit, as well as three arbitrarily selected primer sets that fall into hypothetical proteins, are included in Table 2. A total of 217 strain-specific primer sets (SSPs) were identified for HT1-2. Of those, 89 SSPs targeted annotated coding sequences (CDS), seven of which targeted known genes, and the other 82 SSPs targeted CDSs annotated as hypothetical proteins. Primer sets and predicted products for two SSPs targeting identified genes and two targeting hypothetical proteins are included in Table 2. Table 2. Candidate primer sets including amplicon sequences. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0074] Example 4 (Individual synergistic strains induce higher activity in nitrogen fixing bacteria)

[0075] Acetylene reduction assays of diluted cultures showed that synergistic partners could increase the nitrogen fixation activity of various nitrogen fixers. Figure 1. Although the effect varied from strain to strain, all synergistic partners increased the activity of at least two nitrogen fixers. In Figure 1, the open bars represent the tested nitrogen fixers alone (e.g., WP5, HT1-9, and SherDot2 (SD2)). As shown for the WP5 nitrogen fixer strain, the addition of synergistic partner strains WW5 and HT1-2 (striped bars) produced the greatest synergistic increase in nitrogen fixation, as shown by acetylene reduction to ethylene. Similar to the WP5 nitrogen fixer strain, the addition of synergistic partner strains 11RB and HT1-2 produced the greatest synergistic increase in nitrogen fixation. Similar results were observed for the SD2 nitrogen fixer strain. However, all three synergistic partner strains (eg, WW5, 11RB, and HT1-2) synergistically increased the nitrogen fixation of the SD2 nitrogen fixing strain.

[0076] (Synergistic mixture induces higher acetylene reduction) Acetylene reduction assay of diluted suspensions showed that the mixture of synergistic strains increased the activity of various nitrogen fixing bacteria (white bars). The synergistic mixture was analyzed by OD 600 The synergistic mixture (striped bars) containing the synergistic partner strains HT1-2 and 11RB increased nitrogen fixation as indicated by acetylene reduction to ethylene, as shown in Figure 2. Increased nitrogen fixation was observed in various nitrogen fixing strains (white bars) including WP5, WP4-2-2, HT1-9, SD2, R10, and SD1.

[0077] Examples of nitrogen fixing strains that show increased nitrogen fixation activity are shown in the various nitrogen fixing species selected. Those skilled in the art will recognize that since the synergistic strains (e.g., WW5, 11RB, and HT1-2) increase nitrogen fixation in the various nitrogen fixing strains selected, this result (i.e., the synergistic strains increase nitrogen fixation) is representative of all nitrogen fixing strains. Therefore, the results disclosed in this example are not limited to the specific nitrogen fixing strains disclosed in the embodiments of the present invention, but may apply to all nitrogen fixing strains. (Table 3. Diversity of nitrogen fixing strains) [Table 3]

[0078] (The synergistic strains induced greater activity after incubation with nitrogen fixing bacteria as the concentration increased)

[0079] The synergistic strains induced greater activity as the concentration increased after 3 days of incubation with the nitrogen fixing strains. Acetylene reduction assays of diluted cultures in nitrogen-free medium (NFM) show that as the ratio of synergistic strains to nitrogen fixing bacteria increases, the nitrogen fixation activity also increases. As shown in Figure 3A, (1) incubating synergistic strains (striped bars) (e.g., HT1-2 and 11RB) with nitrogen fixing strains (e.g., WP5 and SD2) for 3 days and (2) increasing the ratio of the synergistic strains to nitrogen fixing strains (white bars) (e.g., 5:1 and 10:1) resulted in an increase in synergistic nitrogen fixation.

[0080] The synergistic strains induced greater activity as the concentration increased after 4 days of incubation with the nitrogen fixing strains. Acetylene reduction assays of diluted suspensions in NFM indicate that as the ratio of synergistic strains to nitrogen fixing bacteria increases, the nitrogen fixation activity also increases. As shown in Figure 3B, (1) incubating synergistic strains (striped bars) (e.g., 11RB and WW5) with nitrogen fixing strains (white bars) (e.g., HT9 and SD2) for 4 days and (2) increasing the ratio of the synergistic strains to nitrogen fixing strains (e.g., 5:1 and 10:1) resulted in an increase in synergistic nitrogen fixation.

[0081] (Synergistic activity is not a common trait in bacteria in general) Effect of strains related to WW5 on acetylene reduction by WP5 and WPB after 3 days of incubation together. Each nitrogen fixing organism was incubated at OD 600 Acetylene reduction assays were performed with a mixture of nitrogen fixing strains treated as a single cell suspension with an acetylene concentration of 0.2 and mixed with various strains related to WW5 shown in FIG. 4. In FIG. 4, various strains related to WW5 were first incubated with the mixture of nitrogen fixing strains for 3 days. As shown in FIG. 3A, incubating the synergistic strains with the nitrogen fixing strains for at least 3 days increased nitrogen fixation. See FIG. 3A and FIG. 3B. However, as shown in FIG. 4, incubation of the WW5 synergistic strain in the mixture of nitrogen fixing strains increased nitrogen fixation as expected, but this result was not observed for the various strains related to this WW5 synergistic strain. These results are important because they indicate that the synergistic activity observed in the disclosed partner strains is unique to these endophytic strains and that this synergistic activity is not a common trait of bacteria in general.

[0082] While exemplary embodiments have been shown and described, it will be understood that various changes can be made in these exemplary embodiments without departing from the spirit and scope of the invention. The embodiments of the invention in which an exclusive property or right is claimed are defined in the appended claims.

Claims

**Claim 1** An inoculant for synergistically increasing nitrogen acquisition in plants that require a synergistic increase in nitrogen acquisition, comprising: An effective amount of a solution derived from a lyophilized preparation containing an effective amount of at least one live, isolated endophytic strain from one or more plants grown in a nutrient-limited and / or water stress environment The inoculant comprising. **Claim 2** The inoculant according to claim 1, wherein the inoculant is administered to a plant in need of administration, and the at least one live, isolated endophytic strain contacts at least one nitrogen-fixing strain associated with the plant, and a high percentage compared to the nitrogen fixation rate of the nitrogen-fixing strain in the absence of the at least one isolated endophytic strain, causes the nitrogen-fixing strain to fix nitrogen. **Claim 3** The inoculant according to claim 1, wherein the at least one live, isolated endophytic strain comprises the 16S nucleic acid sequences shown in SEQ ID NOs: 1, 5, and 10. **Claim 4** The inoculant according to claim 1, wherein the at least one live, isolated endophytic strain comprises the 16S nucleic acid sequence shown in SEQ ID NO:

1. **Claim 5** The inoculant according to claim 1, wherein the at least one live, isolated endophytic strain comprises the 16S nucleic acid sequence shown in SEQ ID NO:

5. **Claim 6** The inoculant according to claim 1, wherein the at least one live, isolated endophytic strain comprises the 16S nucleic acid sequence shown in SEQ ID NO:

10. **Claim 7** The inoculant according to claim 1, wherein the at least one live, isolated endophytic strain comprises a marker comprising a sequence shown in SEQ ID NOs: 2, 3, 4, 6, 7, 8, 9, 11, 12, 13, 14, or any combination thereof. **Claim 8** The inoculant according to claim 4, wherein the at least one live, isolated endophytic strain comprises three markers comprising the sequences shown in SEQ ID NOs: 2-4. **Claim 9** The inoculant according to claim 5, wherein the at least one live, isolated endophytic strain comprises four markers comprising the sequences shown in SEQ ID NOs: 6-9. **Claim 10** The inoculant according to claim 6, wherein the at least one live, isolated endophytic strain comprises four markers comprising the sequences shown in SEQ ID NOs: 11-14. **Claim 11** The inoculant according to claim 8, wherein the at least one live, isolated endophytic strain is of the genus Sphingobium.

12. The inoculant according to claim 9, wherein the at least one live, isolated endophytic strain is of the genus Herbiconiux.

13. The inoculant according to claim 10, wherein the at least one live, isolated endophytic strain is of the genus Sphingobium.

14. The inoculant according to claim 1, wherein the nutrient limitation and / or water stress environment is an initial substrate containing large gravel or sand.

15. The inoculant according to claim 1, wherein the nutrient limitation and / or water stress environment is a lava field, a desert, a dry environment, a semi-dry environment, a carbonized environment, or any combination thereof.

16. The inoculant according to claim 1, wherein the plant requiring a synergistic increase in nitrogen acquisition is a crop plant, a bioenergy crop plant, a forestry tree, a horticultural plant, a spice or medicinal plant, turfgrass, or any combination thereof.

17. The inoculant according to claim 1, wherein the solution derived from the lyophilized preparation further contains at least one live, isolated nitrogen-fixing strain.

18. The inoculant according to claim 17, wherein the ratio of the at least one live, isolated endophytic strain to the at least one live, isolated nitrogen-fixing strain is 1 + n:1 or 1:n + 1, and n is an integer from 1 to 20.

19. A method for synergistically increasing nitrogen fixation of at least one nitrogen-fixing strain, comprising: contacting at least one nitrogen-fixing strain with an effective amount of a solution containing an effective amount of at least one live, isolated endophytic strain according to claim 1, wherein the at least one live, isolated endophytic strain is isolated from one or more plants grown in a nutrient-limited and / or water-stress environment; wherein the step of contacting the live nitrogen-fixing strain with the at least one live, isolated endophytic strain causes the live nitrogen-fixing strain to fix nitrogen at a higher rate compared to the nitrogen fixation rate of the live nitrogen-fixing strain in the absence of the at least one live, isolated endophytic strain.

20. A method for synergistically increasing nitrogen acquisition in a plant requiring a synergistic increase in nitrogen acquisition, comprising: (i)A step of generating an inoculant for field treatment of plants that requires a synergistic increase in nitrogen acquisition, wherein the inoculant comprises a solution containing an effective amount of at least one live, isolated endophytic strain, and the live, isolated endophytic strain is isolated from one or more plants grown in a nutrient-limited and / or water stress environment; and (ii)A step of applying the inoculant to a plant that requires a synergistic increase in nitrogen acquisition, wherein the at least one live, isolated endophytic strain contacts at least one nitrogen-fixing strain associated with the plant and causes the nitrogen-fixing strain to fix nitrogen at a higher rate compared to the nitrogen fixation rate of the nitrogen-fixing strain in the absence of the at least one live, isolated endophytic strain A method comprising.

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