Nitrogen-fixing bacteria

Abstract

The field of the invention relates to nitrogen fixing bacterial strains and compositions thereof.

Description

Title of InventionNitrogen-Fixing BacteriaTechnical Field

[0001] The field of the invention relates to nitrogen fixing bacterial strains and compositions thereof.Background of Invention

[0002] Fertilizers provide essential nutrients for plants to grow and the most significant nutrients required are nitrogen, phosphorus and potassium. Recently there has been increases in the cost of fertilizers that mean farmers must carefully plan their fertilizer use. Furthermore, excess or unused fertilizer on farm, particularly in pasture, may be leached from the soil and provide a number of environmental hazards such as groundwater pollution, eutrophication and the increase in algal blooms in waterways and soil acidity.

[0003] Providing sufficient nutrients to crops and pastures to obtain good yield at an acceptable price is essential for farmers cost effective and productive farming.

[0004] Biofertilizers have recently emerged as an ecofriendly and cost-effective alternative to chemical fertilizers. Plant-associated microbes with growth promoting properties can reduce environmental hazards and cost of chemicals on farm while enhancing plant biomass and crop yield. There is a need for alternative biofertilizers that contribute to cost effective and productive farming even in low nitrogen soils.

[0005] The application of most nitrogen-fixing microorganisms as biofertilizers in agricultural production faces various obstacles, such as harsh growth conditions and low nitrogen-fixation efficiency.Summary of Invention

[0006] In one aspect, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 94.7% identical to any one of SEQ ID NO: I to 21.

[0007] In another aspect, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 95% identical to any one of SEQ ID NO: I to 7.

[0008] In a further aspect, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 95% identical to any one of SEQ ID NO: 8 to 14.

[0009] In a further aspect, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 95% identical to any one of SEQ ID NO: 15 to 21.

[0010] In a further aspect, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 97% identical to any one of SEQ ID NO: I to 7.

[0011] In a further aspect, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 97% identical to any one of SEQ ID NO: 8 to 14.

[0012] In a further aspect, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 97% identical to any one of SEQ ID NO: 15 to 21.

[0013] In a further aspect, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is 100% identical to any one of SEQ ID NO: I to 7.

[0014] In a further aspect, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is 100% identical to any one of SEQID NO: 8 to 14.

[0015] In a further aspect, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is 100% identical to any one of SEQ ID NO: 15 to 21.

[0016] In a further aspect, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises 16S rRNA sequences 100% identical to each of SEQ ID NO: 1 to 7.

[0017] In a further aspect, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises 16S rRNA sequences 100% identical to each of SEQ ID NO: 8 to 14.

[0018] In a further aspect, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises 16S rRNA sequences 100% identical to each of SEQ ID NO: 15 to 21.

[0019] In a further aspect, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 97.6% identical to any one of SEQ ID NO: 22 to 42.

[0020] In a further aspect, the present invention provides a Phytobacter bacterium represented by bacteria deposited under Accession Nos. V24 / 010671 and V24 / 010672 (BC01 and BC03 respectively) on 18 June 2024 with the National Measurement Institute, located at 1 / 153 Bertie Street, Port Melbourne, Victoria, Australia 3207.

[0021] In a further aspect, the present invention provides a Kosakonia bacterium represented by bacteria deposited as under Accession No. V24 / 010673 (BC05) on 18 June 2024 with the National Measurement Institute, located at 1 / 153 Bertie Street, Port Melbourne, Victoria, Australia 3207.

[0022] In a further aspect, the present invention provides a substantially purified or isolated bacteria as described herein.

[0023] In a further aspect, the present invention provides a composition comprising one or more bacteria as described herein.

[0024] In one embodiment, the composition is in the form of a liquid or a solid. In another embodiment, the composition further comprises an agriculturally acceptable carrier. In a further embodiment, the composition is in the form of a seed coating.

[0025] In a further aspect, the present invention provides a method of increasing the amount of nitrogen fixation of a plant, comprising contacting the plant, a part of the plant, seed or soil into which the seed or plant is planted with a bacteria as described herein, or a composition as described herein.

[0026] In a further aspect, the present invention provides a method of increasing biomass in a plant, comprising contacting the plant, a part of the plant, seed or soil into which the seed or plant is planted with a bacteria as described herein, or a composition as described herein.

[0027] In a further aspect, the present invention provides a method of providing biofertilizer to a plant, comprising contacting the plant, a part of the plant, seed or soil into which the seed or plant is planted with a bacteria as described herein, or a composition as described herein.

[0028] In a further aspect, the present invention provides a method of increasing germination of seed of a plant, comprising contacting the seed or soil into which the seed is planted with a bacteria as described herein, or a composition as described herein.

[0029] In a further aspect, the present invention provides a method as described herein, wherein the plant is a crop plant or a forage plant.

[0030] In a further aspect, the present invention provides a plant having improved biomass relative to a control plant, comprising a plant in association with a bacteria as described herein, or a composition as described herein.

[0031] In a further aspect, the present invention provides a plant, or part of a plant, derived from a method as described herein.

[0032] In a further aspect, the present invention provides a seed of a plant coated with a bacteria as described herein, or a composition as described herein.Brief Description of Drawings

[0033] Figure 1 provides representative images of the growth phenotypes of N-fixing bacteria isolated according to Example 1, on Burks nitrogen free media plates.

[0034] Figure 2 shows the n / Operon gene numbers identified in isolates BC01, BC03, and BC05.

[0035] Figure 3 shows a histogram of percentage protein identity encoded by BC03 and BC05 nif Operon gene sequences, when compared to BC01 nif genes.

[0036] Figure 4 illustrates the layout of the nif Operon in BC01 and BC03, compared to Phytobacter diazotrophicus strain UAEU22 (GB id CP051548.1). Arrows and positions above and below lines show the transcription strand of the gene, where bars represent chromosomes of the respective isolates. The location of the transposon insertion position, in an intergenic region between nifM and a hypothetical gene, is illustrated for BC01.

[0037] Figure 5 provides a sequence alignment of BC01, BC03, and BC05 16S rRNA gene sequences across each of the 7 gene copies identified for each isolate, with nucleotides shown for sequences comprising a difference from the consensus sequence.

[0038] Figure 6 illustrates a maximum likelihood phylogenetic tree for 16S rRNA gene sequences of BC01, BC03, and BC05, compared to known sequences. Numbers indicate bootstrap values.

[0039] Figure 7 shows the germination of barley seeds inoculated with either non- microbial control (PBS), BC01, BC03, BC05, or a microbial control (Pseudomonas sp.), accordingto Example 2. A) Germination, out of 130 seeds, for seeds planted into pots soaked in 0.1 mM nitrogen. B) Germination, out of 100 seeds, for seeds planted into pots soaked in 10 mM nitrogen.

[0040] Figure 8 shows the root and shoot growth for germinants of barley seeds inoculated with either control (PBS), BC01, BC03, BC05, according to Example 2. A) Root and shoot length (cm), for seeds planted into pots soaked in 0.1 mM, 1 mM, 10 mM, or 25 mM nitrogen. B) Root and shoot weight (g), for seeds planted into pots soaked in 0.1 mM, 1 mM, 10 mM, or 25 mM nitrogen.

[0041] Figure 9 illustrates the proportion of microbes, identified by 16S sequencing, within the root microbiome of 6-week-old barley plants grown from seeds inoculated with either control (PBS), BC01, BC03, BC05, according to Example 2. Plants were grown in either 0.1 mM or 1 mM nitrogen. Thick, diagonal bars indicate the portions of each microbiome in the 0.1 mM and 1 mM nitrogen conditions likely to be from the inoculants.Detailed Description

[0042] Nitrogenase - which produces NH3 - is a complex oxygen-sensitive metalloenzyme with three isoforms: Fe-Mo nitrogenase (encoded by nif gene cluster), Fe-V nitrogenase (encoded by vnf gene cluster), and Fe-Fe nitrogenase (encoded by anf gene cluster). The present inventors have characterised in Example l two isolates of Phytobacter sp (isolate BC01 and BC03) and one isolate of Kosakonia sp (isolate BC05) isolated from Cenchrus clandestinum and Paspalum dilatatum, respectively, that contain a full nif nitrogen fixation operon.

[0043] NifA is the transcriptional activator protein of the nif gene cluster, while NifL inhibits the activity of NifA via interacting with NifA. The NifL-NifA system is affected by the intracellular redox environment and binding state of the ligand (2-OG, ATP / ADP, FAD). The reversible modification of GlnK by uridylation also has a regulatory effect on the transcription of the NifL-NifA system. When there is excess nitrogen, nitrogen metabolism results in low concentrations of 2-OG and high concentrations of glutamine. Gink, NifA, and NifL can formthe ternary complex to suppress the activity of NifA, which results in the blockage of nitrogenase expression. When nitrogen is limited, the floundering nitrogen metabolism results in the accumulation of 2-OG and the excessive consumption of glutamine, which disrupts the formation of the ternary complex to activate NifA. The active NifA can promote the expression of nitrogenase.

[0044] As a result, in nitrogen-excess conditions, there is a sequence of events leading to inhibition of NifA activity, resulting in the transcriptional silencing of the n / cluster.

[0045] This is consistent with observations that the application of chemical fertilizers and residual nitrogen levels in field soils signal the shut down the biochemical pathway for nitrogen fixation.

[0046] In nitrogen-limiting conditions, free NifA can activate the transcription of nitrogenase gene.

[0047] In Figure 1, the present inventors demonstrate that isolates BC01, BC03, and BC05 are nitrogen fixing. Figures 2, 3 and 4 demonstrate the isolates all contain nitrogen fixing genes. Surprisingly, the present inventors have demonstrated in Example 2 and Figure 8 that the isolates described herein can increase biomass in the presence of high levels of nitrogen.

[0048] The present invention is based in part on the characterisation of nitrogen fixing bacteria that are able to increase germination of seed, increase plant biomass, and persist in plant roots over time. The isolates described herein include two Phytobacter sp and one Kosakonia sp that were isolated from the seeds of warm season grasses Cenchrus clandestinum (Kikuyu grass) and Paspalum dilatum (Da His grass) respectively.

[0049] In one aspect the present invention provides a nitrogen fixing bacteria as described herein.

[0050] The isolates characterised in the Examples are not genetically engineered, and are regarded as wild-type isolates.

[0051] Phyotobacter isolate BC01 was isolated from Cenchrus clandestinum, and as shown in Figure 5, has multiple copies of the 16S rRNA gene encoded in its genome, with 16S rRNA sequences included below as SEQ ID NO: 1 to 7.

[0052] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 95% identical to any one of SEQ ID NO: I to 7.

[0053] As used herein "16S rDNA" or "16S rRNA gene sequence" refers to all or part of the gene encoding the 16S ribosomal RNA (16S rRNA).

[0054] SEQ ID NO: 1: from BC01_contig_l:2775307-2776843AGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCGAUACGGUUAAUAACCGUG UUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGG AGGGUG

[0055] SEQ ID NO: 2: from BC01_contig_l:2922706-2924242AGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCAAUGCGGUUAAUAACCGUG UUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGG AGGGUG

[0056] SEQ ID NO: 3: from BC01_contig_l:cl323651-1322117AGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCGAUACGGUUAAUAACCGUG UUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGG AGGGUG

[0057] SEQ ID NO: 4: from BC01_contig_l:2874724-2876258AGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCGAUACGGUUAAUAACCGUG UUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGG AGGGUG

[0058] SEQ ID NO: 5: from BC01_contig_l:c2064166-2062632AGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCAAUGCGGUUAAUAACCGUG UUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGG AGGGUG

[0059] SEQ ID NO: 6: from BC01_contig_l:2632097-2633631AGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCAAUGCGGUUAAUAACCGUG UUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGG AGGGUG

[0060] SEQ ID NO: 7: from BC01_contig_l:3603090-3604624AGAAGGCCUUCGGGUUGUAAAGUACUUUCAGCGGGGAGGAAGGCAAUGCGGUUAAUAACCGUG UUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGG AGGGUG

[0061] As is shown in Figure 5, the BC01 16S rRNA gene sequence sequences are at least 97.7% identical, over 98.5% identical, or 100% identical over the portion of the 16S rRNA gene sequence shown.

[0062] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 96% identical to any one of SEQ ID NO: I to 7.

[0063] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 97% identical to any one of SEQ ID NO: I to 7.

[0064] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 98% identical to any one of SEQ ID NO: I to 7.

[0065] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 99% identical to any one of SEQ ID NO: I to 7.

[0066] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is 100% identical to any one of SEQ ID NO: I to 7.

[0067] In another embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacterial isolate comprises 16S rRNA sequence 100% identical to each of SEQ ID NO: I to 7.

[0068] A substantially pure culture of Phyotobacter isolate BC01 was deposited on 18 June2024 with the National Measurement Institute, located at 1 / 153 Bertie Street, Port Melbourne, Victoria, Australia 3207, and assigned accession number V24 / 010671. The microbial deposits of the present disclosure were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure (Budapest Treaty).

[0069] Phyotobacter isolate BC03 was isolated from Cenchrus clandestinum, and as shown in Figure 5, has multiple copies of the 16S rRNA gene encoded in its genome, with 16S rRNA sequences included below as SEQ ID NO: 8 to 14.

[0070] SEQ ID NO: 8: from BC03_contig_l:4638455-4639991AGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCGACACGGUUAAUAACCGUG UUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGG AGGGUG

[0071] SEQ ID NO: 9: from BC03_contig_l:5297908-5299444AGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCAAUGCGGUUAAUAACCGUG UUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGG AGGGUG

[0072] SEQ ID NO: 10: from BC03_contig_l:4490768-4492304AGAAGGCCUUCGGGUUGUAAAGUACUUUCAGCGGGGAGGAAGGCAAUGCGGUUAAUAACCGUG UUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGG AGGGUG

[0073] SEQ ID NO: 11: from BC03_contig_l:4590346-4591880AGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCGAUACGGUUAAUAACCGUG UUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGG AGGGUG

[0074] SEQ ID NO: 12: from BC03_contig_l:c3040482-3038948AGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCAAUGCGGUUAAUAACCGUG UUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGG AGGGUG

[0075] SEQ ID NO: 13: from BC03_contig_l:c3781938-3780404AGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCAAUGCGGUUAAUAACCGUG UUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGG AGGGUG

[0076] SEQ ID NO: 14: from BC03_contig_l:4349429-4350963AGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCAAUGCGGUUAAUAACCGUG UUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGG AGGGUG

[0077] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 95% identical to any one of SEQ ID NO: 8 to 14.

[0078] As is shown in Figure 5, the BC03 16s rRNA sequences are at least 97.7% identical, at least 98.5% identical, at least 99.2% identical, or 100% identical over the portion of the 16S rRNA gene sequence shown.

[0079] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 96% identical to any one of SEQ ID NO: 8 to 14.

[0080] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 97% identical to any one of SEQ ID NO: 8 to 14.

[0081] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 98% identical to any one of SEQ ID NO: 8 to 14.

[0082] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 99% identical to any one of SEQ ID NO: 8 to 14.

[0083] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is 100% identical to any one of SEQ ID NO: 8 to 14.

[0084] In another embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacterial isolate comprises 16S rRNA sequence 100% identical to each of SEQ ID NO: 8 to 14.

[0085] A substantially pure culture of Phyotobacter isolate BC03 was deposited on 18 June 2024 with the National Measurement Institute, located at 1 / 153 Bertie Street, Port Melbourne, Victoria, Australia 3207, and assigned accession number V24 / 010672.

[0086] Kosakonia isolate BC05 was isolated from Paspalum dilatatum, and as shown in Figure 5, has multiple copies of the 16S rRNA gene encoded in its genome, with 16S rRNA sequence included below as SEQ ID NO: 15 to 21.

[0087] SEQ ID NO: 15: from BC05_contig_l:c3016259-3014725AGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCAGUACGGUUAAUAACCGUGCUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGG AGGGUG

[0088] SEQ ID NO: 16: from BC05_contig_l 09-1843AGAAGGCCUUUUCGGGUUUUGUUAAAGCACUUUUUUCAGCGGGGAGGAAGGCGGUUACGGUUUUAAUUAACCGUUGCUUGAUUUUGACGUUUUACCCGCAGAAGAAGCACCGGCUUAACUUCCGU UGCCAGCAGCCGCGGUUAAUUACGGAGGGUUG

[0089] SEQ ID NO: 17: from BC05_contig_l:c3861974-3860440AGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCGGUACGGUUAAUAACCGUGCCGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGG AGGGUG

[0090] SEQ ID NO: 18: from BC05_contig_l:4409811-4411345AGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCGGUACGGUUAAUAACCGUGCCGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGG AGGGUG

[0091] SEQ ID NO: 19: from BC05_contig_l:4548057-4549591AGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCGGUACGGUUAAUAACCGUGCCGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGG AGGGUG

[0092] SEQ ID NO: 20: from BC05_contig_l:4640776-4642310AGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCGGUACGGUUAAUAACCGUGCCGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUG

[0093] SEQ ID NO: 21: from BC05_contig_l:4681131-4682665AGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCGGUACGGUUAAUAACCGUG CCGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGG AGGGUG

[0094] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 95% identical to any one of SEQ ID NO: 15 to 21.

[0095] As is shown in Figure 5, the BC05 16s rRNA sequences are at least 98.5% identical, at least 99.2% identical, or 100% identical over the portion of the 16S rRNA gene sequence shown.

[0096] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 96% identical to any one of SEQ ID NO: 15 to 21.

[0097] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 97% identical to any one of SEQ ID NO: 15 to 21.

[0098] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 98% identical to any one of SEQ ID NO: 15 to 21.

[0099] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 99% identical to any one of SEQ ID NO: 15 to 21.

[0100] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is 100% identical to any one of SEQID NO: 15 to 21.

[0101] In another embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacterial isolate comprises 16S rRNA sequence 100% identical to each of SEQ IDNO: 15 to 21.

[0102] A substantially pure culture of Kosakonia isolate BC05 was deposited on 18 June 2024 with the National Measurement Institute, located at 1 / 153 Bertie Street, Port Melbourne, Victoria, Australia 3207, and assigned accession number V24 / 010673. It should be noted that Enterobacter has now been reclassified into several genera including Kosakonia.

[0103] As is shown in Figure 5, the BC01, BC03 and BC05 16s rRNA gene sequences are all at least 94.7% identical to each other, with 7 non-identical nucleotides in 133 nucleotides.

[0104] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 94.7% identical to any one of SEQ ID NO: I to 21.

[0105] The Phyotobacter isolate BC01 16S rRNA gene sequences are included below as SEQ ID NO: 22 to 28.

[0106] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 99.5% identical to any one of SEQ ID NO: 22 to 28.

[0107] SEQ ID NO: 22: BC01_contig_l:2775307-2776843UGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAAC GGUAGCACAGAGGAGCUUGCUCCUCGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGA AACUGCCCGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAAUGUCGCAAGAC CAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCGGAUGUGCCCAGAUGGGAUUAGCUAGUUGGUGAGGUAAUGGCUCACCAAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGA ACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAA GCCUGAUGCAGCCAUGCCGCGUGUGUGAAGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGG GAGGAAGGCGAUACGGUUAAUAACCGUGUUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGAAACUGGCAGGCUUGAGUCUUGUAGAGGGGGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCCCCUGGACAAAGACUGACGCUCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGUCGACUUGGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAGUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACGGAAUUCGGCAGAGAUGCCUUAGUGCCUUCGGGAACCGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGGCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCAUAUACAAAGAGAAGCACCUCGCGAGAGCAAGCGGACCUCAUAAAGUAUGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUGGAUCAGAAUGCCACGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUU

[0108] SEQ ID NO: 23: BC01_contig_l:2922706-2924242UGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAACGGUAGCACAGAGGAGCUUGCUCCUCGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGAAACUGCCCGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAAUGUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCGGAUGUGCCCAGAUGGGAUUAGCUUGUUGGUGAGGUAAUGGCUCACCAAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUGUGAAGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCAAUGCGGUUAAUAACCGUGUUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGAAACUGGCAGGCUUGAGUCUUGUAGAGGGGGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCCCCUGGACAAAGACUGACGCUCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGUCGACUUGGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAGUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACGGAAUUUGGCAGAGAUGCCUUAGUGCCUUCGGGAGCCGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGGCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCAUAUACAAAGAGAAGCGACCUCGCGAGAGCAAGCGGACCUCAUAAAGUAUGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUGGAUCAGAAUGCCACGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUU

[0109] SEQ ID NO: 24; BC01_contig_l:cl323651-1322117UGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAACGGUAGCACAGAGAGCUUGCUCUCGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGAAACUGCCCGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAAUGUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCGGAUGUGCCCAGAUGGGAUUAGCUAGUUGGUGAGGUAAUGGCUCACCAAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUGUGAAGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCGAUACGGUUAAUAACCGUGUUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGAAACUGGCAGGCUUGAGUCUUGUAGAGGGGGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCCCCUGGACAAAGACUGACGCUCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGUCGACUUGGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAGUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACGGAAUUUGGCAGAGAUGCCUUAGUGCCUUCGGGAGCCGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGGCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCAUAUACAAAGAGAAGCGACCUCGCGAGAGCAAGCGGACCUCAUAAAGUAUGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUGGAUCAGAAUGCCACGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUU

[0110] SEQ ID NO: 25: BC01_contig_l:2874724-2876258UGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAACGGUAGCACAGAGAGCUUGCUCUCGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGAAACUGCCCGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAAUGUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCGGAUGUGCCCAGAUGGGAUUAGCUAGUUGGUGAGGUAAUGGCUCACCAAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUGUGAAGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCGAUACGGUUAAUAACCGUGUUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGAAACUGGCAGGCUUGAGUCUUGUAGAGGGGGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCCCCUGGACAAAGACUGACGCUCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGUCGACUUGGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAGUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACGGAAUUCGGCAGAGAUGCCUUAGUGCCUUCGGGAACCGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGGCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCAUAUACAAAGAGAAGCGACCUCGCGAGAGCAAGCGGACCUCAUAAAGUAUGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUGGAUCAGAAUGCCACGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUU

[0111] SEQ ID NO: 26: BC01_contig_l:c2064166-2062632UGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAACGGUAGCACAGAGAGCUUGCUCUCGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGAAACUGCCCGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAAUGUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCGGAUGUGCCCAGAUGGGAUUAGCUUGUUGGUGAGGUAAUGGCUCACCAAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUGUGAAGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCAAUGCGGUUAAUAACCGUGUUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGAAACUGGCAGGCUUGAGUCUUGUAGAGGGGGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCCCCUGGACAAAGACUGACGCUCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGUCGACUUGGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAGUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACGGAAUUUGGCAGAGAUGCCUUAGUGCCUUCGGGAACCGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGGCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCAUAUACAAAGAGAAGCGACCUCGCGAGAGCAAGCGGACCUCAUAAAGUAUGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUGGAUCAGAAUGCCACGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUU

[0112] SEQ ID NO: 27; BC01_contig_l:2632097-2633631UGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAACGGUAGCACAGAGAGCUUGCUCUCGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGAAACUGCCCGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAAUGUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCGGAUGUGCCCAGAUGGGAUUAGCUUGUUGGUGAGGUAAUGGCUCACCAAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUGUGAAGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCAAUGCGGUUAAUAACCGUGUUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGAAACUGGCAGGCUUGAGUCUUGUAGAGGGGGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCCCCUGGACAAAGACUGACGCUCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGUCGACUUGGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAGUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACGGAAUUUGGCAGAGAUGCCUUAGUGCCUUCGGGAACCGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGGCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCAUAUACAAAGAGAAGCGACCUCGCGAGAGCAAGCGGACCUCAUAAAGUAUGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUGGAUCAGAAUGCCACGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUU

[0113] SEQ ID NO: 28: BC01_contig_l:3603090-3604624UGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAACGGUAGCACAGAGAGCUUGCUCUCGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGAAACUGCCCGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAAUGUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCGGAUGUGCCCAGAUGGGAUUAGCUUGUUGGUGAGGUAAUGGCUCACCAAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUAUGAAGAAGGCCUUCGGGUUGUAAAGUACUUUCAGCGGGGAGGAAGGCAAUGCGGUUAAUAACCGUGUUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGAAACUGGCAGGCUUGAGUCUUGUAGAGGGGGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCCCCUGGACAAAGACUGACGCUCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGUCGACUUGGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAGUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACGGAAUUCGGCAGAGAUGCCUUAGUGCCUUCGGGAACCGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGGCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCAUAUACAAAGAGAAGCGACCUCGCGAGAGCAAGCGGACCUCAUAAAGUAUGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUGGAUCAGAAUGCCACGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUU

[0114] The BC01 16S rRNA gene sequence sequences (SEQ IS NO: 22 to 28) are at least 99.5% identical, at least 99.6%% identical, at least 99.7% identical, or at least 99.9% identical.

[0115] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 99.5% identical to any one of SEQ ID NO: 22 to 28.

[0116] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 99.6% identical to any one of SEQ ID NO: 22 to 28.

[0117] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 99.7% identical to any one of SEQ ID NO: 22 to 28.

[0118] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 99.9% identical to any one of SEQ ID NO: 22 to 28.

[0119] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is 100% identical to any one of SEQ ID NO: 22 to 28.

[0120] In another embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacterial isolate comprises 16S rRNA sequence 100% identical to each of SEQ ID of SEQ ID NO: 22 to 28.

[0121] Phyotobacter isolate BC03 16S rRNA sequences are included below as SEQ ID NO: 29 to 35.

[0122] SEQ ID NO: 29: BC03_contig_l:4638455-4639991UGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAAC GGUAGCACAGAGGAGCUUGCUCCUCGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGA AACUGCCCGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAAUGUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCGGAUGUGCCCAGAUGGGAUUAGCUAGUUGGUGAGGUAAUGGCUCACCAAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUGUGAAGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCGACACGGUUAAUAACCGUGUUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGAAACUGGCAGGCUUGAGUCUUGUAGAGGGGGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCCCCUGGACAAAGACUGACGCUCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGUCGACUUGGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAGUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACGGAAUUUGGCAGAGAUGCCUUAGUGCCUUCGGGAGCCGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGGCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCAUAUACAAAGAGAAGCGACCUCGCGAGAGCAAGCGGACCUCAUAAAGUAUGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUGGAUCAGAAUGCCACGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUU

[0123] SEQ ID NO: 30: BC03_contig_l:5297908-5299444UGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAACGGUAGCACAGAGGAGCUUGCUCCUCGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGAAACUGCCCGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAAUGUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCGGAUGUGCCCAGAUGGGAUUAGCUAGUUGGUGAGGUAAUGGCUCACCAAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUGUGAAGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCAAUGCGGUUAAUAACCGUGUUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGAAACUGGCAGGCUUGAGUCUUGUAGAGGGGGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCCCCUGGACAAAGACUGACGCUCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGUCGACUUGGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAGUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACGGAAUUUGGCAGAGAUGCCUUAGUGCCUUCGGGAACCGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGGCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCAUAUACAAAGAGAAGCGACCUCGCGAGAGCAAGCGGACCUCAUAAAGUAUGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUGGAUCAGAAUGCCACGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUU

[0124] SEQ ID NO: 31: BC03_contig_l:4490768-4492304UGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAACGGUAGCACAGAGGAGCUUGCUCCUUGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGAAACUGCCCGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAAUGUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCGGAUGUGCCCAGAUGGGAUUAGCUUGUUGGUGAGGUAAUGGCUCACCAAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUAUGAAGAAGGCCUUCGGGUUGUAAAGUACUUUCAGCGGGGAGGAAGGCAAUGCGGUUAAUAACCGUGUUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGAAACUGGCAGGCUUGAGUCUUGUAGAGGGGGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCCCCUGGACAAAGACUGACGCUCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGUCGACUUGGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAGUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACGGAAUUUGGCAGAGAUGCCUUAGUGCCUUCGGGAGCCGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGGCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCAUAUACAAAGAGAAGCGACCUCGCGAGAGCAAGCGGACCUCAUAAAGUAUGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUGGAUCAGAAUGCCACGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUU

[0125] SEQ ID NO: 32: BC03_contig_l:4590346-4591880TGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAACGGUAGCACAGAGAGCUUGCUCUCGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGAAACUGCCCGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAAUGUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCGGAUGUGCCCAGAUGGGAUUAGCUAGUUGGUGAGGUAAUGGCUCACCAAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUGUGAAGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCGAUACGGUUAAUAACCGUGUUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGAAACUGGCAGGCUUGAGUCUUGUAGAGGGGGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCCCCUGGACAAAGACUGACGCUCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGUCGACUUGGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAGUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACGGAAUUUGGCAGAGAUGCCUUAGUGCCUUCGGGAGCCGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGGCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCAUAUACAAAGAGAAGCGACCUCGCGAGAGCAAGCGGACCUCAUAAAGUAUGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUGGAUCAGAAUGCCACGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUU

[0126] SEQ ID NO: 33: BC03_contig_l:c3040482-3038948UGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAACGGUAGCACAGAGAGCUUGCUCUCGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGAAACUGCCCGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAAUGUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCGGAUGUGCCCAGAUGGGAUUAGCUUGUUGGUGAGGUAAUGGCUCACCAAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUGUGAAGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCAAUGCGGUUAAUAACCGUGUUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGAAACUGGCAGGCUUGAGUCUUGUAGAGGGGGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCCCCUGGACAAAGACUGACGCUCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGUCGACUUGGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAGUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACGGAAUUUGGCAGAGAUGCCUUAGUGCCUUCGGGAACCGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGGCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCAUAUACAAAGAGAAGCGACCUCGCGAGAGCAAGCGGACCUCAUAAAGUAUGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUGGAUCAGAAUGCCACGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUU

[0127] SEQ ID NO: 34: BC03_contig_l:c3781938-3780404UGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAACGGUAGCACAGAGAGCUUGCUCUCGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGAAACUGCCCGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAAUGUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCGGAUGUGCCCAGAUGGGAUUAGCUUGUUGGUGAGGUAAUGGCUCACCAAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUGUGAAGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCAAUGCGGUUAAUAACCGUGUUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGAAACUGGCAGGCUUGAGUCUUGUAGAGGGGGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCCCCUGGACAAAGACUGACGCUCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGUCGACUUGGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAGUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACGGAAUUUGGCAGAGAUGCCUUAGUGCCUUCGGGAACCGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGGCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCAUAUACAAAGAGAAGCGACCUCGCGAGAGCAAGCGGACCUCAUAAAGUAUGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUGGAUCAGAAUGCCACGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUU

[0128] SEQ ID NO: 35: BC03_contig_l:4349429-4350963UGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAACGGUAGCACAGAGAGCUUGCUCUCGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGAAACUGCCCGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAAUGUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCGGAUGUGCCCAGAUGGGAUUAGCUUGUUGGUGAGGUAAUGGCUCACCAAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUGUGAAGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCAAUGCGGUUAAUAACCGUGUUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGAAACUGGCAGGCUUGAGUCUUGUAGAGGGGGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCCCCUGGACAAAGACUGACGCUCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGUCGACUUGGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAGUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACGGAAUUUGGCAGAGAUGCCUUAGUGCCUUCGGGAGCCGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGGCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCAUAUACAAAGAGAAGCGACCUCGCGAGAGCAAGCGGACCUCAUAAAGUAUGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUGGAUCAGAAUGCCACGGUGAAUACGUUCCCGGGCCUUGUA CACACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAG GGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAAC CUGCGGUUGGAUCACCUCCUU

[0129] The BC03 16s rRNA sequences (SEQ IS NO: 29 to 35) are at least 99.5% identical, at least 99.6%% identical, at least 99.7% identical, at least 99.8% identical, or at least 99.9% identical.

[0130] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 99.5% identical to any one of SEQ ID NO: 29 to 35.

[0131] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 99.6% identical to any one of SEQ ID NO: 29 to 35.

[0132] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 99.7% identical to any one of SEQ ID NO: 29 to 35.

[0133] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 99.8% identical to any one of SEQ ID NO: 29 to 35.

[0134] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 99.9% identical to any one of SEQ ID NO: 29 to 35.

[0135] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is 100% identical to any one of of SEQ ID NO: 29 to 35.

[0136] In another embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacterial isolate comprises 16S rRNA sequence 100% identical to each of SEQ ID NO: 29 to 35.

[0137] Kosakonia isolate BC05 w 16S rRNA sequences are included below as SEQ ID NO: 36 to 42.

[0138] SEQ ID NO: 36: BC05_contig_l:c3016259-3014725UGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAAC GGUAGCACAGAGAGCUUGCUCUCGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGAAA CUGCCUGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAACGUCGCAAGACCA AAGAGGGGGACCUUCGGGCCUCUUGCCAUCAGAUGUGCCCAGAUGGGAUUAGCUGGUAGGCGG GGUAAUGGCCCACCUAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAAC UGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCC UGAUGCAGCCAUGCCGCGUGUGUGAAGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAG GAAGGCAGUACGGUUAAUAACCGUGCUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUC CGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGC GCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGA AACUGGCAGGCUGGAGUCUCGUAGAGGGAGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUA GAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCUCCUGGACGAAGACUGACGCUCAGGUGCG AAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCUGUAAACGAUGUCGAUUU GGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAAUCGACCGCCUGGGGAGU ACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGG UUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACAGAACUUGCCAGAGAUG GCUUGGUGCCUUCGGGAACUGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGA AAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGG GAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUG GCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCGCAUACAAAGAGAAGCGACCUCGCGAG AGCAAGCGGACCUCAUAAAGUGCGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAA GUCGGAAUCGCUAGUAAUCGUGAAUCAGAAUGUCACGGUGAAUACGUUCCCGGGCCUUGUACA CACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUU

[0139] SEQ ID NO: 37: BC05_contig_l:309-1843UGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAACGGUAGCACAGAGAGCUUGCUCUCGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGAAACUGCCUGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAACGUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCAGAUGUGCCCAGAUGGGAUUAGCUGGUAGGCGGGGUAACGGCCCACCUAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUGUGAAGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCGGUACGGUUAAUAACCGUGCUGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGAAACUGGCAGGCUGGAGUCUCGUAGAGGGAGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCUCCUGGACGAAGACUGACGCUCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCUGUAAACGAUGUCGAUUUGGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAAUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACAGAACUUUCCAGAGAUGGAUUGGUGCCUUCGGGAACUGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGGCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCGCAUACAAAGAGAAGCGACCUCGCGAGAGCAAGCGGACCUCAUAAAGUGCGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUGAAUCAGAAUGUCACGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUU

[0140] SEQ ID NO: 38: BC05_contig_l:c3861974-3860440UGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAACGGUAGCACAGAGAGCUUGCUCUCGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGAAACUGCCUGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAACGUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCAGAUGUGCCCAGAUGGGAUUAGCUGGUAGGCGGGGUAAUGGCCCACCUAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUGUGAAGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCGGUACGGUUAAUAACCGUGCCGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGAAACUGGCAGGCUGGAGUCUCGUAGAGGGAGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCUCCUGGACGAAGACUGACGCUCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCUGUAAACGAUGUCGAUUUGGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAAUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACAGAACUUUCCAGAGAUGGAUUGGUGCCUUCGGGAACUGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGGCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCGCAUACAAAGAGAAGCGACCUCGCGAGAGCAAGCGGACCUCAUAAAGUGCGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUGAAUCAGAAUGUCACGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUU

[0141] SEQ ID NO: 39: BC05_contig_l:4409811-4411345UGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAACGGUAGCACAGAGAGCUUGCUCUCGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGAAACUGCCUGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAACGUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCAGAUGUGCCCAGAUGGGAUUAGCUGGUAGGCGGGGUAAUGGCCCACCUAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUGUGAAGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCGGUACGGUUAAUAACCGUGCCGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGAAACUGGCAGGCUGGAGUCUCGUAGAGGGAGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCUCCUGGACGAAGACUGACGCUCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCUGUAAACGAUGUCGAUUUGGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAAUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACAGAACUUUCCAGAGAUGGAUUGGUGCCUUCGGGAACUGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGGCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCGCAUACAAAGAGAAGCGACCUCGCGAGAGCAAGCGGACCUCAUAAAGUGCGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUGAAUCAGAAUGUCACGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUU

[0142] SEQ ID NO: 40: BC05_contig_l:4548057-4549591UGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAACGGUAGCACAGAGAGCUUGCUCUCGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGAAACUGCCUGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAACGUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCAGAUGUGCCCAGAUGGGAUUAGCUGGUAGGCGGGGUAAUGGCCCACCUAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUGUGAAGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCGGUACGGUUAAUAACCGUGCCGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGAAACUGGCAGGCUGGAGUCUCGUAGAGGGAGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCUCCUGGACGAAGACUGACGCUCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCUGUAAACGAUGUCGAUUUGGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAAUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACAGAACUUUCCAGAGAUGGAUUGGUGCCUUCGGGAACUGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGGCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCGCAUACAAAGAGAAGCGACCUCGCGAGAGCAAGCGGACCUCAUAAAGUGCGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUGAAUCAGAAUGUCACGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUU

[0143] SEQ ID NO: 41: BC05_contig_l:4640776-4642310UGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAACGGUAGCACAGAGAGCUUGCUCUCGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGAAACUGCCUGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAACGUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCAGAUGUGCCCAGAUGGGAUUAGCUGGUAGGCGGGGUAAUGGCCCACCUAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUGUGAAGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCGGUACGGUUAAUAACCGUGCCGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGAAACUGGCAGGCUGGAGUCUCGUAGAGGGAGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCUCCUGGACGAAGACUGACGCUCAGGUGCGAAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCUGUAAACGAUGUCGAUUUGGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAAUCGACCGCCUGGGGAGUACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACAGAACUUUCCAGAGAUGGAUUGGUGCCUUCGGGAACUGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGAAAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGGGAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGGCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCGCAUACAAAGAGAAGCGACCUCGCGAGAGCAAGCGGACCUCAUAAAGUGCGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAAGUCGGAAUCGCUAGUAAUCGUGAAUCAGAAUGUCACGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAGGGCGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAACCUGCGGUUGGAUCACCUCCUU

[0144] SEQ ID NO: 42: BC05_contig_l:4681131-4682665UGAAGAGUUUGAUCAUGGCUCAGAUUGAACGCUGGCGGCAGGCCUAACACAUGCAAGUCGAACGGUAGCACAGAGAGCUUGCUCUCGGGUGACGAGUGGCGGACGGGUGAGUAAUGUCUGGGAAACUGCCUGAUGGAGGGGGAUAACUACUGGAAACGGUAGCUAAUACCGCAUAACGUCGCAAGACCAAAGAGGGGGACCUUCGGGCCUCUUGCCAUCAGAUGUGCCCAGAUGGGAUUAGCUGGUAGGCGGGGUAAUGGCCCACCUAGGCGACGAUCCCUAGCUGGUCUGAGAGGAUGACCAGCCACACUGGAACUGAGACACGGUCCAGACUCCUACGGGAGGCAGCAGUGGGGAAUAUUGCACAAUGGGCGCAAGCCUGAUGCAGCCAUGCCGCGUGUGUGAAGAAGGCCUUCGGGUUGUAAAGCACUUUCAGCGGGGAGGAAGGCGGUACGGUUAAUAACCGUGCCGAUUGACGUUACCCGCAGAAGAAGCACCGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGAGGGUGCAAGCGUUAAUCGGAAUUACUGGGCGUAAAGCGCACGCAGGCGGUCUGUCAAGUCGGAUGUGAAAUCCCCGGGCUCAACCUGGGAACUGCAUUCGAAACUGGCAGGCUGGAGUCUCGUAGAGGGAGGUAGAAUUCCAGGUGUAGCGGUGAAAUGCGUAGAGAUCUGGAGGAAUACCGGUGGCGAAGGCGGCCUCCUGGACGAAGACUGACGCUCAGGUGCG AAAGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCUGUAAACGAUGUCGAUUU GGAGGUUGUGCCCUUGAGGCGUGGCUUCCGGAGCUAACGCGUUAAAUCGACCGCCUGGGGAGU ACGGCCGCAAGGUUAAAACUCAAAUGAAUUGACGGGGGCCCGCACAAGCGGUGGAGCAUGUGG UUUAAUUCGAUGCAACGCGAAGAACCUUACCUGGUCUUGACAUCCACAGAACUUUCCAGAGAUG GAUUGGUGCCUUCGGGAACUGUGAGACAGGUGCUGCAUGGCUGUCGUCAGCUCGUGUUGUGA AAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUAUCCUUUGUUGCCAGCGGUCCGGCCGG GAACUCAAAGGAGACUGCCAGUGAUAAACUGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUG GCCCUUACGACCAGGGCUACACACGUGCUACAAUGGCGCAUACAAAGAGAAGCGACCUCGCGAG AGCAAGCGGACCUCAUAAAGUGCGUCGUAGUCCGGAUUGGAGUCUGCAACUCGACUCCAUGAA GUCGGAAUCGCUAGUAAUCGUGAAUCAGAAUGUCACGGUGAAUACGUUCCCGGGCCUUGUACA CACCGCCCGUCACACCAUGGGAGUGGGUUGCAAAAGAAGUAGGUAGCUUAACCUUCGGGAGGG CGCUUACCACUUUGUGAUUCAUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGAACCU GCGGUUGGAUCACCUCCUU

[0145] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 95% identical to any one of SEQ ID NO: 36 to 42.

[0146] The BC03 16s rRNA sequences (SEQ IS NO: 36 to 42) are at least 99.7% identical.

[0147] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 99.7% identical to any one of SEQ ID NO: 36 to 42.

[0148] In another embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacterial isolate comprises 16S rRNA sequence 100% identical to each of SEQ ID NO: 36 to 42.

[0149] The BC01, BC03 and BC05 16s rRNA gene sequences (SEQ ID NO:22 to 42) are at least 97.6 % identical to each other.

[0150] In one embodiment, the present invention provides a nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 97.6% identical to any one of SEQ ID NO: 22 to 42.

[0151] As used herein the term 'nitrogen fixation' refers to combining molecular dinitrogen (N2) with hydrogen in planta. Diazotrophs (i.e., bacteria and archaea that fix atmospheric nitrogen gas) have evolved sophisticated and tight regulation of a nif gene cluster in response to environmental oxygen and available nitrogen, nif genes encode enzymes involved in nitrogen fixation, such as the nitrogenase complex, and proteins that regulate nitrogen fixation.

[0152] Figure 4 shows an insertion of a transposon in between a hypothetical protein and nifM. Without wishing to be bound by theory, the presence of the transposon may alter nif gene expression in isolate BC01.

[0153] In some embodiments, the present invention provides isolated and substantially pure bacteria with applications in agriculture. The disclosed microorganisms can be utilized in their isolated and substantially pure states, as well as being formulated into compositions, as is discussed below. Furthermore, the present invention provides compositions containing at least one isolated and substantially pure bacteria as described herein, as well as methods of utilizing said bacterial compositions. Furthermore, the disclosure provides for methods of modulating nitrogen fixation in plants via the utilization of the disclosed isolated and substantially pure bacteria, or compositions, as described herein.

[0154] In some embodiments, the present invention provides a strain, child, mutant, or derivative, of a bacteria as described herein.

[0155] In some embodiments, the present invention provides a substantially purified or isolated bacteria of a bacteria as described herein.

[0156] As used herein, "isolated" "isolate," "isolated bacteria" refers to the bacteria having been separated from at least one of the materials with which it is associated in aparticular environment (for example soil, water, plant tissue, etc.). The term strain is used interchangeably with "isolate".

[0157] By "substantially pure" is meant that the nitrogen fixing bacteria is free of other organisms. The term includes, for example, a N-fixing bacteria in axenic culture. Preferably, the N-fixing bacteria is at least approximately 90% pure, more preferably at least approximately 95% pure, even more preferably at least approximately 98% pure, even more preferably at least approximately 99% pure.

[0158] As used herein, "in planta" may refer to in the plant, on the plant, or intimately associated with the plant, depending upon context of usage (e.g. endophytic, epiphytic, or rhizospheric associations). The plant may comprise plant parts, tissue, leaves, roots, root hairs, rhizomes, stems, seed, ovules, pollen, flowers, fruit, etc. As used herein the term "plant" can include plant parts, tissue, leaves, roots, root hairs, rhizomes, stems, seeds, ovules, pollen, flowers, fruit, etc.

[0159] In another aspect, the present invention provides a composition comprising one or more bacteria as described herein.

[0160] Compositions comprising bacteria or bacterial populations produced according to methods described herein and / or having characteristics as described herein can be in the form of a liquid or a solid product. For example, a composition comprising one or more bacteria as described herein may be in the form of a liquid suitable for spraying, in the form of a solid that could be mixed with soil, a dry powder, a slurry of powder and water, or a seed coating or treatment. The compositions comprising one or more bacteria as described herein may be coated on a surface of a seed, and may be in liquid form.

[0161] Compositions comprising one or more bacteria as described herein may be formulated separately using one bacteria as described herein and combined, and / or formulated together.

[0162] In another embodiment, the composition further comprises an agriculturally acceptable carrier.

[0163] As used herein, "carrier," "acceptable carrier," or "agriculturally acceptable carrier" includes a diluent, adjuvant, excipient, or vehicle with which the one or more bacteria as described herein can be administered, which does not detrimentally effect the one or more bacteria as described herein.

[0164] The carrier can be a solid carrier or liquid carrier, and in various forms including microspheres, powders, emulsions and the like. The carrier may be any one or more of a number of carriers that confer a variety of properties, such as increased stability, wettability, or dispersibility. Wetting agents such as natural or synthetic surfactants, which can be nonionic or ionic surfactants, or a combination thereof can be included in the composition. Water-in-oil emulsions can also be used to formulate a composition that includes the bacteria as described herein.

[0165] In one embodiment, the agriculturally acceptable carrier is selected from the group consisting of an adhesion agent, a microbial stabiliser and a fertiliser.

[0166] In one embodiment, the composition comprises an adhesion agent to help bind other active agents to a substance (e.g., a surface of a seed). Such agents are useful for combining bacteria with carriers that can contain other compounds to yield a coating composition. Coating compositions help create coatings around the plant or seed to maintain contact between the at least one bacteria and other agents with the plant or plant part.

[0167] In one embodiment, the composition comprises a microbial stabilizer to promote the ability of the bacteria to survive application on the plant or seeds, and to survive desiccation.

[0168] In one embodiment, the composition comprises a fertiliser, for example, to help promote the growth or provide nutrients to a seed, seedling, or plant.

[0169] In another embodiment, the agriculturally acceptable carrier is selected from the group consisting of a fungicide, a preservative, a stabilizer, a surfactant, an anti-complex agent, a pesticide, a herbicide, a nematicide, an insecticide, a plant growth regulator, a fertilizer, a dessicant, a nutrient, or any combination thereof.

[0170] In one embodiment, the agriculturally acceptable carrier may be soil, or a plant growth medium.

[0171] In liquid form, for example, solutions or suspensions, the one or more bacteria as described herein can be mixed or suspended in water or in aqueous solutions. Suitable liquid diluents or carriers include water, aqueous solutions, petroleum distillates, or other liquid carriers. In some embodiments, the liquid form may be or comprise fermentation broth that supports the growth of the microbe, such as Lysogeny broth or Nutrient broth. These broths may for example include nutrients as supplied by components such as tryptone (5-15 g / L), peptone (3-15 g / L) yeast extract (2-10 g / L), beef extract (2-10 g / L), sodium chloride (50 g / L for 5% to 200g / L for 20% and all amounts in between). The broths are aqueous and contain water making up to the required volume.

[0172] In solid form, solid compositions can be prepared by dispersing the one or more bacteria as described herein in and on an appropriately divided solid carrier, such as peat, wheat bran, vermiculite, clay, talc, bentonite, diatomaceous earth, fuller's earth, pasteurized soil, lime, biochar, chitosan, methyl cellulose, carboxymethylcellulose, gum arabic, polysaccharide Pelgel®, xanthan gum and alginate and the like. When such compositions are used as wettable powders, biologically compatible dispersing agents such as non-ionic, anionic, amphoteric, or cationic dispersing and emulsifying agents can be used.

[0173] The bacteria or compositions described herein can be coated onto a seed. Accordingly, in one embodiment, the present invention provides a composition as described herein, wherein the composition is in the form of a seed coating.

[0174] In one embodiment, the bacteria or compositions described herein may be coated onto a surface of a seed. In another embodiment, the bacteria or compositions described herein may be coated as a layer above a surface of a seed. In another embodiment, the bacteria or compositions described herein that is coated onto a seed may be in liquid form, in solid form, in a form of a slurry of powder and water, or in a flowable seed treatment. In one embodiment, the seed may be treated with an aqueous composition comprising alginate and the N-fixing bacteria followed by treatment with a complexing agent. For example, the N-fixingbacteria may be in a composition comprising sodium alginate and after coating the seed with sufficient composition, a complexing agent such as calcium chloride may be added so solidify the alginate polymer thereby coating the seed. The sodium alginate may be present in the composition at a concentration of 1 to about 10% w / volume in water, especially 3 to 5% w / v. The calcium chloride solution may be at any concentration suitable to result in polymerisation of the alginate, for example, 1-1000 mM, 20-500 mM or 50-300 mM. The thickness of the seed coating once solidified may be in the range of about 0.1 to about 5 mm, especially about 0.25 to about 1.5 mm. In some embodiments, a second coating may be applied to the seed, this outer coating not including the N-fixing bacteria. In another embodiment, the bacteria or compositions described herein may be applied to a seed and / or seedling by spraying, immersing, coating, encapsulating, and / or dusting the seed and / or seedling.

[0175] In another embodiment, the bacteria or compositions described herein may be coated onto a seedling.

[0176] In another embodiment, the present invention provides a bacteria or composition as described herein, wherein the composition is applied to the planting area, crop area, plant or part thereof. For example, the bacteria or composition may be sprayed on the plant aerial parts, or applied to the roots by inserting into furrows in which the plant seeds are planted, watering to the soil, or dipping the roots in a suspension of the bacteria or composition.

[0177] In another embodiment, the present invention provides a composition as described herein, wherein the composition is applied to the planting area, crop area, plant or part thereof, simultaneously or in succession, with other compounds. These compounds can be fertilizers, herbicides, and pesticides etc.

[0178] Importantly, the present inventors have demonstrated that the bacteria described herein can - when contacted with seeds - be used to increase germination of plants. The present inventors have also demonstrated that the bacteria described herein can be used to increase biomass of plants. The bacteria and compositions described herein therefore may also be used to improve plant traits.

[0179] Figure 7 shows that germination is increased under conditions of low nitrogen(O.lmM).

[0180] Figure 8 shows that root and shoot biomass is increased under conditions of high nitrogen (25mM). Without wishing to be bound by theory, in nitrogen excess, GHKL domain of NifL protein interacts with NifA protein to inhibit NifA. Gink can interact with NifL, enhancing the inhibition of NifA activity by NifL, resulting in the transcriptional silencing of n / cluster, and so it is surprising that the bacteria described herein can increase biomass under conditions of high nitrogen.

[0181] Examples of traits that may be improved include germination, germination at low nitrogen, root biomass, shoot biomass, root length, height, shoot length, leaf number, water use efficiency, overall biomass, yield, fruit size, grain size etc.

[0182] The traits, including germination, germination at low nitrogen, root biomass, shoot biomass, root length, height, shoot length, leaf number, water use efficiency, overall biomass, yield, fruit size, grain size etc, or any combination thereof, can be compared with reference (e.g. control) plants not contacted with a bacteria as described herein, or a composition as described herein grown under identical conditions.

[0183] In one embodiment, the present invention provides a method of increasing the amount of nitrogen fixation of a plant, comprising contacting the plant, a part of the plant, or soil into which the plant is planted with a bacteria as described herein, or a composition as described herein.

[0184] As used herein the term "contacting" includes any suitable method for bringing a bacteria or composition as described herein, in contact with the plant, a part of the plant, or soil into which the plant is planted. Such methods include, but are not limited to, dipping, dripping, spraying, root dipping, soil drenching, dabbing, painting, coating, blotting, fumigating, irrigating, atomizing, or soil injection. Contacting can refer to application to the plant, a part of the plant or seed directly. For example, a plant, a part of the plant or seed can be dipped or otherwise contacted with one or more bacteria or compositions. Alternatively, the contacting can be applied to the plant or seed indirectly. For example, a soil or otherplanting media can be contacted with one or more bacteria or products thereof, and the plant contacted with the soil or other planting media. In some embodiments, the plant, part of the plant or soil may be free of said bacteria as described herein before contact occurs.

[0185] In one embodiment, the present invention provides a method of improving a trait of a plant, comprising contacting the plant, a part of the plant, or soil into which the plant is planted with a bacteria as described herein, or a composition as described herein.

[0186] The traits improved can be compared with reference (e.g. control) plants not contacted with a bacteria as described herein, or a composition as described herein, grown under identical conditions.

[0187] In some embodiments, a plant resulting from the methods described herein exhibits an improvement in the trait that is at least about 5%, for example an improvement of at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 75%, at least about 80%, at least about 80%, at least about 90%, or at least 100%, at least about 200%, at least about 300%, at least about 400% or more, than a plants not contacted with a bacteria as described herein, or a composition as described herein, grown under identical conditions.

[0188] In additional examples, a plant resulting from the methods described herein exhibits a difference in the trait that is at least about 5% , for example at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 75%, at least about 80%, at least about 80%, at least about 90%, or at least 100%, at least about 200%, at least about 300%, at least about 400% or more, than a plant not contacted with a bacteria as described herein, or a composition as described herein, grown under identical conditions.

[0189] The traits improved can be compared with reference to the plant or plants prior to contact with a bacteria as described herein, or a composition as described herein.

[0190] In another embodiment, the present invention provides a method of increasing the amount of nitrogen in a plant, comprising contacting the plant, a part of the plant, or soil into which the plant is planted with a bacteria as described herein, or a composition as described herein.

[0191] Methods for measuring nitrogen fixation are known in the art, including using in vitro acetylene reduction assays (ARA).

[0192] In another embodiment, the present invention provides a method of increasing biomass in a plant, comprising contacting the plant, a part of the plant, or soil into which the plant is planted with a bacteria as described herein, or a composition as described herein.

[0193] As used herein, the term biomass refers to the mass of a plant (e.g. overall biomass), or the mass of a part of the plant (e.g. or and / or shoot biomass).

[0194] In another embodiment, the present invention provides a method of increasing germination of seed of a plant, comprising contacting the seed or soil into which the seed is planted with a bacteria as described herein, or a composition as described herein

[0195] In another embodiment, the present invention provides a method of providing biofertilizer to a plant, comprising contacting the plant, a part of the plant, or soil into which the plant is planted with a bacteria as described herein, or a composition as described herein

[0196] The present inventors have demonstrated that a bacteria as described herein, or a composition as described herein, isolated from grass plants (Paspalum dilatatum; Da His grass, and Cenchrus clandestinus; Kikuyu grass) are able to increase germination and biomass of barley. Figure 9 demonstrates that the bacteria of the present invention persist in plant material (roots) 6 weeks after germination.

[0197] Furthermore, the amount of bacteria present is dependent on the amount of nitrogen present in the growth environment, as is shown in Figure 9.

[0198] Accordingly, in one embodiment, the present invention provides a method as described herein, wherein the plant is a crop plant or a forage plant.

[0199] In one embodiment, the plant is a commercially important agricultural crop, for example, barley, sorghum, canola, tomato, strawberry, rice, maize, wheat, cereals, and oilseeds.

[0200] Accordingly, in one embodiment, the present invention provides a plant having improved biomass relative to a control plant, comprising a plant in association with a bacteria as described herein, or a composition as described herein, produced by contacting the plant with a bacteria as described herein, or a composition as described herein.

[0201] In another embodiment, the present invention also provides a plant, or part of a plant, derived from a method as described herein.

[0202] In another embodiment, the present invention also provides a seed of a plant coated with a bacteria as described herein, or a composition as described herein.ExamplesExample 1: Isolation of N-fixing bacteria.

[0203] 14 batches of seed from Cenchrus clandestinum and Paspalum dilatatum were ground and applied to liquid, semisolid (1.6 g / L of agar) and solid (15 g / L of agar) Burks nitrogen free media in order. Three batches of seed produced bacterial growth and single bacteria were isolated as separate colonies from this growth. The genomes of these bacteria were sequenced using Nanopore technology whole genome sequencing, as described in Li T et al. Sci Rep. 2021 Jul 30;ll(l):15545. In the bioinformatics pipeline, the tool Kracken2 assigned the closest species matches which indicated that two of the microbes were Phytobacter species (BC01 and BC03) and one was a Kosakonia species (BC05) as shown in Table 1.

[0204] Table 1 - Species identification of isolated bacteria

[0205] When grown on Burks nitrogen free media plates, the two Phytobacter isolates looked similar and the Kosakonia isolate was visually different (Figure 1).

[0206] The genomes of all three isolates included the complete colinear nitrogen fixing operon (nif Operon) with the genes being present in each genome in the same order (Figure 2). The percent protein identity also showed that these microbes were not identical, indicating that they comprised three different n / operons (Figure 3).

[0207] BC01 included a transposon insertion between NifM and a hypothetical protein not present in the same position in BC03 or BC05 (Figure 2). The position of the transposon insertion for BC01, in comparison to BC03, is shown in Table 2, and illustrated in comparison to Phytobacter diazotrophicus strain UAEU22 (GB id CP051548.1) in Figure 4.

[0208] Table 2 - Position of nif Operon genes in BC01 and BC03

[0209] Alignment with the n / fOperon of Phytobacter diazotrophicus strain UAEU22 (GB id CP051548.1) demonstrated that the BC01 transposon insertion was present in an intergenic region (Figure 4), so would not disrupt coding of the n / fOperon. Hypothetical and AAA ATPase genes seen in Phytobacter diazotrophicus are not present in BC01 and BC03 n / fOperons (Figure 4). The BC01 insertion sequence was analysed using BLASTX, which gave a 100% sequence identity match with transposase InsO family protein (GB id PXW44122.1) (data not shown).

[0210] 16S amplicon sequencing identified that each of BC01, BC03, and BC05 had seven copies of the 16S rRNA gene encoded in their genomes, which were non-identical (Figure 5). Alignment of 16S rRNA gene sequences was used to generate a phylogenetic tree using the maximum likelihood method (Figure 6).Example 2: In planta assay - germination under different nitrogen conditions

[0211] Barley seeds were inoculated in bacteria suspended in PBS at an OD 600 ~ 1 by shaking for 2 hours in the suspension. Control seeds were shaken for 2 hours in PBS. The liquid was decanted from the seed and the seeds were immediately planted in pots containing a mixture of fine perlite, coarse perlite and coarse vermiculite at a volumetric ratio of 2:2:1. The pots were then soaked in a nutrient solution containing 0.48 g / L of Magnesium Sulphate, 0.298 g / L of Potassium Chloride, 0.55 g / L of Calcium Chloride, 0.408 g / L of Potassium Dihydrogen Phosphate, 34.4 mg / L of Iron-EDTA, 1.26 mg / L of Manganese Chloride, 1.62 mg / L of Zinc Sulphate, 0.32 mg / L of Copper Sulphate, 3.09 mg / L of Dihydrogen Borate and 41.18 pg / L of Sodium Molybdate. Potassium nitrate was added to this mixture in the concentration of 0.1 mM, 1 mM, 10 mM, or 25 mM, to provide assess the effects of nitrogen concentration.

[0212] Seedling germination was counted after one week. The inventors found that the three isolates BC01, BC03 and BC05 all improved germination at low nitrogen treatment (0.1 mM potassium nitrate) compared to the no microbe control (grey) and the non-N-fixing control microbe, Pseudomonas sp (Figure 7A). In comparison, only BC05 maintained a slight advantage over the control at higher nitrogen levels (10 mM potassium nitrate, Figure 7B).

[0213] The germinants were allowed to grow for 6 weeks and were then destructively harvested. While there was no statistical difference in the lengths of roots and shoots betweenthe three microbe treatments and control (Figure 8A), root and shoot weight for BC03 and root weight for BC01 were statistically higher than control at 25 mM nitrogen (Figure 8B).

[0214] The roots of 6-week-old barley plants were isolated and the DNA was extracted and analysed for the microbes in their microbiomes through 16S rRNA sequences. The results are shown in Figure 9 where thick bars show portions of microbiome likely to be from the inoculants, where the proportion of inoculant is higher at lower nitrogen levels (0.1 mM) compared to the proportion at higher nitrogen levels (1 mM). The genus Phytobacter is part of the Enterobacteriaceae family and the amplicon sequence variant (ASV) of Enterobacteriaceae contains the same 16S rRNA sequence to the two Phytobacter isolates, so identification of Enterobacteriaceae was taken to indicate Phytobacter microbes. The 16S rRNA sequence of Enterobacteriaceae / Phytobacter were present in reasonably high abundance in both the BC01 and BC03 treated samples, while the Kosakonia species was present in even higher abundance in the BC05 treated sample which also contained the same 16S rRNA sequence as the Kosakonia isolate (Figure 5). These results show that BC01, BC03 and BC05 were present in the plant material at 6 weeks after germination.

Claims

The claims defining the invention are as follows:

1. A nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 94.7% identical to any one of SEQ ID NO: 1 to 21.

2. A nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 95% identical to any one of SEQ ID NO: 1 to 7.

3. A nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 95% identical to any one of SEQ ID NO: 8 to 14.

4. A nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 95% identical to any one of SEQ ID NO: 15 to 21.

5. A nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 97% identical to any one of SEQ ID NO: 1 to 7.

6. A nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 97% identical to any one of SEQ ID NO: 8 to 14.

7. A nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 97% identical to any one of SEQ ID NO: 15 to 21.

8. A nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is 100% identical to any one of SEQ ID NO: 1 to 7.

9. A nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is 100% identical to any one of SEQ ID NO: 8 to 14.

10. A nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is 100% identical to any one of SEQ ID NO: 15 to 21.

11. A nitrogen fixing bacteria wherein the bacteria comprises 16S rRNA sequences 100% identical to each of SEQ ID NO: 1 to 7.

12. A nitrogen fixing bacteria wherein the bacteria comprises 16S rRNA sequences 100% identical to each of SEQ ID NO: 8 to 14.

13. A nitrogen fixing bacteria wherein the bacteria comprises 16S rRNA sequences 100% identical to each of SEQ ID NO: 15 to 21.

14. A nitrogen fixing bacteria wherein the bacteria comprises a 16S rRNA sequence that is at least 97.6% identical to any one of SEQ ID NO: 22 to 42.

15. A Phytobacter bacterium represented by bacteria deposited under Accession Nos. V24 / 010671 and V24 / 010672 (BC01 and BC03 respectively) on 18 June 2024 with the National Measurement Institute, located at 1 / 153 Bertie Street, Port Melbourne, Victoria, Australia 3207.

16. A Kosakonia bacterium represented by bacteria deposited as under Accession No. V24 / 010673 (BC05) on 18 June 2024 with the National Measurement Institute, located at 1 / 153 Bertie Street, Port Melbourne, Victoria, Australia 3207.

17. A substantially purified or isolated bacteria according to any one of claims 1 to 16.

18. A composition comprising one or more bacteria according to any one of claims 1 to 17.

19. A composition according to claim 18, wherein the composition is in the form of a liquid or a solid.

20. A composition according to claim 18 or claim 19, wherein the composition further comprises an agriculturally acceptable carrier.

21. A composition according to claim 18 or claim 19, wherein the composition is in the form of a seed coating.

22. A method of increasing the amount of nitrogen fixation of a plant, comprising contacting the plant, a part of the plant, seed or soil into which the seed or plant is planted with a bacteria according to any one of claims 1 to 16, or a composition according to any one of claims 18 to 21.

23. A method of increasing biomass in a plant, comprising contacting the plant, a part of the plant, seed or soil into which the seed or plant is planted with a bacteria according to any one of claims 1 to 17, or a composition according to any one of claims 18 to 21.

24. A method of providing biofertilizer to a plant, comprising contacting the plant, a part of the plant, seed or soil into which the seed or plant is planted with a bacteria according to any one of claims 1 to 17, or a composition according to any one of claims 18 to 21.

25. A method of increasing germination of seed of a plant, comprising contacting the seed or soil into which the seed is planted with a bacteria according to any one of claims 1 to 17, or a composition according to any one of claims 18 to 21.

26. A method according to any one of claims 22 to 24, wherein the plant is a crop plant or a forage plant.

27. A plant having improved biomass relative to a control plant, comprising a plant in association with a bacteria according to any one of claims 1 to 17, or a composition according to any one of claims 18 to 21.

28. A plant, or part of a plant, derived from a method according to any one of claims 22 to 26.

29. Seed of a plant coated with a bacteria according to any one of claims 1 to 17, or a composition according to any one of claims 18 to 21.

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