Methods and compositions for engineering microorganisms for improved nitrogen fixation

Abstract

Methods and systems are provided for producing and utilizing bacterial compositions comprising at least one genetically engineered bacterial strain that fixes atmospheric nitrogen in agricultural systems that have been fertilized with more than 20 pounds of nitrogen per acre.

Description

[0001] Cross Reference

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 577,148, filed on October 25, 2017, which is incorporated herein by reference in its entirety.

[0003] Statement Regarding Federally Sponsored Research

[0004] This invention was made with U.S. Government support under SBIR Grant No. 1520545 awarded by the National Science Foundation. The Government has certain rights in the subject matter disclosed. Background Art

[0005] Plants are associated with microbiomes through a shared metabolome. The multidimensional relationship between specific crop traits and the underlying metabolome is characterized by a landscape with many local maxima. For a variety of reasons (such as for crop optimization), it may be desirable to optimize from a poor local maximum to another local maximum representing a better trait by changing the effect of the microbiome on the metabolome. Economically, environmentally and socially sustainable agricultural and food production methods are needed to meet the needs of a growing global population. The United Nations' Food and Agriculture Organization predicts that by 2050, total food production must increase by 70% to meet the needs of a growing population, a challenge that is exacerbated by many factors, including reduced freshwater resources, increased competition for arable land, rising energy prices, increased input costs, and the pressure that crops may need to adapt to a drier, hotter and more extreme global climate.

[0006] One area of ​​focus is the improvement of nitrogen fixation. 2 ) is a major component of the Earth's atmosphere. In addition, the element nitrogen (N) is an important component of many compounds that make up living organisms. However, many organisms cannot directly use N 2 To synthesize chemicals used in physiological processes such as growth and reproduction. 2 , N must be 2 Combine with hydrogen. Hydrogen and N 2The combination is called nitrogen fixation. Whether achieved chemically or biologically, nitrogen fixation requires a large energy investment. In biological systems, enzymes called nitrogenases catalyze the reaction that causes nitrogen fixation. An important goal of nitrogen fixation research is to extend this phenotype to non-leguminous plants, especially important agronomic grasses such as wheat, rice and corn. Although great progress has been made in understanding the establishment of nitrogen-fixing symbiosis between rhizobia and leguminous plants, the pathways for inducing nitrogen-fixing nodules on non-leguminous crops using this knowledge remain unclear. At the same time, as the demand for increased food production continues to increase, the challenge of providing adequate supplemental nitrogen sources (such as in fertilizers) will continue to increase. Summary of the invention

[0007] In some embodiments, the present disclosure provides a genetically engineered bacterium comprising an insertion sequence having at least about 80% sequence identity to a sequence selected from the group consisting of: SEQ ID No.1-72. In some cases, the insertion sequence replaces the natural promoter sequence. In some cases, the insertion sequence comprises at least about 85% sequence identity to a sequence selected from the group consisting of: SEQ ID No.1-72. In some cases, the insertion sequence comprises at least about 90% sequence identity to a sequence selected from the group consisting of: SEQ ID No.1-72. In some cases, the insertion sequence comprises at least about 95% sequence identity to a sequence selected from the group consisting of: SEQ ID No.1-72. In some cases, the insertion sequence comprises at least about 97% sequence identity to a sequence selected from the group consisting of: SEQ ID No.1-72. In some cases, the insertion sequence comprises at least about 98% sequence identity to a sequence selected from the group consisting of: SEQ ID No.1-72. In some cases, the insert sequence comprises at least about 99% sequence identity to a sequence selected from the group consisting of: SEQ ID Nos. 1-72.

[0008] In some embodiments, the present disclosure provides a genetically engineered bacterium comprising a natural coding sequence operably linked to an insertion sequence, the insertion sequence having at least 80% sequence identity with a sequence selected from the group consisting of: SEQ ID No.1-72. In some cases, the insertion sequence replaces the natural promoter sequence. In some cases, the insertion sequence comprises at least about 85% sequence identity with a sequence selected from the group consisting of: SEQ ID No.1-72. In some cases, the sequence comprises at least about 90% sequence identity with a sequence selected from the group consisting of: SEQ ID No.1-72. In some cases, the insertion sequence comprises at least about 95% sequence identity with a sequence selected from the group consisting of: SEQ ID No.1-72. In some cases, the insertion sequence comprises at least about 97% sequence identity with a sequence selected from the group consisting of: SEQ ID No.1-72. In some cases, the insertion sequence comprises at least about 98% sequence identity with a sequence selected from the group consisting of: SEQ ID No.1-72. In some cases, the insert sequence comprises at least about 99% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the native coding sequence is selected from the group consisting of: cysZ, otsB, bcs genes, and treZ. In some cases, the native coding sequence is selected from the group consisting of: a transporter gene, an ion transporter gene, an exopolysaccharide biosynthesis gene, a cellulose biosynthesis gene, and a trehalose biosynthesis gene.

[0009] In some embodiments, the present disclosure provides a genetically engineered bacterium comprising a nitrogen fixation or nitrogen assimilation coding sequence operably linked to an insertion sequence, the insertion sequence having at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID No.1-72. In some cases, the insertion sequence having at least about 80% sequence identity to a sequence selected from the group consisting of SEQ ID No.1-72 replaces a native promoter sequence. In some cases, the insertion sequence comprises at least about 85% sequence identity to a sequence selected from the group consisting of SEQ ID No.1-72. In some cases, the insertion sequence comprises at least about 90% sequence identity to a sequence selected from the group consisting of SEQ ID No.1-72. In some cases, the insertion sequence comprises at least about 95% sequence identity to a sequence selected from the group consisting of SEQ ID No.1-72. In some cases, the insertion sequence comprises at least about 97% sequence identity to a sequence selected from the group consisting of SEQ ID No.1-72. In some cases, the inserted sequence comprises at least about 98% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the inserted sequence comprises at least about 99% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the nitrogen fixation or nitrogen assimilation coding sequence is selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotides encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotides encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, and genes associated with the biosynthesis of nitrogenase. In some cases, the genetically engineered bacteria are genetically engineered nitrogen-fixing bacteria. In some cases, the genetically engineered bacteria are non-intergeneric. In some cases, the genetically engineered bacteria are intergeneric. In some cases, the genetically engineered bacteria fix atmospheric nitrogen under non-nitrogen limiting conditions. In some cases, the genetically engineered bacteria fix more atmospheric nitrogen than non-engineered bacteria of the same species.In some cases, the genetically engineered bacteria is selected from the group consisting of: Rahnella aquatilis, Klebsiella variicola, Kosakonia pseudosacchari, Kluyvera intermedia, Klebsiella sp., Enterobacter sp., and Kosakonia sacchari. In some cases, the genetically engineered bacteria belongs to the genus Rahnella, and the inserted sequence comprises at least about 80% sequence identity with a sequence selected from the group consisting of: SEQ ID No. 41-59 and 63-66. In some cases, the genetically engineered bacteria is Rahnella aquatilis, and the inserted sequence comprises at least about 80% sequence identity with a sequence selected from the group consisting of: SEQ ID No. 41-59 and 63-66. In some cases, the genetically engineered bacteria belongs to the genus Microcera, and the inserted sequence comprises at least about 80% sequence identity with a sequence selected from the group consisting of: SEQ ID No.1-10. In some cases, the genetically engineered bacteria is Microcera sucrosei, and the inserted sequence comprises at least about 80% sequence identity with a sequence selected from the group consisting of: SEQ ID No.1-10. In some cases, the genetically engineered bacteria belongs to the genus Klebsiella, and the inserted sequence comprises at least about 80% sequence identity with a sequence selected from the group consisting of: SEQ ID No.11-40. In some cases, the genetically engineered bacteria is Klebsiella variegata, and the inserted sequence comprises at least about 80% sequence identity with a sequence selected from the group consisting of: SEQ ID No.11-40. In some cases, the genetically engineered bacteria belongs to the genus Kluyvera, and the inserted sequence comprises at least about 80% sequence identity with SEQ ID No.60. In some cases, the genetically engineered bacteria is Kluyvera intermedia, and the inserted sequence comprises at least about 80% sequence identity to SEQ ID No. 60. In some cases, the genetically engineered bacteria is Oxycopersicon spp., and the inserted sequence comprises at least about 80% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 61 and 70-72. In some cases, the genetically engineered bacteria is an Enterobacter species, and the inserted sequence comprises at least about 80% sequence identity to SEQ ID No. 62. In some cases, the genetically engineered bacteria is a Klebsiella species, and the inserted sequence comprises at least about 80% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 67-69.In some cases, the inserted sequence is a native sequence inserted into a non-native context.

[0010] In some embodiments, the present disclosure provides a composition comprising a plant seed and a genetically engineered bacterium described herein. In some cases, the plant seed is selected from the group consisting of: corn seeds, wheat seeds, rice seeds, barley seeds, soybean seeds, sorghum seeds, and rye seeds.

[0011] In some embodiments, the disclosure provides a composition comprising a plant and a genetically engineered bacterium provided herein. In some cases, the plant is a seedling. In some cases, the plant is selected from the group consisting of: corn, wheat, rice, barley, rye, soybean and sorghum.

[0012] In some embodiments, the present disclosure provides a method for increasing microbial gene expression in a microorganism by replacing the native promoter sequence of the microbial gene with a promoter sequence, the promoter sequence comprising at least 80% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the promoter sequence comprises at least 85% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the promoter sequence comprises at least 90% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the promoter sequence comprises at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the promoter sequence comprises at least 97% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the promoter sequence comprises at least 98% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the promoter sequence comprises at least 99% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, increasing expression of the microbial gene increases ammonium excretion by the microbe. In some cases, increasing expression of the microbial gene increases nitrogen fixation by the microbe. In some cases, increasing expression of the microbial gene increases colonization of a plant by the microbe.

[0013] In some embodiments, the present disclosure provides polynucleotides comprising a coding sequence for a protein associated with nitrogen fixation or nitrogen assimilation and a sequence comprising at least 80% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the coding sequence for a protein associated with nitrogen fixation or nitrogen assimilation is selected from the group consisting of: nifA, nifL, ntrB, ntrC, a polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, a polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, and genes associated with the biosynthesis of nitrogenase.

[0014] In some embodiments, the present disclosure provides a polynucleotide comprising a coding sequence of a protein of interest and a sequence comprising at least 80% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the protein of interest is selected from the group consisting of: a transporter gene, an ion transporter gene, an exopolysaccharide biosynthesis gene, a cellulose biosynthesis gene, and a trehalose biosynthesis gene. In some cases, the protein of interest is selected from the group consisting of: a CysZ gene, a bcs gene, a treZ gene, and an otsB gene. In some cases, the polynucleotide comprises a sequence having at least 85% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the polynucleotide comprises a sequence having at least 90% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the polynucleotide comprises a sequence having at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the polynucleotide comprises a sequence having at least 97% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the polynucleotide comprises a sequence having at least 98% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the polynucleotide comprises a sequence having at least 99% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72.

[0015] In some embodiments, the present disclosure provides a method of increasing the amount of atmospheric nitrogen in a plant, comprising contacting the plant with a genetically engineered bacterium, wherein the genetically engineered bacterium comprises a nitrogen fixation coding sequence operably linked to a promoter, the promoter comprising at least 80% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the promoter comprises at least 85% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the promoter comprises at least 90% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the promoter comprises at least 95% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the promoter comprises at least 97% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the promoter comprises at least 98% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, increasing the expression of the microbial gene increases ammonium excretion by the microorganism. In some cases, increasing the expression of the microbial gene increases nitrogen fixation by the microorganism. In some cases, increasing the expression of the microbial gene increases colonization of plants by the microorganism.

[0016] In some cases, the promoter comprises at least 99% sequence identity to a sequence selected from the group consisting of: SEQ ID No. 1-72. In some cases, the genetically engineered bacteria is selected from the group consisting of: Rahnella aquatica, Klebsiella variegata, Achromobacter spiritinus, Achromobacter marplatensis, Microbacterium murale, Kluyvera intermedia, Pseudocercosus micrococcus, Enterobacter, Azospirillum lipoferum, and Azospirillum sucrose micrococcus.

[0017] In some embodiments, the present disclosure provides a method of reducing the amount of nitrogen fertilizer required between planting and harvesting of a crop, the method comprising inoculating the crop with a genetically engineered bacterium described herein.

[0018] Incorporated by Reference

[0019] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description and the accompanying drawings, which set forth illustrative embodiments utilizing the principles of the present invention, wherein:

[0021] Figure 1A -B depicts the enrichment and isolation of nitrogen-fixing bacteria. (A) Nfb agar plates were used to isolate single colonies of nitrogen-fixing bacteria. (B) Semi-solid Nfb agar poured in a Balch tube. Arrows point to the surface membrane of enriched nitrogen-fixing bacteria.

[0022] Figure 2 A representative nifH PCR screen is depicted. In this screen, positive bands were observed at approximately 350 bp for two colonies. The lower band represents primer dimers.

[0023] Figure 3 An example of PCR screening of mutagenized colonies from CRISPR-Cas selection is depicted. CI006 colonies were screened with primers specific to the nifL locus. The wild-type PCR product is expected at approximately 2.2 kb, while the mutant is expected at approximately 1.1 kb. Seven of the ten colonies screened clearly showed the desired deletion.

[0024] Figure 4A -D depicts the in vitro phenotypes of various strains. Mutants of strain CI010 grown in nitrogen-fixing medium supplemented with 0 to 10 mM glutamine ( Figure 4A ) and mutants of strain CI006 ( Figure 4B )'s acetylene reduction assay (ARA) activity. Figure 4C The ARA activity of other strains is shown in Figure 4D Ammonium excretion profiles of the two strains across time are shown in .

[0025] Figure 5 Depicted are the culture expression profiles of 9 different genes involved in nitrogen fixation in strain CI006. Numbers represent counts for each transcript. Various conditions are indicated (0, 1, 10 mM glutamine and 0%, 10%, 20% atmosphere in N2).

[0026] Figure 6 Depicted is CI006 colonization of maize roots. Maize seedlings were inoculated with CI006 carrying an RFP expression plasmid. After two weeks of growth and maintenance of the plasmid by irrigation with appropriate antibiotics, roots were harvested and imaged by fluorescence microscopy. Colonization of the intercellular spaces of the roots was observed.

[0027] Figure 7Nitrogen derived from microbial levels in WT (CI050) and optimized (CM002) strains is depicted.

[0028] Figure 8 The experimental setup for quality analysis of Micro-Tom results is shown.

[0029] Fig. 9 A screen of 10 strains for increasing the quality of Micro-Tom plant fruit is shown. Results of six replicates are presented. For column 3, p=0.07. For column 7, p=0.05.

[0030] Fig. 10A -C depicts additional results of ARA activities of candidate microorganisms and corresponding candidate mutants grown in nitrogen-fixing medium supplemented with 0 to 10 mM glutamine.

[0031] Fig.11 Depicted are double mutants that exhibit higher ammonia excretion than the single mutants from which they were derived.

[0032] Fig.12 Depicted are NDFA obtained from a 15N gas uptake experiment used to measure NDFA in corn under fertilized conditions (back extrapolated using exposure days).

[0033] Fig.13 Depicted are NDFA values ​​obtained from a 15N gas uptake experiment used to measure NDFA in Setaria plants under fertilized conditions (back extrapolated using exposure days).

[0034] Fig.14A The incorporation rate of 15N gas is depicted. Plants inoculated with the evolved strain showed increased 15N gas incorporation compared to non-inoculated plants.

[0035] Fig. 14B It is depicted that 4 weeks after planting, up to 7% of the nitrogen in plants inoculated with the evolved strain originated from microbially fixed nitrogen.

[0036] Fig. 14C Depicted are increases in leaf area (and other biomass measures, data not shown) in plants inoculated with the evolved strains compared to uninoculated or wild-type inoculated plants.

[0037] Fig.15A Evolved strains are depicted that showed significantly higher nifH production in root tissue as measured in plant transcriptomic studies.

[0038] Fig. 15B The rates of fixed nitrogen found in plant tissues are plotted in relation to the rates at which a particular organism is colonized by the HoME-optimized strain.

[0039] Fig.16A Soil texture maps of various field soils tested for colonization are depicted. The soils of original origin of several microorganisms are indicated as asterisks.

[0040] Fig. 16B Depicted are the colonization rates of strain 1 and strain 5 tested across four different soil types (circles). Both strains showed relatively robust colonization profiles across different soil types.

[0041] Fig. 16C Depicted is the colonization of strain 1 as tested in a field trial over the span of the growing season. Strain 1 persisted in corn tissue up to 12 weeks after planting and began to show a decline in colonization thereafter.

[0042] Fig.17 Depicted is a schematic diagram of microbial breeding according to an embodiment.

[0043] Fig.18 Describes Fig.17 Zoomed-in views of microbiome composition measurements are shown.

[0044] Fig.19A Several examples of promoter insertions are shown, showing an increase in nifA transcription in O. sucroseus. According to some embodiments, NifA transcription was measured by qPCR, replicated 3 times, and helD was used as a housekeeping gene to normalize transcript counts. Cells were cultured in minimal nitrogen-free medium.

[0045] Fig.19B Several examples of promoter insertion upstream of the nifA gene leading to increased nitrogenase activity in Oscillopora sucrosei are shown. According to some embodiments, the activity is measured in minimal medium supplemented with 5 mM glutamine as the sole N source in the ARA assay.

[0046] Fig. 20A An example of a promoter insertion showing increased nifA transcription in K. variabilis is shown. According to some embodiments, NifA transcription was measured by qPCR using helD as a housekeeping gene to normalize transcript counts. Cells were cultured in minimal medium supplemented with 10 mM glutamine as the sole N source.

[0047] Fig. 20B Shows Fig. 20A Modification of leads to increased nitrogenase activity in Klebsiella mutans. According to some embodiments, the activity is measured in minimal medium supplemented with 10 mM glutamine as the sole N source in an ARA assay.

[0048] Fig.21 and Fig. 22Other examples of promoter insertions are shown, showing increased nifA transcription in K. variabilis. According to some embodiments, NifA transcription was measured by qPCR using helD as a housekeeping gene to normalize transcript counts. Cells were cultured in minimal medium supplemented with 10 mM glutamine as the sole N source.

[0049] Fig.23 Shows Fig. 22 Modification of causes an increase in nitrogenase activity in Klebsiella mutans. According to some embodiments, the activity is measured in minimal medium supplemented with 10 mM glutamine as the sole N source in the ARA assay. Error bars represent standard deviation of the mean.

[0050] Fig.24A and Fig. 24B An example of promoter insertion upstream of the nifA gene, which results in increased nitrogenase activity in Klebsiella mutans, is shown according to some embodiments. A scatter plot of two biological replicates, measured in minimal medium supplemented with 5 mM ammonium phosphate in an ARA assay, is shown.

[0051] Fig.25A An example of promoter insertion upstream of the nifA gene, which results in increased nitrogenase activity in Rahnella aquatica, according to some embodiments, is shown. The activity was measured in minimal medium supplemented with 5 mM ammonium phosphate in the ARA assay.

[0052] Fig.25B An example of promoter insertion upstream of the nifA gene, which results in increased nitrogenase activity in Rahnella aquatica, according to some embodiments, is shown. The activity was measured in minimal medium supplemented with 10 mM ammonium phosphate in the ARA assay.

[0053] Fig.26 An example of promoter insertion upstream of the nifA gene, which results in increased nitrogenase activity in Osseotheca pseudosucrose, according to some embodiments, is shown. The activity was measured in minimal medium supplemented with 5 mM glutamine in the ARA assay.

[0054] Fig. 27 An example of promoter insertion upstream of the nifA gene, which results in increased nitrogenase activity in Enterobacter sp., is shown, according to some embodiments. The activity was measured in minimal medium supplemented with 5 mM ammonium phosphate in the ARA assay.

[0055] Fig.28 An example of promoter insertion upstream of the nifA gene, which results in increased nitrogenase activity in Klebsiella sp., according to some embodiments, is shown. The activity was measured in minimal medium supplemented with 5 mM ammonium phosphate in the ARA assay.

[0056] Fig.29 Two examples of promoter insertion upstream of the nifA gene, which results in increased nitrogenase activity, are shown according to some embodiments. The activity was measured in minimal medium supplemented with 5 mM glutamine in the ARA assay.

[0057] Fig.30 An example of a promoter insertion showing increased bcsI transcription in O. sucroseus is shown. According to some embodiments, BcsI transcription was measured by qPCR using helD as a housekeeping gene to normalize transcript counts. Cells were cultured in minimal nitrogen-free medium.

[0058] Fig.31A An example of a promoter insertion showing increased cysZ transcription in O. sucroseus is shown. According to some embodiments, CysZ transcription is measured by qPCR using helD as a housekeeping gene to normalize transcript counts. Cells were cultured in minimal medium supplemented with 5 mM glutamine.

[0059] Fig.31B An example of a promoter insertion is shown showing increased transcription of otsB in O. sucroseus. According to some embodiments, OtsB transcription was measured by qPCR using helD as a housekeeping gene to normalize transcript counts. Cells were cultured in minimal medium supplemented with 5 mM glutamine.

[0060] Fig.31C An example of a promoter insertion is shown, showing an increase in treZ transcription in O. sucroseus. According to some embodiments, TreZ transcription is measured by qPCR, and helD is used as a housekeeping gene to normalize transcript counts. Cells were cultured in minimal medium supplemented with 5 mM glutamine.

[0061] Fig.32A Shows Figure 20A-23 The promoter insertion method used in .

[0062] Fig.32B Shows Fig.19A , 19B and the promoter insertion method used in 24A-31C. DETAILED DESCRIPTION

[0063] The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides) of any length or their analogs. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci (loci / locus) determined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA with any sequence, isolated RNA with any sequence, nucleic acid probes and primers. Polynucleotides may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after polymer assembly. The nucleotide sequence may be interspersed with non-nucleotide components. The polynucleotides may be further modified after polymerization, such as by conjugation with a labeling component.

[0064] "Hybridization" refers to the reaction of one or more polynucleotide reactions to form a stable complex by hydrogen bonding between the bases of nucleotide residues. Hydrogen bonding can occur by Watson Crick base pairing, Hoogstein binding or in any other sequence-specific manner according to base complementarity. Complex can include two chains forming a duplex structure, three or more chains forming a multi-chain complex, a single self-hybridizing chain or any combination of these. Hybridization reaction can constitute a step in a wider process, and the process is such as initial PCR or enzymatic cutting of polynucleotides by endonuclease. The second sequence complementary to the first sequence is referred to as the "complementary sequence" of the first sequence. The term "hybridizable" as applied to polynucleotides refers to the ability of polynucleotides to form a stable complex by hydrogen bonding between the bases of nucleotide residues in hybridization reactions.

[0065] "Complementarity" refers to the ability of a nucleic acid to form (multiple) hydrogen bonds with another nucleic acid sequence by traditional Watson-Crick or other non-traditional types. The complementarity percentage indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). "Complete complementarity" means that all consecutive residues of a nucleic acid sequence will hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or to two nucleic acids that hybridize under stringent conditions. Sequence identity (e.g., for the purpose of assessing complementarity percentage) can be measured by any suitable alignment algorithm, including, but not limited to, the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needle alignment available at www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html, optionally with default settings), the BLAST algorithm (see, e.g., the BLAST alignment tool available at blast.ncbi.nlm.nih.gov / Blast.cgi, optionally with default settings), or the Smith-Waterman algorithm (see, e.g., the EMBOSS Water alignment available at www.ebi.ac.uk / Tools / psa / emboss_water / nucleotide.html, optionally with default settings). Optimal alignment can be assessed using any suitable parameters of the selected algorithm, including the default parameters.

[0066] In general, the "stringent conditions" of hybridization refer to conditions in which the nucleic acid having complementarity with the target sequence mainly hybridizes with the target sequence, and does not hybridize with non-target sequences in general. Stringent conditions are generally sequence-dependent and vary according to several factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes with its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), "Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes", Part I, Chapter II "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, NY.

[0067] In general, "sequence identity" refers to the precise nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences. Typically, the technology for determining sequence identity includes determining the nucleotide sequence of the polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences with a second nucleotide or amino acid sequence. It can be compared by determining the "percent identity" of two or more sequences (polynucleotides or amino acids). The percent identity of two sequences (whether nucleic acid sequences or amino acid sequences) can be calculated by dividing the exact match number between the two comparison sequences by the length of the shorter sequence and multiplying by 100. In some cases, the percent identity of a test sequence and a reference sequence (whether nucleic acid sequences or amino acid sequences) can be calculated by dividing the exact match number between the two comparison sequences by the length of the reference sequence and multiplying by 100. For example, the percent identity can also be determined, for example, by using the advanced BLAST computer program (including version 2.2.9) comparative sequence information available from the National Institutes of Health (National Institutes of Health). The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical alignment symbols (generally nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The program can be used to determine the percent identity over the entire length of the compared proteins. Default parameters are provided to optimize searches of short query sequences, such as in the case of the blastp program. The program also allows the use of SEG filters to mask segments of the query sequence, as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). The desired degree of sequence identity ranges from approximately 80% to 100% and integer values ​​therebetween. Typically, the percent identity between the disclosed sequence and the claimed sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%.

[0068] As used herein, "expression" refers to the process of transcribing a polynucleotide from a DNA template (e.g., into mRNA or other RNA transcripts) and / or the process of subsequently translating the transcribed mRNA into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression may include splicing mRNA in eukaryotic cells.

[0069] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interspersed with non-amino acids. The term also encompasses amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein, the term "amino acid" includes natural and / or non-natural or synthetic amino acids, including glycine and both D or L optical isomers, as well as amino acid analogs and peptide mimetics.

[0070] As used herein, the term “about” is used synonymously with the term “substantially.” Illustratively, the term “about” is used with respect to an amount to indicate a value slightly above the recited value, for example, plus or minus 0.1% to 10%.

[0071] The term "biologically pure culture" or "substantially pure culture" refers to a culture of a bacterial species described herein that is free of other bacterial species in sufficient quantities to interfere with replication of the culture or detection by ordinary bacteriological techniques.

[0072] "Plant productivity" generally refers to any aspect of plant growth or development that is responsible for plant growth. For food crops, such as grains or vegetables, "plant productivity" can refer to the yield of grains or fruits harvested from a particular crop. As used herein, increased plant productivity broadly refers to increased yield of grains, fruits, flowers or other plant parts harvested for various purposes; improved growth of plant parts, including stems, leaves and roots; increased plant growth; maintenance of high chlorophyll content in leaves; increased fruit or seed number; increased fruit or seed unit weight; reduced NO due to reduced nitrogen fertilizer use; and increased nitrogen production. 2 emissions; and similar improvements in plant growth and development.

[0073] Microbes in and around food crops can influence traits of those crops. Plant traits that may be affected by microbes include: yield (e.g., grain production, biomass production, fruit development, flower formation); nutrition (e.g., nitrogen, phosphorus, potassium, iron, micronutrient acquisition); abiotic stress management (e.g., drought tolerance, salt tolerance, heat tolerance); and biotic stress management (e.g., pests, weeds, insects, fungi, and bacteria). Strategies to alter crop traits include: increasing key metabolite concentrations; changing the temporal dynamics of microbial effects on key metabolites; linking microbial metabolite production / degradation to novel environmental cues; reducing negative metabolites; and improving the balance of metabolites or underlying proteins.

[0074] As used herein, "control sequence" refers to an operator, promoter, silencer, or terminator.

[0075] In some embodiments, the native or endogenous control sequences of a gene of the present disclosure are replaced by one or more endogenous control sequences.

[0076] As used herein, "introduced" means introduced through modern biotechnology, and not introduced naturally.

[0077] In some embodiments, the bacteria of the present disclosure have been modified such that they are not naturally occurring bacteria.

[0078] In some embodiments, the bacteria of the present disclosure are present at a concentration of at least 10 g of plant fresh weight or dry weight. 3 cfu, 10 4 cfu, 10 5 cfu, 10 6 cfu, 10 7 cfu, 10 8 cfu, 10 9 cfu, 10 10 cfu, 10 11 cfu or 10 12 In some embodiments, the bacteria of the present disclosure are present in the plant at a concentration of at least about 10 cfu per gram of fresh or dry weight of the plant. 3 cfu, about 10 4 cfu, about 10 5 cfu, about 10 6 cfu, about 10 7 cfu, about 10 8 cfu, about 10 9 cfu, about 10 10 cfu, about 10 11 cfu or about 10 12 In some embodiments, the bacteria of the present disclosure are present in the plant at a concentration of at least 10 cfu per gram of fresh or dry weight of the plant. 3to 10 9 , 10 3 to 10 7 , 10 3 to 10 5 , 10 5 to 10 9 , 10 5 to 10 7 , 10 6 to 10 10 , 10 6 to 10 7 The amount of cfu present in the plant.

[0079] The fertilizers and exogenous nitrogen of the present disclosure may include the following nitrogen-containing molecules: ammonium, nitrate, nitrite, ammonia, glutamine, etc. The nitrogen sources of the present disclosure may include anhydrous ammonia, ammonium sulfate, urea, diammonium phosphate, urea-form, monoammonium phosphate, ammonium nitrate, nitrogen solution, calcium nitrate, potassium nitrate, sodium nitrate, etc.

[0080] As used herein, "exogenous nitrogen" refers to non-atmospheric nitrogen that is readily available in the soil, field, or growing medium that exists under non-nitrogen limiting conditions, including ammonia, ammonium, nitrate, nitrite, urea, uric acid, ammonium acid, and the like.

[0081] As used herein, "non-nitrogen limiting conditions" refers to non-atmospheric nitrogen available in soil, fields, media, at a concentration greater than about 4 mM nitrogen, as disclosed by Kant et al. (2010. Journal of Experimental Biology (J. Exp. Biol.) 62(4): 1499-1509), which is incorporated herein by reference.

[0082] As used herein, "introduced genetic material" means genetic material that is added to the genome of a recipient and remains as a component of the genome.

[0083] In some embodiments, the gene regulatory network for nitrogen fixation and assimilation comprises polynucleotide encoding genes and non-coding sequences that guide, regulate and / or modulate microbial nitrogen fixation and / or assimilation, and may comprise nif cluster (e.g., nifA, nifB, nifC ... nifZ) polynucleotide sequences, polynucleotides encoding nitrogen regulatory protein C, polynucleotides encoding nitrogen regulatory protein B, polynucleotide sequences of the gln cluster (e.g., glnA and glnD), draT, and ammonia transporters / permeases. In some cases, the Nif cluster may comprise NifB, NifH, NifD, NifK, NifE, NifN, NifX, hesa, and NifV. In some cases, the Nif cluster may comprise subclasses of NifB, NifH, NifD, NifK, NifE, NifN, NifX, hesa, and NifV.

[0084] In some embodiments, the fertilizer of the present disclosure comprises at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110 %, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% nitrogen.

[0085] In some embodiments, the fertilizer of the present disclosure comprises at least about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about About 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75 %, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% nitrogen.

[0086] In some embodiments, the fertilizer of the present disclosure comprises about 5% to 50%, about 5% to 75%, about 10% to 50%, about 10% to 75%, about 15% to 50%, about 15% to 75%, about 20% to 50%, about 20% to 75%, about 25% to 50%, about 25% to 75%, about 30% to 50%, about 30% to 75%, about 35% to 50%, about 35% to 75%, about 40% to 50%, about 40% to 75%, about 45% to 50%, about 45% to 75%, or about 50% to 75% nitrogen by weight.

[0087] In some embodiments, an increase in nitrogen fixation and / or production of 1% or more nitrogen in the plant is measured relative to a control plant not exposed to the bacteria of the present disclosure. All increases or decreases in bacteria are measured relative to control bacteria. All increases or decreases in plants are measured relative to control plants.

[0088] As used herein, a "constitutive promoter" is a promoter that is active under most conditions and / or during most stages of development. There are several advantages to using constitutive promoters in expression vectors used in biotechnology, such as: high production levels of proteins for selecting transgenic cells or organisms; high expression levels of reporter proteins or scorable markers, making them easy to detect and quantify; high production levels of transcription factors that are part of a regulatory transcription system; production of compounds that require ubiquitous activity in an organism; and production of compounds required at all stages of development. Non-limiting exemplary constitutive promoters include CaMV 35S promoter, opine promoter, ubiquitin promoter, alcohol dehydrogenase promoter, etc.

[0089] As used herein, a "non-constitutive promoter" is a promoter that is active under certain conditions, in certain types of cells, and / or during certain developmental stages. For example, tissue-specific, tissue-preferential, cell-type-specific, cell-type-preferential, inducible promoters, and promoters under developmental control are non-constitutive promoters. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues.

[0090] As used herein, an "inducible" or "repressible" promoter is a promoter under the control of a chemical or environmental factor. Examples of environmental conditions that may affect transcription of an inducible promoter include anaerobic conditions, certain chemicals, the presence of light, acidic or alkaline conditions, etc.

[0091] As used herein, a "tissue-specific" promoter is a promoter that initiates transcription only in certain tissues. Unlike constitutive expression of a gene, tissue-specific expression is the result of gene regulation at several interacting levels. Therefore, in the art, it is sometimes preferred to use promoters from homologous or closely related species to achieve efficient and reliable expression of transgenes in specific tissues. This is one of the main reasons why a large number of tissue-specific promoters isolated from specific tissues are found in the scientific and patent literature.

[0092] As used herein, the term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one nucleic acid sequence is regulated by another. For example, a promoter is operably linked to a coding sequence when it is capable of regulating the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in a sense or antisense direction. In another example, a complementary RNA region of the present disclosure can be operably linked to a target mRNA at the 5' end (directly or indirectly), or operably linked to a target mRNA at the 3' end, or within a target mRNA, or for a target mRNA, the first complementary region is 5' and its complementary sequence is 3'.

[0093] Nitrogen fixation regulation

[0094] One trait that can be a target for regulation of the methods described herein is nitrogen fixation. Nitrogen fertilizer is the largest operating expense on farms and is the largest driver of higher yields for row crops such as corn and wheat. Microbial products that can deliver renewable forms of nitrogen in non-leguminous crops are described herein. Although some endophytes have the genetics required for nitrogen fixation in pure culture, the fundamental technical challenge is that wild-type endophytes of cereals and grasses stop fixing nitrogen in fertilized fields. Fertilizers are applied and residual nitrogen levels in field soils signal microorganisms to shut down the biochemical pathways for nitrogen fixation.

[0095] The transcriptional and post-translational levels of nitrogen fixation regulatory networks need to be altered to develop microorganisms that can fix nitrogen in the presence of fertilizers and transfer nitrogen to corn. To this end, host-microbe evolution (HoME) technology is described herein to accurately evolve regulatory networks and induce new phenotypes. A unique proprietary library of nitrogen-fixing endophytes isolated from corn is also described herein, which is paired with extensive omics data around the interaction of microorganisms with host plants under different environmental conditions (such as nitrogen stress and excess). This technology enables the precise evolution of the gene regulatory network of endophytes to produce microorganisms that actively fix nitrogen even in the presence of fertilizers in the field. The potential assessment of the technology of microorganisms that colonize corn root tissue and produce nitrogen for fertilized plants, and the assessment of the compatibility of endophytes with standard formulation practices and different soils, are also described herein to determine the feasibility of integrating microorganisms into modern nitrogen management strategies.

[0096] In order to utilize elemental nitrogen (N) for chemical synthesis, life forms use nitrogen gas (N) available in the atmosphere. 2 ) is combined with hydrogen in a process called nitrogen fixation. Due to the energy-intensive nature of biological nitrogen fixation, diazotrophs (bacteria and archaea that fix atmospheric nitrogen) have evolved precise and strict regulation of nif gene clusters in response to ambient oxygen and available nitrogen. Nif genes encode enzymes involved in nitrogen fixation (such as nitrogenase complexes) and proteins that regulate nitrogen fixation. Shamseldin (2013. "Global J. Biotechnol. Biochem. " 8 (4): 84-94) discloses a detailed description of nif genes and their products, and is incorporated herein by reference. This article describes a method for producing plants with improved traits, which includes isolating bacteria from a first plant, introducing genetic variation into the nif gene of the isolated bacteria, exposing a second plant to a variant bacterium, isolating bacteria from a second plant with improved traits relative to the first plant, and repeating the steps with bacteria isolated from the second plant.

[0097] In Proteobacteria, nitrogen fixation is regulated by σ 54The nifLA-dependent enhancer-binding protein NifA, a positive transcriptional regulator of the nif cluster, is centered. The level of intracellular active NifA is controlled by two key factors: transcription of the nifLA operon, and inhibition of NifA activity through protein-protein interactions with NifL. Both processes respond to intracellular glutamine levels through the PII protein signaling cascade. This cascade is mediated by GlnD, which directly senses glutamine and catalyzes the uridylylation or deuridylylation of two PII regulatory proteins, GlnB and GlnK, respectively, in response to the presence or absence of bound glutamine. Under conditions of nitrogen excess, unmodified GlnB signals the nifLA promoter to be inactivated. However, under conditions of nitrogen limitation, GlnB is post-translationally modified, which inhibits its activity and causes transcription of the nifLA operon. In this way, nifLA transcription is tightly controlled in response to environmental nitrogen through the PII protein signaling cascade. At the post-translational level of NifA regulation, GlnK inhibits NifL / NifA interaction, depending on the total level of intracellular free GlnK.

[0098] NifA is transcribed from the nifLA operon, whose promoter is phosphorylated by NTRC, another σ 54 Dependent regulator activation. The phosphorylation state of NtrC is mediated by histidine kinase NtrB, which interacts with deuridylylated GlnB rather than uridylylated GlnB. Under conditions of excessive nitrogen, the high glutamine level in the cell causes the deuridylylation of GlnB, and then GlnB interacts with NtrB to inactivate its phosphorylation activity and activate its phosphatase activity, causing NtrC dephosphorylation and nifLA promoter inactivation. However, under conditions of nitrogen restriction, low levels of intracellular glutamine cause GlnB uridylylation, which inhibits the interaction of GlnB and NtrB, and allows NtrC phosphorylation and nifLA operon transcription. In this way, nifLA expression is tightly controlled by the PII protein signaling cascade in response to environmental nitrogen. nifA, ntrB, ntrC and glnB are all genes that can be mutated in the methods described herein. These processes may also have in response to intracellular or extracellular ammonia, urea or nitrate levels.

[0099] The activity of NifA is also regulated post-translationally in response to environmental nitrogen, most typically by inhibition of NifA activity mediated by NifL. In general, the interaction of NiilL and NifA is affected by the PII protein signaling cascade via GlnK, but the nature of the interaction between GlnK and NifL / NifA varies greatly between nitrogen-fixing organisms. In Klebsiella pneumoniae, both forms of GlnK inhibit the NifL / NifA interaction, and the interaction between GlnK and NifL / NifA is determined by the overall level of free GlnK in the cell. Under conditions of excess nitrogen, deuridylylated GlnK interacts with the ammonium transporter AmtB, which is used to block AmtB from absorbing ammonium and chelate GlnK to the membrane, thereby allowing NifA to be inhibited by NifL. On the other hand, in Azotobacter vinelandii, NifL / NifA interaction and NifA inhibition require interaction with deuridylylated GlnK, while uridylylation of GlnK inhibits its interaction with NifL. In nitrogen-fixing organisms lacking the nifL gene, there is evidence that NifA activity is directly inhibited by interaction with the deuridylylated forms of both GlnK and GlnB under conditions of nitrogen excess. In some bacteria, Nif clusters may be regulated by glnR, and in some cases, this may also include negative regulation. Regardless of the mechanism, post-translational inhibition of NifA is an important regulator of nif clusters in most known nitrogen-fixing organisms. In addition, nifL, amtB, glnK and glnR are genes that can be mutated in the methods described herein.

[0100] In addition to regulating transcription of the nif gene cluster, many diazotrophs have evolved mechanisms for direct post-translational modification and inhibition of the nitrogenase enzyme itself, known as nitrogenase shutoff. This is mediated by ADP-ribosylation of the Fe protein (NifH) under conditions of nitrogen excess, which disrupts the interaction of the Fe protein with the MoFe protein complex (NifDK) and abolishes nitrogenase activity. DraT catalyzes ADP-ribosylation of the Fe protein and shutoff of the nitrogenase, while DraG catalyzes the removal of ADP-ribose and reactivation of the nitrogenase. As with nifLA transcription and NifA inhibition, nitrogenase shutoff is also regulated by the PII protein signaling cascade. Under conditions of nitrogen excess, deuridylylated GlnB interacts with and activates DraT, while deuridylylated GlnK interacts with both DraG and AmtB to form a complex that sequesters DraG to the membrane. Under nitrogen limiting conditions, the uridylylated forms of GlnB and GlnK do not interact with DraT and DraG, respectively, resulting in inactivation of DraT and diffusion of DraG to the Fc protein, where DraG removes ADP-ribose and activates nitrogenase. The methods described herein also encompass the introduction of genetic variation into the nifH, nifD, nifK, and draT genes.

[0101] Although some endophytes are capable of nitrogen fixation in vitro, genetics are often silenced in the field by high levels of exogenous fertilizers. The induction of exogenous nitrogen can be decoupled from the expression of nitrogenase to promote field-based nitrogen fixation. The integration of increasing nitrogenase activity across time is further used to increase nitrogen production for crop utilization. The specific targets of genetic variation that promote field-based nitrogen fixation using the methods described herein include one or more genes selected from the group consisting of: nifA, nifL, ntrB, ntrC, glnA, glnB, glnK, draT, amtB, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB and nifQ.

[0102] Another target for genetic variation to promote field-based nitrogen fixation using the methods described herein is the NifA protein. The NifA protein is typically an activator of nitrogen fixation gene expression. Increasing NifA production (constitutively or during high ammonia conditions) avoids the natural ammonia sensing pathway. In addition, reducing the production of the NifL protein (a known NifA inhibitor) also causes an increase in free active NifA levels. In addition, increasing the transcription level of the nifAL operon (constitutively or during high ammonia conditions) also causes an overall higher level of NifA protein. Increased nifAL expression levels are achieved by changing the promoter itself or by reducing the expression of NtrB (part of the ntrB and ntrC signaling cascade that would otherwise cause the nifAL operon to shut down during high nitrogen conditions). High levels of NifA achieved by these or any other methods described herein increase the nitrogen fixation activity of endophytes.

[0103] Another target for genetic variation to promote field-based nitrogen fixation using the methods described herein is the GlnD / GlnB / GlnK PII signaling cascade. Intracellular glutamine levels are sensed by the GlnD / GlnB / GlnK PII signaling cascade. Active site mutations in GlnD that abolish the uridylic acid removal activity of GlnD disrupt the nitrogen sensing cascade. In addition, reducing the GlnB concentration short-circuits the glutamine sensing cascade. These mutations "cheat" cells into sensing nitrogen-limited states, thereby increasing nitrogen fixation activity. These processes may also be responsive to intracellular or extracellular ammonia, urea, or nitrate levels.

[0104] The amtB protein is also a target for genetic variation to promote field-based nitrogen fixation using the methods described herein. Ammonia uptake from the environment can be reduced by reducing the expression level of the amtB protein. Without intracellular ammonia, the endophyte cannot sense high levels of ammonia, thereby preventing the downregulation of nitrogen fixation genes. Any ammonia that manages to enter the intracellular compartment is converted to glutamine. Intracellular glutamine levels are the main currency for nitrogen sensing. Reducing intracellular glutamine levels prevents cells from sensing high ammonium levels in the environment. This effect can be achieved by increasing the expression level of glutaminase, an enzyme that converts glutamine to glutamate. In addition, intracellular glutamine can also be reduced by reducing glutamine synthase, an enzyme that converts ammonia to glutamine. In nitrogen-fixing organisms, fixed ammonia is rapidly assimilated into glutamine and glutamate for use in cellular processes. Disrupting ammonia assimilation can enable the transfer of fixed nitrogen to be exported from the cell in the form of ammonia. Fixed ammonia is mainly assimilated into glutamine by glutamine synthetase (GS) encoded by glnA, and then assimilated into glutamine by glutamine ketoglutarate transaminase (GOGAT). In some instances, glnS encodes glutamine synthetase. GS is regulated by GS adenylyltransferase (GlnE) translationally, and GlnE is a bifunctional enzyme encoded by glnE, which catalyzes both adenylylation and deadenylylation of GS through the activity of its adenylyltransferase (AT) and adenylyl removal (AR) domains, respectively. Under nitrogen limiting conditions, glnA is expressed, and the AR domain of GlnE deadensylate GS, thereby making GS active. Under conditions of excessive nitrogen, glnA expression is turned off, and the AT domain of GlnE is allosterically activated by glutamine, thereby causing adenylylation and inactivation of GS.

[0105] In addition, the draT gene can also be a target for genetic variation to promote field-based nitrogen fixation using the methods described herein. Once nitrogenase is produced by the cell, nitrogenase shutoff represents another level of downregulation of fixation activity by the cell under high nitrogen conditions. This shutoff can be removed by reducing the expression level of DraT.

[0106] The method of conferring a new microbial phenotype can be carried out at the transcriptional, translational and post-translational levels. The transcriptional level includes changes at the promoter (e.g., changing the affinity of the sigma factor or the binding site of the transcription factor, including all or part of the promoter deletion) or changing the transcription terminator and attenuator. The translational level includes changes at the ribosome binding site and changing the mRNA degradation signal. The post-translational level includes mutating the active site of the enzyme and changing the protein-protein interaction. These changes can be achieved in a variety of ways. The reduction in expression level (or complete abolition) can be achieved by exchanging the natural ribosome binding site (RBS) or promoter with another one with lower strength / efficiency. The ATG start site can be exchanged for GTG, TTG or CTG start codons, which causes the translation activity of the coding region to decrease. Complete abolition of expression can be accomplished by knocking out (deleting) the coding region of the gene. Shifting the open reading frame (ORF) may cause premature stop codons along the ORF, resulting in non-functional truncated products. Inserting an in-frame stop codon will also similarly produce non-functional truncated products. Degradation tags can also be added to the N or C terminus to reduce the effective concentration of a specific gene.

[0107] In some embodiments, the expression level of gene described herein can be realized by using stronger promoter.In order to ensure that high promoter activity is present during high nitrogen level condition (or any other condition), the transcription spectrum of full genome under high nitrogen level condition can be obtained, and the active promoter with required transcription level can be selected from that data set to replace weak promoter.Weak start codon can be exchanged with ATG start codon, to obtain better translation initiation efficiency.Weak ribosome bind site (RBS) can also be exchanged with different RBS with higher translation initiation efficiency.In addition, site-specific mutagenesis can also be carried out to change the activity of enzyme.

[0108] Examples of promoters that can be used to drive gene expression as described herein include the promoters in Table 8. Additional details about these sequences are provided in Table 9. Table 9 lists the species of origin of each sequence, as well as the native gene and native gene function of some of the sequences. In addition, some of the sequences have been confirmed as promoters in in vitro transcription assays, in plant transcription assays, or both, see Example 8.

[0109] Increasing the level of nitrogen fixation in plants can result in a reduction in the amount of fertilizer required for crop production and reduce greenhouse gas emissions (eg, nitrous oxide).

[0110] Generation of bacterial populations

[0111] Isolation of bacteria

[0112] Microorganisms suitable for the methods and compositions disclosed herein can be obtained by extracting microorganisms from the surface or tissue of natural plants. Microorganisms can be obtained by grinding seeds to separate microorganisms. Microorganisms can be obtained by planting seeds in different soil samples and recovering microorganisms from tissues. In addition, microorganisms can be obtained by inoculating plants with exogenous microorganisms and determining which microorganisms appear in plant tissues. Non-limiting examples of plant tissues can include seeds, seedlings, leaves, cuttings, plants, bulbs or tubers.

[0113] The method for obtaining microorganisms can be by separating bacteria from soil. Bacteria can be collected from various soil types. In some instances, the characteristics of soil can be such as high or low fertility, moisture level, mineral level and various farming practices. For example, soil may participate in crop rotation, in which different crops are planted in the same soil in successive planting seasons. The sequential growth of different crops on the same soil can prevent the disproportionate depletion of certain minerals. Bacteria can be isolated from plants grown in selected soil. Seedling plants can be harvested after growing for 2-6 weeks. For example, at least 400 isolates can be collected in one round of harvesting. Soil and plant type show plant phenotype and conditions, and the conditions allow downstream enrichment of certain phenotypes.

[0114] Microorganisms can be isolated from plant tissues to assess microbial traits. Parameters used to process tissue samples can be varied to isolate different types of associated microorganisms, such as rhizobacteria, epiphytes, or endophytes. Isolates can be cultured in nitrogen-free media to enrich for bacteria that fix nitrogen. Alternatively, microorganisms can be obtained from global strain libraries.

[0115] Analysis is performed in plants to assess microbial traits. In certain embodiments, plant tissues can be processed so that DNA and RNA screening can be performed by high-throughput processing. In addition, non-invasive measurements can be used to assess colonization characteristics, such as colonization. Measurements of wild microorganisms can be obtained plant-by-plant. Medium-throughput methods can also be used to obtain measurements of wild microorganisms in the field. Measurements can be performed continuously over time. Model plant systems can be used, including but not limited to Setaria.

[0116] Microorganisms in plant systems can be screened by performing transcriptional profiling of the microorganisms in the plant system. Examples of screening by transcriptional profiling are methods using quantitative polymerase chain reaction (qPCR), molecular barcoding for transcript detection, next generation sequencing, and microbial labeling with fluorescent markers. Influencing factors can be measured to assess colonization in a greenhouse, including but not limited to the microbiome, abiotic factors, soil conditions, oxygen, moisture, temperature, inoculum status, and root positioning. As described herein, nitrogen fixation in bacteria can be assessed by measuring 15N gas / fertilizer (dilution) with IRMS or NanoSIMS. NanoSIMS is a high-resolution secondary ion mass spectrometry technique. NanoSIMS technology is a way to study the chemical activity of biological samples. Reduction catalysis of oxidation reactions that drive microbial metabolism can be studied at the cellular, subcellular, molecular, and elemental levels. NanoSIMS can provide a high spatial resolution of greater than 0.1 μm. NanoSIMS can detect isotopic tracers, such as 13 C. 15 N and 18 O usage. Therefore, NanoSIMS can be used to measure nitrogen fixation activity in cells.

[0117] Automated greenhouses can be used for plant analysis. Plant indicators in response to microbial exposure include but are not limited to biomass, chloroplast analysis, CCD cameras, volume tomography measurements.

[0118] One way to enrich microbial populations is based on genotype. For example, polymerase chain reaction (PCR) analysis using targeted primers or specific primers. Primers designed for the nifH gene can be used to identify nitrogen-fixing organisms because nitrogen-fixing organisms express the nifH gene during nitrogen fixation. Microbial populations can also be enriched by methods that are independent of single cell culture and chemotaxis-guided separation methods. Alternatively, targeted separation of microorganisms can be performed by culturing microorganisms on selective culture media. Pre-planned methods for enriching microbial populations for desired traits can be guided by bioinformatics data and are described in this article.

[0119] Enrichment of microorganisms with nitrogen fixation ability using bioinformatics

[0120] Bioinformatics tools can be used to identify and isolate plant growth promoting rhizobacteria (PGPR), which are selected based on their ability to fix nitrogen. Microorganisms with high nitrogen fixation ability can promote favorable traits in plants. Bioinformatics analysis modes used to identify PGPR include, but are not limited to, genomics, metagenomics, targeted isolation, gene sequencing, transcriptome sequencing, and modeling.

[0121] Genomic analysis can be used to identify PGPRs and confirm the presence of mutations using next generation sequencing and microbial version control methods as described herein.

[0122] Metagenomics can be used to identify and isolate PGPRs using colonization prediction algorithms. Metadata can also be used to identify the presence of engineered strains in environmental and greenhouse samples.

[0123] Transcriptomic sequencing can be used to predict the genotype that causes the PGPR phenotype. In addition, transcriptomic data are used to identify promoters that alter gene expression. Transcriptomic data can be combined with whole genome sequence (WGS) analysis to generate metabolic and gene regulatory network models.

[0124] Microbial domestication

[0125] Microorganisms isolated from nature can undergo a domestication process, in which microorganisms are converted into forms that can be genetically tracked and identified. One way to domesticate microorganisms is to engineer microorganisms with antibiotic resistance. The process of engineering antibiotic resistance can begin by determining the antibiotic sensitivity in wild-type microbial strains. If the bacteria are sensitive to antibiotics, the antibiotics can be used as good candidates for antibiotic resistance engineering. Subsequently, recombinant engineering methods can be used to incorporate antibiotic resistance genes or reverse selectable (counterselectable) suicide vectors into the genome of the microorganism. Reverse selectable suicide vectors can be composed of missing genes of interest, selectable markers, and reverse selectable marker sacB. Reverse selection can be used to exchange natural microbial DNA sequences with antibiotic resistance genes. Medium throughput methods can be used to evaluate multiple microorganisms simultaneously, allowing parallel domestication. Alternative domestication methods include using homing nucleases to prevent suicide vector sequences from looping out or obtaining intermediate vector sequences.

[0126] DNA vectors can be introduced into bacteria by several methods, including electroporation and chemical transformation. Transformation can be performed using a standard vector library. An example of a gene editing method is CRISPR, and there is a previous Cas9 test to ensure the activity of Cas9 in microorganisms.

[0127] Non-GMO Engineering of Microorganisms

[0128] Microbial populations with favorable traits can be obtained by directed evolution. Directed evolution is a method that mimics the natural selection process to evolve proteins or nucleic acids toward custom goals. An example of directed evolution is when random mutations are introduced into a microbial population, microorganisms with the most favorable traits are selected, and the growth of the selected microorganisms is continued. The most favorable trait in growth-promoting rhizobacteria (PGPR) can be nitrogen fixation. Based on the selection process after each iteration, the directed evolution method can be iterative and adaptive.

[0129] Plant growth promoting rhizobacteria (PGPR) with high nitrogen fixation ability can be generated. The evolution of PGPR can be carried out by introducing genetic mutations. Genetic mutations can be introduced by: polymerase chain reaction mutagenesis, oligonucleotide directed mutagenesis, saturation mutagenesis, fragment shuffling mutagenesis, homologous recombination, CRISPR / Cas9 system, chemical mutagenesis and combinations thereof. These methods can introduce random mutations into microbial populations. For example, mutants can be generated using synthetic DNA or RNA by oligonucleotide directed mutagenesis. Mutants can be generated using tools contained on plasmids, which are later cured.

[0130] In some cases, a mutant or heterologous sequence endogenous to the host cell can be introduced without introducing any sequence exogenous to the host cell. In some cases, a heterologous sequence from a cell of the same species as the host cell can be introduced without introducing any sequence exogenous to the host cell species. Any plasmid used to construct such a cell can then be eliminated to produce a genetically engineered cell containing genetic material exogenous to the cell. In some cases, a heterologous sequence from a cell of the same genus as the host cell can be introduced without introducing any sequence exogenous to the genus of the host cell. Any plasmid used to construct such a cell can then be eliminated to produce a genetically engineered cell containing genetic material exogenous to the genus of the host cell, thereby producing a non-intergenic cell.

[0131] Libraries from other species with improved traits can be used to identify genes of interest, including but not limited to improved PGPR properties, improved cereal colonization, increased oxygen sensitivity, increased nitrogen fixation, and increased ammonium excretion. Software such as Geneious or Platypus design software can be used to design genes within the genus based on these libraries. Mutations can be designed with the aid of machine learning. Mutations can be designed with the aid of metabolic models. Automated design of mutations can be accomplished using a la Platypus, and RNA for Cas directed mutagenesis can be directed.

[0132] Genes within the genera can be transferred into host microorganisms. In addition, reporter systems can also be transferred into microorganisms. Reporter systems characterize promoters, determine transformation success, screen for mutants, and serve as negative screening tools.

[0133] Microorganisms carrying mutations can be cultured through serial subculture. Microbial colonies contain a single variant of the microorganism. Microbial colonies are screened with the aid of an automated colony picker and liquid handler. Mutants with gene duplication and increased copy number express higher genotypes of the desired trait.

[0134] Selection of plant growth-promoting microorganisms based on nitrogen fixation

[0135] Microbial colonies can be screened using various analyses that assess nitrogen fixation. One way to measure nitrogen fixation is by a single fermentation analysis that measures nitrogen excretion. An alternative is acetylene reduction analysis (ARA), which samples online over time. ARA can be performed in a high-throughput plate in a microtube array. ARA can be performed with living plants and plant tissues. The culture medium formulation and culture medium oxygen concentration can be changed in the ARA analysis. Another method for screening microbial variants is by using a biosensor. The use of NanoSIMS and Raman microspectroscopy can be used to study the activity of microorganisms. In some cases, bacteria can also be cultured and amplified using a method of fermentation in a bioreactor. Bioreactors are designed to increase the robustness of bacterial growth and reduce bacterial sensitivity to oxygen. Micro-fermenters based on medium to high TP plates are used to assess oxygen sensitivity, nutritional requirements, nitrogen fixation, and nitrogen excretion. Bacteria can also be co-cultured with competitive microorganisms or beneficial microorganisms to elucidate hidden pathways. Flow cytometry can be used to screen bacteria that produce high levels of nitrogen using chemical, colorimetric, or fluorescent indicators. Bacteria can be cultured in the presence or absence of a nitrogen source. For example, the bacteria can be cultured with glutamine, ammonia, urea or nitrates.

[0136] Microbial breeding

[0137] Microbial breeding is a method to systematically identify and improve the role of species within the crop microbiome. The method involves three steps: 1) selecting candidate species by mapping plant-microbe interactions and predicting regulatory networks associated with specific phenotypes, 2) practical and predictable improvement of microbial phenotypes through intraspecific hybridization of regulatory networks and gene clusters, and 3) screening and selecting new microbial genotypes that produce desired crop phenotypes. In order to systematically assess strain improvements, a model was created that links the colonization dynamics of microbial communities to the genetic activity of key species. The model was used to predict genetic target breeding and increase the frequency of selecting microbiome-encoded agronomically relevant trait improvements.

[0138] The production of bacteria for improving plant traits (e.g., nitrogen fixation) can be achieved by continuous propagation. In addition to identifying bacteria and / or compositions that can confer one or more improved traits on one or more plants, the production of such bacteria can also be achieved by selecting plants with specific improved traits affected by microbial flora. A method for producing bacteria for improving plant traits comprises the following steps: (a) isolating bacteria from the tissue or soil of a first plant; (b) introducing genetic variation into one or more of the bacteria to produce one or more variant bacteria; (c) exposing a plurality of plants to the variant bacteria; (d) isolating bacteria from the tissue or soil of one of the plurality of plants, wherein the plant from which the bacteria is isolated has improved traits relative to other plants in the plurality of plants; (e) repeating steps (b) to (d) with bacteria isolated from plants with improved traits (step (d)). Steps (b) to (d) can be repeated any number of times (e.g., once, twice, three times, four times, five times, ten times or more) until the improved traits in the plant reach the desired level. Furthermore, the plurality of plants can be more than two plants, such as 10 to 20 plants, or 20 or more, 50 or more, 100 or more, 300 or more, 500 or more, or 1000 or more plants.

[0139] In addition to obtaining plants with improved traits, a bacterial population is also obtained, which includes bacteria that include one or more gene variations introduced into one or more genes (for example, genes regulating nitrogen fixation). By repeating the steps described above, a bacterial population including the most appropriate member in a population related to the plant traits of interest can be obtained. The bacteria in this population can be identified and its beneficial properties can be determined as by genetic and / or phenotypic analysis. In step (a), isolated bacteria may be subjected to genetic analysis. Phenotypic and / or genotypic information can be obtained using the following technology: high-throughput screening of chemical components of plant origin, sequencing technology including high-throughput sequencing of genetic material, differential display technology (including DDRT-PCR and DD-PCR), nucleic acid microarray technology, RNA sequencing (whole transcriptome shotgun sequencing (Whole Transcriptome Shotgun Sequencing)) and qRT-PCR (quantitative real-time PCR). The information obtained can be used to obtain the identity and activity of the community spectrum analysis information of the bacteria present, such as phylogenetic analysis or nucleic acid screening of the encoding rRNA operon component or other taxonomic information loci based on microarray. The example of taxonomic information loci includes 16S rRNA gene, 23S rRNA gene, 5S rRNA gene, 5.8S rRNA gene, 12S rRNA gene, 18S rRNA gene, 28S rRNA gene, gyrB gene, rpoB gene, fusA gene, recA gene, coxl gene, nifD gene. The example method of taxonomic spectrum analysis to determine the taxon present in the population is described in US20140155283. Bacterial identification can include characterizing one or more genes or one or more signal transduction pathways, such as the activity of genes related to the nitrogen fixation pathway. There may also be synergistic interactions between different bacterial species in the bacterial population (wherein the degree to which the desired effect is increased by two components due to their combination exceeds the cumulative amount).

[0140] The gene variation may be a gene selected from the group consisting of nifA, nifL, ntrB, ntrC, glnA, glnB, glnK, draT, amtB, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, and nifQ. The gene variation may be a variation in a gene encoding a protein having a function selected from the group consisting of glutamine synthetase, glutaminase, glutamine synthetase adenylyltransferase, transcription activator, anti-transcription activator, pyruvate flavodoxin oxidoreductase, flavodoxin, or NAD+-diazo-reductase aDP-D-ribosyltransferase. The genetic variation may be a mutation that causes one or more of the following: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased adenylyl removal activity of GlnE; or decreased uridylyl removal activity of GlnD. The introduction of genetic variation may comprise insertion and / or deletion of one or more nucleotides, such as 1, 2, 3, 4, 5, 10, 25, 50, 100, 250, 500 or more nucleotides, at the target site. The genetic variation introduced into one or more bacteria of the methods disclosed herein may be a knockout mutation (e.g., deletion of a promoter, insertion or deletion to produce a premature stop codon, deletion of an entire gene), or may be a point mutation that eliminates or abolishes the activity of a protein domain (e.g., a point mutation that affects an active site, or a portion of a gene encoding a relevant portion of a protein product), or it may alter or abolish the regulatory sequence of a target gene. One or more regulatory sequences can also be inserted, including heterologous regulatory sequences and regulatory sequences found in the genome of bacterial species or genus corresponding to the bacterium introduced with genetic variation. In addition, regulatory sequences can be selected based on the gene expression level in bacterial culture or in plant tissue. In some cases, the regulatory sequence inserted can be selected from SEQ ID NO.: 1-72. Genetic variation can be a predetermined genetic variation specifically introduced into the target site. Genetic variation can be a random mutation in the target site. Genetic variation can be the insertion or deletion of one or more nucleotides. In some cases, before the bacterium is exposed to plant to assess the improvement of properties, a variety of different genetic variations (for example, 2,3,4,5,10 kinds or more) are introduced into one or more separated bacteria. A variety of genetic variations can be any one of the above types, the same or different types and any combination. In some cases, a variety of different genetic variations are continuously introduced, the first genetic variation is introduced after the first separation step, the second genetic variation is introduced after the second separation step, and so on, so as to accumulate a variety of genetic variations in bacteria, giving the properties of the related plants gradually improved.

[0141] In some embodiments, the microorganism can be genetically altered by introducing a regulatory sequence. For example, a sequence selected from SEQ ID NO.: 1-72, or a sequence comprising a fragment of one of SEQ ID NO.: 1-72, or a sequence having at least about 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with a sequence selected from SEQ ID NO.: 1-72. In some cases, the regulatory sequence can comprise a sequence having at least about 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with a fragment of a sequence selected from SEQ ID NO.: 1-72.

[0142] In some cases, the genetically engineered microorganism as described herein can include an insertion sequence. In some cases, the insertion sequence can include a native sequence inserted under a non-natural background. For example, the insertion sequence can include a promoter from a first gene of the genetically engineered microorganism, and the promoter is inserted in the position of the promoter of a second gene of the microorganism. In some cases, the insertion sequence can include a non-natural promoter inserted under a non-natural background. In some cases, the insertion sequence can include a non-natural promoter inserted in a position equivalent to its natural background. For example, the microorganism of the first species can include a gene operably connected to a natural promoter of the gene in the second species.

[0143] In some cases, the regulatory sequence can be inserted into the upstream of the gene to be operably connected to the gene. In some cases, inserting the regulatory sequence upstream of the gene may also involve the natural regulatory sequence of the missing gene. In some cases, the regulatory sequence can be inserted into the upstream of the Nif cluster gene to control the Nif cluster gene. In some cases, the regulatory sequence can be inserted into the upstream of the transporter gene to control the transporter gene. In some cases, the regulatory sequence can be inserted into the upstream of the ion transporter gene to control the ion transporter gene. In some cases, the regulatory sequence can be inserted into the upstream of the CysZ gene to control the CysZ gene. In some cases, the regulatory sequence can be inserted into the upstream of the exopolysaccharide biosynthesis gene to control the exopolysaccharide biosynthesis gene. In some cases, the regulatory sequence can be inserted into the upstream of the cellulose biosynthesis gene to control the cellulose biosynthesis gene. In some cases, the regulatory sequence can be inserted into the upstream of the bcs gene to control the bcs gene. In some cases, the regulatory sequence can be inserted into the upstream of the trehalose biosynthesis gene to control the trehalose biosynthesis gene. In some cases, a regulatory sequence can be inserted upstream of the treZ gene to control the treZ gene. In some cases, a regulatory sequence can be inserted to increase the expression of a gene having a desired phenotype. Desired phenotypes can include nitrogen fixation, ammonium excretion, transport of components required for nitrogenase cofactors, biofilm formation, plant colonization, rhizosphere adaptability or competitiveness, scavenging of reactive oxygen species, expression of plant cell wall degrading enzymes, and root attachment.

[0144] In some cases, the regulatory sequence can be selected so that the genus of the origin microorganism of the regulatory sequence is the same as the genus of the microorganism into which it is inserted. In some cases, the regulatory sequence can be selected so that the origin species of the regulatory sequence is the same as the species into which it is inserted. For example, the regulatory element identified in the first sucrose ... In some cases, a sequence selected from the group consisting of: SEQ ID No. 11-40 is used to modify Klebsiella variegata. In some cases, SEQ ID NO. 60 is used to modify Kluyvera intermedia. In some cases, a sequence selected from the group consisting of: SEQ ID No. 1-10 is used to modify Osteria pseudosucrose: SEQ ID No. 61 and 70-72. In some cases, SEQ ID NO. 62 is used to modify Enterobacter bacteria. In some cases, a sequence selected from the group consisting of: SEQ ID No. 67-69 is used to modify Klebsiella bacteria. In some cases, the regulatory sequence can be selected so that the species of origin of the regulatory sequence is the same as the species into which it is inserted, but the regulatory sequence can contain one of more mutations.

[0145] In some cases, regulatory sequences can be selected so that the species of origin of the regulatory sequence is different from the species into which it is inserted. For example, regulatory elements identified in the bacterium Oxycoperdon sucrose were inserted into Klebsiella variabilis, thereby generating a genetically modified Klebsiella variabilis containing the intergenic DNA sequence.

[0146] In general, the term "genetic variation" refers to any change introduced into a polynucleotide sequence relative to a reference polynucleotide, such as a reference genome or a portion thereof, or a reference gene or a portion thereof. Genetic variation may be referred to as a "mutation", and a sequence or organism comprising a genetic variation may be referred to as a "genetic variant" or a "mutant". Genetic variation may have any number of effects, such as a biological activity, including an increase or decrease in gene expression, metabolism, and cell signaling. Genetic variation may be specifically introduced into a target site, or introduced randomly. A variety of molecular tools and methods may be used to introduce genetic variation. For example, genetic variation may be introduced by: polymerase chain reaction mutagenesis, oligonucleotide directed mutagenesis, saturation mutagenesis, fragment shuffling mutagenesis, homologous recombination, recombineering, lambda red-mediated recombination, CRISPR / Cas9 system, chemical mutagenesis, and combinations thereof. Chemical methods for introducing genetic mutations include exposing DNA to chemical mutagens, such as ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-nitrosourea (ENU), N-methyl-N-nitro-N'-nitrosoguanidine, 4-nitroquinoline N-oxide, diethyl sulfate, benzopyrene, cyclophosphamide, bleomycin, triethylmelamine, acrylamide monomer, nitrogen mustard, vincristine, diepoxyalkanes (e.g., diepoxybutylene), ICR-170, formaldehyde, procarbazine hydrochloride, ethylene oxide, dimethylnitrosamine, 7,12-dimethylbenz(a)anthracene, chlorambucil, hexamethylphosphoramide, busulfan, etc. Radiation mutation inducers include ultraviolet radiation, gamma radiation, X-rays, and fast neutron bombardment. Genetic mutations can also be introduced into nucleic acids using, for example, trimethylpsoralen accompanied by ultraviolet light. Mobile DNA elements, such as random or targeted insertion of transposable elements (transposable element) are another suitable method for generating gene variation.Gene variation can be introduced into nucleic acid during amplification in a cell-free in vitro system, for example, using polymerase chain reaction (PCR) techniques such as error-prone PCR.Gene variation can be introduced into nucleic acid in vitro using DNA shuffling techniques (e.g., exon shuffling, domain swapping, etc.).Due to the lack of DNA repair enzymes in cells, gene variation can also be introduced into nucleic acid, for example, it is expected that there are mutant genes encoding mutant DNA repair enzymes in cells that will generate high-frequency mutations (i.e., about 1 mutation / 100 genes to 1 mutation / 10,000 genes) in the cell genome.Examples of genes encoding DNA repair enzymes include, but are not limited to, Mut H, Mut S, Mut L and Mut U, and their homologues in other species (e.g., MSH 1 6, PMS1 2, MLH 1, GTBP, ERCC-1, etc.).Examples of various methods for introducing genetic variation are described in, for example, Stemple (2004) Nature 5: 1-7; Chiang et al. (1993) PCR Methods Appl 2(3): 210-217; Stemmer (1994) Proceedings of the National Academy of Sciences of the United States of America 91: 10747-10751; and U.S. Pat. Nos. 6,033,861 and 6,773,900.

[0147] The genetic variation introduced into a microorganism can be classified as transgenic, cisgenic, intragenomic, intrageneric, intergeneric, synthetic, evolutionary, rearrangement, or SNP.

[0148] Genetic variation can be introduced into many metabolic pathways within microorganisms to induce improvements in the traits described above. Representative pathways include sulfur uptake pathways, glycogen biosynthesis, glutamine regulatory pathways, molybdenum uptake pathways, nitrogen fixation pathways, ammonia assimilation, ammonia excretion or secretion, nitrogen uptake, glutamine biosynthesis, anaerobic ammonia oxidation (annamox), phosphate solubilization, organic acid transport, organic acid production, lectin production, reactive oxygen radical scavenging genes, indoleacetic acid biosynthesis, trehalose biosynthesis, plant cell wall degrading enzymes or pathways, root attachment genes, exopolysaccharide secretion, glutamate synthase pathways, iron uptake pathways, siderophore pathways, chitinase pathways, ACC deaminase, glutathione biosynthesis, phosphorus signaling genes, quorum quenching pathways, cytochrome pathways, hemoglobin pathways, bacterial hemoglobin-like pathways, small RNA rsmZ, rhizobium toxin biosynthesis, lapA adhesion proteins, AHL quorum sensing pathways, phenazine biosynthesis, cyclolipopeptide biosynthesis, and antibiotic production.

[0149] The CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) / CRISPR-associated (Cas) system can be used to introduce desired mutations. CRISPR / Cas9 provides bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNA (crRNA) to guide the silencing of invading nucleic acids. The Cas9 protein (or its functional equivalents and / or variants, i.e., Cas9-like proteins) naturally contains DNA endonuclease activity, which relies on the protein's association with two naturally occurring or synthetic RNA molecules called crRNA and tracrRNA (also called guide RNA). In some cases, the two molecules are covalently linked to form a single molecule (also referred to as a single guide RNA ("sgRNA"). Thus, the Cas9 or Cas9-like protein is associated with a DNA targeting RNA (the term encompasses both dual-molecule guide RNA configurations and single-molecule guide RNA configurations), which activates the Cas9 or Cas9-like protein and guides the protein to the target nucleic acid sequence. If the Cas9 or Cas9-like protein retains its natural enzymatic function, it will cut the target DNA to produce a double-strand break, which can result in genomic changes (i.e., edits: deletions, insertions (when a donor polynucleotide is present), substitutions, etc.), thereby altering gene expression. Some variants of Cas9 (which are encompassed by the term Cas9-like) have been altered so that they have reduced DNA cleavage activity (in some cases, they cut a single strand instead of both strands of the target DNA, and in other cases, they are severely reduced to no DNA cleavage activity). Other exemplary descriptions of CRISPR systems for introducing genetic variations can be found, for example, in US8795965.

[0150] As a cyclic amplification technique, polymerase chain reaction (PCR) mutagenesis uses mutagenic primers to introduce desired mutations. PCR is performed through cycles of denaturation, ligation, and extension. After PCR amplification, selection of mutant DNA and removal of parental plasmid DNA can be achieved by: 1) replacing dCTP with hydroxymethylated-dCTP during PCR, followed by restriction enzyme digestion to remove only non-hydroxymethylated parental DNA; 2) mutagenesis of antibiotic resistance genes and the gene of interest simultaneously, changing the plasmid to a different antibiotic resistance, which helps to select the desired mutations thereafter; 3) after the desired mutations are introduced, digesting the parental methylated template DNA with the restriction enzyme Dpnl that only cuts methylated DNA, thereby recovering the mutagenized unmethylated chain; or 4) circularizing the mutant PCR product in an additional ligation reaction to increase the conversion efficiency of the mutant DNA. Further description of exemplary methods can be found in, for example, US7132265, US6713285, US6673610, US6391548, US5789166, US5780270, US5354670, US5071743, and US20100267147.

[0151] Oligonucleotide directed mutagenesis, also referred to as site-directed mutagenesis, typically utilizes synthetic DNA primers.This synthetic primer contains the desired mutation, and is complementary to the template DNA around the mutation site, so it can be hybridized with the DNA in the gene of interest.The mutation can be a single base change (point mutation), a plurality of base changes, a deletion or an insertion or a combination of these.Then, the DNA polymerase extension single-stranded primer is used to replicate the remainder of the gene.The gene so replicated contains the mutation site, and then it can be introduced into a host cell and cloned as a vector.Finally, a mutant can be selected by DNA sequencing to check that it contains the desired mutation.

[0152] Genetic variation can be introduced using error-prone PCR. In this technique, a DNA polymerase is used to amplify a gene of interest in the absence of fidelity for sequence replication. The result is that the amplified product contains at least one error in the sequence. When a gene is amplified and the resulting (multiple) products of the reaction contain one or more changes in sequence when compared to the template molecule, the resulting product is mutagenized compared to the template. Another means of introducing random mutations is to expose the cells to chemical mutagens, such as nitrosoguanidine or ethyl methanesulfonate (Nestmann, Mutat Res, June 1975; 28(3): 323-30), and then isolate the vector containing the gene from the host.

[0153] Saturation mutagenesis is another form of random mutagenesis, in which people attempt to generate all or nearly all possible mutations at a specific site or narrow region of a gene. In a broad sense, saturation mutagenesis comprises a complete set of mutagenic cassettes (wherein the length of the sequence to be mutagenized is, for example, 15 to 100,000 bases) in a polynucleotide sequence to be mutagenized that is determined by mutagenesis (wherein the length of the sequence to be mutagenized is, for example, 15 to 100,000 bases). Therefore, a group of mutations (for example, within the range of 1 to 100 mutations) is introduced into each box to be mutagenized. In the process of applying a round of saturation mutagenesis, the grouping of the mutation to be introduced into a box can be different or identical from the second grouping of the mutation to be introduced into the second box. Such grouping is exemplified by the grouping of deletions, additions, specific codons, and specific nucleotide boxes.

[0154] Fragment shuffling mutagenesis, also referred to as DNA shuffling, is a mode of rapid propagation of beneficial mutations. In an example of the shuffling process, DNA enzyme is used to fragment a group of parental genes into fragments of length, for example, about 50-100bp. This is followed by polymerase chain reaction (PCR) without primers-DNA fragments with enough overlapping homologous sequences will be bonded to each other, and then extended by DNA polymerase. After some of the DNA molecules reach the size of the parental gene, it is allowed to carry out several rounds of this PCR extension. These genes can then be amplified with another PCR, this time adding a primer designed to be complementary to the end of the chain. Primer can have the additional sequence of addition at its 5 ' end, such as being connected to the sequence of the restriction enzyme recognition site required in the cloning vector. Other examples of shuffling techniques are provided in US20050266541.

[0155] Homologous recombination mutagenesis involves the recombination between an exogenous DNA fragment and a targeted polynucleotide sequence. After a double-strand break occurs, a DNA segment near the 5' end of the break is excised in a process referred to as excision. In the subsequent chain invasion step, the protruding 3' end of the broken DNA molecule then "invades" an unbroken similar or identical DNA molecule. The method can be used to delete genes, remove exons, add genes, and introduce point mutations. Homologous recombination mutagenesis can be permanent or conditional. Typically, a recombination template is also provided. The recombination template can be a component of another vector, contained in a separate vector, or provided as a separate polynucleotide. In certain embodiments, the recombination template is designed to serve as a template in homologous recombination, such as in or near a target sequence nicked or cut by a site-specific nuclease. The template polynucleotide can have any suitable length, such as about or greater than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000 or more nucleotides in length. In certain embodiments, the template polynucleotide is complementary to a portion of a polynucleotide comprising a target sequence. When optimally aligned, the template polynucleotide may overlap with one or more nucleotides (e.g., about or greater than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides) of the target sequence. In certain embodiments, when the template sequence and the polynucleotides comprising the target sequence are optimally aligned, the closest nucleotides of the template polynucleotide are within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000 or more nucleotides of the target sequence. Non-limiting examples of site-directed nucleases applicable in homologous recombination methods include zinc finger nucleases, CRISPR nucleases, TALE nucleases, and meganucleases. For further description of the use of such nucleases, see, for example, US8795965 and US20140301990.

[0156] Mutagens that primarily produce point mutations and short deletions, insertions, transversions and / or conversions, including chemical mutagens or radiation, can be used to produce genetic variation. Mutagens include, but are not limited to, ethyl methanesulfonate, methyl methanesulfonate, N-ethyl-N-nitrosourea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N'-nitro-nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12-dimethyl-benzo(a)anthracene, ethylene oxide, hexamethylphosphoramide, busulfan, diepoxyalkanes (diepoxyoctane, diepoxybutane, etc.), 2-methoxy-6-chloro-9[3-(ethyl-2-chloro-ethyl)aminopropylamino]acridine dihydrochloride, and formaldehyde.

[0157] Introducing gene variation may be an incomplete process, so that some bacteria in the treated bacterial population carry the desired mutation, while other bacteria do not. In some cases, it is desirable to apply selection pressure in order to enrich bacteria carrying the desired gene variation. Traditionally, the selection of successful gene variants involves selecting for or against certain functions that gene variation confer or abolishes, such as inserting antibiotic resistance genes or abolishing the metabolic activity that can convert non-lethal compounds into lethal metabolites. It is also possible to apply selection pressure based on the polynucleotide sequence itself, so that only the required gene variation (for example, no selectable marker is required in addition) needs to be introduced. In this case, the selection pressure can include cutting the genome lacking the gene variation introduced into the target site, so that the selection is effectively directed to the reference sequence seeking to introduce the gene variation therein. Typically, cutting occurs within 100 nucleotides of the target site (for example, within 75, 50, 25, 10 or less nucleotides from the target site, including cutting at or within the target site). Cutting can be guided by a site-specific nuclease selected from the group consisting of: zinc finger nuclease, CRISPR nuclease, TALE nuclease (TALEN) or meganuclease. Such a process is similar to that used to enhance homologous recombination at a target site, except that no template is provided for homologous recombination. As a result, bacteria lacking the desired genetic variation are more likely to undergo cleavage, which, if not repaired, will result in cell death. Bacteria that survive the selection can then be isolated for exposure to plants to assess the conferment of improved traits.

[0158] CRISPR nucleases can be used as site-specific nucleases to guide the cutting of target sites. Improved selection of mutant microorganisms can be obtained by killing non-mutated cells using Cas9. Plants are then inoculated with mutant microorganisms to reconfirm symbiosis and generate evolutionary pressure to select effective symbionts. Microorganisms can then be re-isolated from plant tissues. The CRISPR nuclease system for selecting non-variants can use elements similar to those described above for introducing genetic mutations, except that a template for homologous recombination is not provided. Therefore, cutting at the target site promotes the death of affected cells.

[0159] Other options for specific induction of cutting at the target site can be used, such as zinc finger nuclease, TALE nuclease (TALEN) system and meganuclease. Zinc finger nuclease (ZFN) is an artificial DNA endonuclease produced by fusing a zinc finger DNA binding domain with a DNA cleavage domain. ZFN can be engineered to target the desired DNA sequence, and this enables zinc finger nuclease to cut a unique target sequence. When introduced into a cell, ZFN can be used to edit the target DNA in a cell (e.g., a cell genome) by inducing double-strand breaks. Transcription activator-like effector nuclease (TALEN) is an artificial DNA endonuclease generated by fusing a TAL (transcription activator-like) effector DNA binding domain to a DNA cleavage domain. TALEN can be rapidly engineered to bind to almost any desired DNA sequence, and when introduced into a cell, TALEN can be used to edit the target DNA in a cell (e.g., a cell genome) by inducing double-strand breaks. Meganucleases (homing endonucleases) are endodeoxyribonucleases characterized by large recognition sites (double-stranded DNA sequences of 12 to 40 base pairs. Meganucleases can be used to replace, eliminate or modify sequences in a highly targeted manner. The targeting sequence can be altered by modifying its recognition sequence through protein engineering. Meganucleases can be used to modify all genome types, whether bacterial, plant or animal, and are generally grouped into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cyst box family and the HNH family. Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII.

[0160] Method disclosed herein can be used for introducing or improving one or more in multiple desired proterties.The example of the proterties that can be introduced or improved comprises: root biomass, root length, height, bud length, leaf number, water use efficiency, total biomass, output, fruit size, grain size, photosynthesis rate, drought tolerance, heat tolerance, salt tolerance, resistance to nematode stress, resistance to fungal pathogens, resistance to bacterial pathogens, resistance to viral pathogens, metabolite level and protein group expression.Desired proterties, comprise height, total biomass, root and / or bud biomass, seed germination, seedling survival, photosynthetic efficiency, transpiration rate, seed / fruit number or quality, plant grain or fruit yield, leaf chlorophyll content, photosynthetic rate, root length or its any combination can be used for measuring growth, and compare with the growth rate of reference agricultural plant (for example, plant without improved proterties) grown under the same conditions.

[0161] As described herein, a preferred trait to be introduced or improved is nitrogen fixation. In some cases, plants produced by the methods described herein exhibit a trait difference that is at least about 5% greater, such as 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 greater, compared to a reference agricultural plant grown in soil under the same conditions. In other examples, plants produced by the methods described herein exhibit a trait difference that is at least about 5% greater, such as 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 greater, compared to a reference agricultural plant grown in soil under similar conditions.

[0162] The trait to be improved can be assessed under conditions including application of one or more biological or abiotic stress sources. Examples of stress sources include abiotic stress (such as heat stress, salt stress, drought stress, cold stress, and low nutrient stress) and biotic stress (such as nematode stress, insect herbivory stress, fungal pathogen stress, bacterial pathogen stress, and viral pathogen stress).

[0163] The trait improved by the methods and compositions of the present disclosure can be nitrogen fixation, including in plants that could not previously fix nitrogen. In some cases, the bacteria isolated according to the methods described herein produce 1% or more (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20% or more) of plant nitrogen compared to the bacteria isolated from the first plant before introducing any genetic variation, which can represent an increase in nitrogen fixation capacity by at least 2 times (e.g., 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 50 times, 100 times, 1000 times or more). In some cases, bacteria produce 5% or more of plant nitrogen. After repeating the steps of introducing genetic variation, being exposed to a variety of plants and isolating bacteria from plants with improved traits one or more times (e.g., 1, 2, 3, 4, 5, 10, 15, 25 or more times), desired levels of nitrogen fixation can be achieved. In some cases, increased levels of nitrogen fixation are achieved in the presence of fertilizers supplemented with glutamine, ammonia or other chemical nitrogen sources.Methods of assessing the extent of nitrogen fixation are known, examples of which are described herein.

[0164] Microbial breeding is a method to systematically identify and improve the role of species within the crop microbiome. The method involves three steps: 1) selecting candidate species by mapping plant-microbe interactions and predicting regulatory networks associated with specific phenotypes, 2) practical and predictable improvement of microbial phenotypes through intraspecific hybridization of regulatory networks and gene clusters, and 3) screening and selecting new microbial genotypes that produce desired crop phenotypes. In order to systematically assess strain improvements, a model was created that links the colonization dynamics of microbial communities to the genetic activity of key species. The model was used to predict genetic target breeding and increase the frequency of selecting microbiome-encoded agronomically relevant trait improvements.

[0165] Nitrogen fixation

[0166] Described herein is a method for increasing nitrogen fixation in plants, comprising exposing the plant to bacteria comprising one or more genetic variations introduced into one or more genes regulating nitrogen fixation, wherein the bacteria produce 1% or more nitrogen (e.g., 2%, 5%, 10% or more) in the plant, which can represent at least 2 times the nitrogen fixation capacity compared to the plant in the absence of bacteria. The bacteria can produce nitrogen in the presence of fertilizers supplemented with glutamine, urea, nitrates, or ammonia. The genetic variation can be any number and any combination of any genetic variations described herein (including the examples provided above). The genetic variation may be introduced into a gene selected from the group consisting of: nifA, nifL, ntrB, ntrC, glutamine synthetase, glnA, glnB, glnK, draT, amtB, glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, and nifQ. The genetic variation may be a mutation that causes one or more of: increased expression or activity of ni nifA or glutaminase; decreased expression or activity of nifL, ntrB glutamine synthetase, glnB, glnK, draT, amtB; decreased adenylyl removal activity of GlnE; or decreased uridylyl removal activity of GlnD. The genetic variation introduced into one or more bacteria of the methods disclosed herein can be a knockout mutation or it can abolish the regulatory sequence of the target gene, or it can comprise the insertion of a heterologous regulatory sequence, for example, a regulatory sequence found in the genome of the same bacterial species or genus. Regulatory sequences can be selected based on the gene expression level in bacterial culture or in plant tissue. Genetic variation can be produced by chemical mutagenesis. Plants grown in step (c) can be exposed to biological or abiotic stress sources.

[0167] The amount of nitrogen fixation occurring in the plants described herein can be measured in several ways, such as by acetylene reduction (AR) analysis. Acetylene reduction analysis can be performed in vitro or in vivo. Evidence that a particular bacterium provides fixed nitrogen to a plant may include: 1) a significant increase in total plant N after inoculation, preferably accompanied by an increase in N concentration in the plant; 2) a reduction in nitrogen deficiency symptoms (which should include an increase in dry matter) under N-limited conditions after inoculation; 3) an increase in nitrogen deficiency symptoms (which should include an increase in dry matter) after inoculation; 4) an increase in nitrogen deficiency symptoms (which should include an increase in dry matter) after inoculation; 5) an increase in nitrogen deficiency symptoms (which should include an increase in dry matter) after inoculation; 6) an increase in nitrogen deficiency symptoms (which should include an increase in nitrogen deficiency) after inoculation; 7) an increase in nitrogen deficiency symptoms (which should include an increase in nitrogen deficiency) after inoculation; 8) an increase in nitrogen deficiency symptoms (which should include an increase in nitrogen deficiency) after inoculation; 9) an increase in nitrogen deficiency symptoms (which should include an increase in nitrogen deficiency) after inoculation; 10) an increase in nitrogen deficiency symptoms (which should include an increase in nitrogen deficiency) after inoculation; 11) an increase in nitrogen deficiency symptoms (which should include an increase in nitrogen deficiency) after inoculation; 12) an increase in nitrogen deficiency symptoms (which should include an increase in nitrogen deficiency) after inoculation; 13) an increase in nitrogen deficiency symptoms (which should include an increase in nitrogen deficiency) after inoculation; 14) an increase in nitrogen deficiency symptoms (which should include an increase in nitrogen deficiency) after inoculation; 15 15 N method (which may be an isotope dilution experiment, 15 N 2 Restore analysis or 15 N natural abundance analysis) records N 2 fixation; 4) the fixed N is incorporated into plant proteins or metabolites; 5) all of these effects were not seen in uninoculated plants or inoculated plants with mutants of the inoculated strain.

[0168] The wild-type nitrogen fixation regulatory cascade can be represented as a digital logic circuit, where the input O 2 and NH 4 + The ATP is passed through the NOR gate, and the output of the NOR gate enters the AND gate in addition to the ATP. In some embodiments, the methods disclosed herein interfere with NH at multiple points in the regulatory cascade. 4 + Influencing this circuit allows microorganisms to produce nitrogen even in fertilized fields. However, the methods disclosed herein also contemplate altering ATP or O 2 The influence on the circuit system, or replace the circuit system with other control cascades in the cell, or change the genetic circuit except nitrogen fixation. The gene cluster can be re-engineered to generate functional products under the control of a heterologous control system. By eliminating the natural control elements outside and inside the gene cluster coding sequence, and replacing it with an alternative control system, the functional products of the composite genetic operon and other gene clusters can be controlled and / or the products can be moved to heterologous cells, including cells of other species except the species from which the natural gene is derived. Once re-engineered, the synthetic gene cluster can be controlled by genetic circuits or other inducible control systems, thereby controlling the expression of the product as needed. The expression cassette can be designed to serve as a logic gate, a pulse generator, an oscillator, a switch or a memory device. The control expression cassette can be connected to a promoter so that the expression cassette serves as an environmental sensor, such as oxygen, temperature, contact, osmotic stress, membrane stress or a redox sensor.

[0169] For example, nifL, nifA, nifT and nifX genes can be eliminated from the nif gene cluster. Synthetic genes can be designed by randomizing the codons of the DNA encoding each amino acid sequence. Codon selection is performed to specify that the codon usage is as different as possible from the codon usage in the natural gene. Any undesirable features in the proposed sequence are scanned, such as restriction enzyme recognition sites, transposon recognition sites, repetitive sequences, σ54 σ70 promoters, hidden ribosome binding sites and non-rho dependent terminators. Synthetic ribosome binding sites are selected to match the intensity of each corresponding natural ribosome binding site, such as by constructing a fluorescent reporter plasmid, in which 150bp (-60 to +90) around the gene start codon is fused with a fluorescent gene. This chimera can be expressed under the control of the Ptac promoter, and fluorescence is measured by flow cytometry. In order to generate a synthetic ribosome binding site, a 150bp (-60 to +90) reporter plasmid library using a synthetic expression cassette is generated. In simple terms, the synthetic expression cassette can be composed of a random DNA spacer, a degenerate sequence encoding a RBS library, and the coding sequence of each synthetic gene. Multiple clones are screened to identify the synthetic ribosome binding site that best matches the natural ribosome binding site. Therefore, a synthetic operon consisting of the same genes as the natural operon is constructed, and its functional complementarity is tested. A further exemplary description of a synthetic operon is provided in US20140329326.

[0170] Bacterial species

[0171] Microorganisms suitable for methods and compositions disclosed herein can be obtained from any source. In some cases, the microorganism can be a bacterium, an archaebacterium, a protozoa or a fungus. The microorganism disclosed herein can be a nitrogen-fixing microorganism, such as a nitrogen-fixing bacterium, a nitrogen-fixing archaebacterium, a nitrogen-fixing fungus, a nitrogen-fixing yeast or a nitrogen-fixing protozoa. Microorganisms suitable for methods and compositions disclosed herein can be spore-forming microorganisms, such as spore-forming bacteria. In some cases, bacteria suitable for methods and compositions disclosed herein can be gram-positive bacteria or gram-negative bacteria. In some cases, bacteria can be endospore-producing bacteria of the Firmicutes phylum. In some cases, bacteria can be nitrogen-fixing organisms. In some cases, bacteria may not be nitrogen-fixing organisms.

[0172] The methods and compositions of the present disclosure can be used with Archaea (eg, Methanothermobacter thermoautotrophicus).

[0173] In some cases, potentially useful bacteria include, but are not limited to, Agrobacterium radiobacter, Bacillus acidocaldarius, Bacillus acidoterrestris, Bacillus agri, Bacillus aizawai, Bacillus albolactis, Bacillus alcalophilus, Bacillus alvei, Bacillus aminoglucosidicus, Bacillus aminovorans, Bacillus amylolyticus (also known as Paenibacillus amylolyticus), Bacillus amyloliquefaciens, Bacillus aneurinolyticus, Bacillus atrophaeus, Bacillus thiamine spores ... atrophaeus, Bacillus azotoformans, Bacillus badius, Bacillus cereus (synonyms: Bacillus endorhythmos, Bacillus medusa), Bacillus chitinosporus, Bacillus circulans, Bacillus coagulans, Bacillus endoparasiticus, Bacillus fastidiosus, Bacillus firmus, Bacillus kurstaki, Bacillus lacticola, Bacillus lactimorbus, Bacillus lactis, Bacillus laterosporus laterosporus (also known as Brevibacilluslaterosporus), Bacilluslautus, Bacillus lentimorbus, Bacillus lentus, Bacillus licheniformis, Bacillus maroccanus, Bacillus megaterium, Bacillus metiens, Bacillus mycoides, Bacillus natto, Bacillusnematocida, Bacillus nigrificans, Bacillus nigrum, Bacillus pantothenticus, Bacillus popillae, Bacillus psychrosaccharolyticus, Bacillus brevis pumilus), Bacillus siamensis, Bacillus smithii, Bacillus sphaericus, Bacillus subtilis, Bacillus thuringiensis, Bacillus uniflagellatus, Bradyrhizobium japonicum, Brevibacillus brevis, Brevibacillus laterosporus (Bacillus laterosporus), Chromobacterium subtsugae, Delftia acidovorans, Lactobacillus acidophilus, Lysobacter antibioticus), Lysobacterenzymogenes, Paenibacillus alvei, Paenibacilluspolymyxa, Paenibacillusjapanensispopilliae (formerly Bacillus japonicus), Pantoea agglomerans, Pasteuria penetrans (formerly Bacillus penetrans), Pasteuria usgae, Pectobacterium carotovorum (formerly Erwinia carotovora), Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas cepacia (formerly Burkholderia cepacia), Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas root-supporting bacteria (formerly Burkholderia cepacia). proradix), Pseudomonas putida, Pseudomonas syringae, Serratia entomophila, Serratia marcescens, Streptomyces colombiensis, Streptomyces galbus, Streptomyces goshikiensis, Streptomyces griseoviridis, Streptomyces lavendulae, Streptomyces prasinus, Streptomyces saraceticus, Streptomyces venezuelae, Xanthomonas campestris, Xenorhabdus luminescens), Xenorhabdus nematophila, Rhodococcus globerulus AQ719 (NRRL deposit number B-21663), Bacillus AQ175 (ATCC deposit number 55608), Bacillus AQIn some cases, the bacterium may be Azotobacter chroococcum, Methanosarcina barkeri, Klebsiella pneumoniae, Azotobacter vinelandii, Rhodobacter spharoides, Rhodobacter capsulatus, Rhodobacter palustris, Rhodosporillum rubrum, Rhizobium leguminosarum, or Rhizobium etli.

[0174] In some cases, the bacterium can be a Clostridium species, such as Clostridium pasteurianum, Clostridium beijerinckii, Clostridium perfringens, Clostridium tetani, Clostridium acetobutylicum.

[0175] In some cases, the bacteria used with the methods and compositions of the present disclosure can be cyanobacteria. Examples of cyanobacteria include Anabaena (e.g., Anabaena PCC7120), Nostoc (e.g., Nostoc punctiforme), or Synechochocystis (e.g., Synechochocystis PCC6803).

[0176] In some cases, bacteria used with the methods and compositions of the present disclosure may belong to the phylum Chlorobi, such as Chlorobium tepidum.

[0177] In some cases, the microorganisms used with the methods and compositions of the present disclosure may comprise genes homologous to known NifH genes. Sequences of known NifH genes can be found, for example, in the Zehr lab NifH database (wwwzehr.pmc.ucsc.edu / nifH_Database_Public / , April 4, 2014), or in the Buckley lab NifH database (www.css.cornell.edu / faculty / buckley / nifh.htm, and Gaby, John Christian and Daniel H. Buckley. "A comprehensive aligned nifH gene database: a multipurpose tool for studies of nitrogen-fixing bacteria." Database 2014 (2014): bau001.). In some cases, a microorganism used with the methods and compositions of the present disclosure may comprise a sequence encoding a polypeptide having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 96%, 98%, 99%, or more than 99% sequence identity to a sequence from the Zehr Laboratory NifH Database (https: / / wwwzehr.pmc.ucsc.edu / nifH_Database_Public / , April 4, 2014). In some cases, a microorganism used with the methods and compositions of the present disclosure may comprise a sequence encoding a polypeptide having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 96%, 98%, 99%, or more than 99% sequence identity to a sequence from the Buckley Laboratory NifH database (Gaby, John Christian, and Daniel H. Buckley. "Comprehensive alignment of the nifH gene database: a versatile tool for studying nitrogen-fixing bacteria." Database 2014 (2014): bau001.).

[0178] Microorganisms suitable for the methods and compositions disclosed herein can be obtained by extracting microorganisms from the surface or tissue of natural plants; grinding seeds to isolate microorganisms; planting seeds in different soil samples and recovering microorganisms from tissues; or inoculating plants with exogenous microorganisms and determining which microorganisms appear in plant tissues. Non-limiting examples of plant tissues include seeds, seedlings, leaves, cuttings, plants, bulbs or tubers. In some cases, bacteria are isolated from seeds. The parameters used to process samples can be changed to separate different types of associated microorganisms, such as rhizobacteria, epiphytes or endophytes. Bacteria can also be obtained from a repository such as an environmental strain collection, rather than initially isolated from the first plant. Microorganisms can be genotyped and phenotyped by sequencing the genome of isolated microorganisms; performing community composition profile analysis in plants; characterizing the transcriptomic function of communities or isolated microorganisms; or using selective or phenotypic media to screen microbial characteristics (e.g., nitrogen fixation or phosphate solubility phenotypes). The selected candidate strains or populations can be obtained by: sequence data; phenotypic data; plant data (e.g., genomic, phenotypic and / or yield data); soil data (e.g., pH, N / P / K content and / or bulk soil biome); or any combination of these.

[0179] Bacteria described herein and methods for producing bacteria can be applied to bacteria that can effectively self-propagate on the surface of leaves, the surface of roots or inside plant tissues without inducing harmful plant defense responses, or bacteria that are resistant to plant defense responses. Bacteria described herein can be isolated by culturing plant tissue extracts or leaf surface washings in a medium without the addition of nitrogen. However, bacteria may be uncultivable, that is, it is not known that standard methods known in the art can be used for cultivation, or it is difficult to cultivate using standard methods known in the art. Bacteria described herein may be endophytes or epiphytes or bacteria that live in the rhizosphere of plants (rhizobacteria). Repeating the steps of introducing genetic variation, exposure to a variety of plants, and isolating bacteria from plants with improved traits once or multiple times (e.g., 1, 2, 3, 4, 5, 10, 15, 25 times or more) to obtain bacteria may be endophytes, epiphytes or rhizobacteria. Endophytes are organisms that enter the interior of plants without causing disease symptoms or inducing the formation of symbiotic structures, and are concerned by agronomy because they can enhance plant growth and improve plant nutrition (e.g., by nitrogen fixation). The bacterium can be a seed-borne endophyte. Seed-borne endophytes include bacteria associated with or derived from grass or plant seeds, such as those found in mature, dry, undamaged (e.g., without cracks, visible fungal infection, or premature germination) seeds. Seed-borne bacterial endophytes can be associated with or derived from the surface of the seed; alternatively or in addition, they can be associated with or derived from internal seed compartments (e.g., surface-sterilized seeds). In some cases, seed-borne bacterial endophytes are able to replicate within plant tissues, such as inside seeds. Furthermore, in some cases, seed-borne bacterial endophytes are able to withstand dehydration.

[0180] Bacteria separated according to the method of the present invention, or bacteria used in the method or composition of the present invention, can include a combination of multiple different bacterial taxa. As an example, bacteria can include Proteobacteria (such as Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azotobacter, Duganella, Delftia, etc.). The bacteria used in the methods and compositions of the present invention may include nitrogen-fixing bacteria consortia of two or more species. In some cases, one or more bacteria of the bacterial consortia may be capable of fixing nitrogen. In some cases, one or more species of the bacterial consortia may promote or enhance the ability of other bacteria to fix nitrogen. The bacteria that fix nitrogen and the bacteria that enhance the nitrogen-fixing ability of other bacteria may be the same or different. In some instances, a bacterial strain may be able to fix nitrogen when combined with different bacterial strains or in a bacterial consortium, but may not be able to fix nitrogen in a single culture. Examples of genera of bacteria that can be found in nitrogen-fixing bacterial consortia include, but are not limited to, Grass Spirillum, Azospirillum, Enterobacter, and Bacillus.

[0181] Bacteria that can be produced by the methods disclosed herein include Azotobacter, Brachyrhizobium, Klebsiella and Sinorhizobium. In some cases, bacteria can be selected from the group consisting of: Azotobacter vinelandii, Chronic soybean rhizobium, Klebsiella pneumoniae and Sinorhizobium meliloti. In some cases, bacteria can be Enterobacter or Rahnella. In some cases, the bacteria can be Frankia or Clostridium. Examples of Clostridium bacteria include but are not limited to Clostridium acetobutylicum, Clostridium pasteurianum, Clostridium beijerinckii, Clostridium perfringens and Clostridium tetani. In some cases, the bacteria can be of the genus Paenibacillus, such as Paenibacillus azotofixans, Paenibacillus borealis, Paenibacillus durus, Paenibacillus macerans, Paenibacillus polymyxa, Paenibacillus alvei, Paenibacillus amylolyticus, Paenibacillus campinasensis, Paenibacillus chibensis, Paenibacillus glucanolyticus, Paenibacillus sillinoisensis, Paenibacillus larvae subsp. Larvae, Paenibacillus larvae subsp. subsp. Pulvifaciens), Paenibacillus lautus, Paenibacillus macerans, Paenibacillus macquariensis, Paenibacillus pabuli, Paenibacillus peoriae or Paenibacillus polymyxa.

[0182] In some examples, the bacteria isolated according to the methods of the present disclosure can be members of one or more of the following taxonomic groups: Achromobacter, Acidithiobacillus, Acidovorax, Acidovoraz, Acinetobacter, Actinoplanes, Adlercreutzia, Aerococcus, Aeromonas, Afipia, Agromyces, Arthrobacter, Atopostipes, Azospirillum, Bacillus, Bdellovibrio, Beijerinckia, Bosea, Brevibacillus, Brevibacillus, Brevundimonas, Burkholderia, Candidatus Haloredivivus, Caulobacter, Cellulomonas, Cellvibrio ), Chryseobacterium, Citrobacter, Clostridium, Coraliomargarita, Corynebacterium, Cupriavidus, Brevibacter, Curvibacter, Deinococcus, Delftia, Desemzia, Devosia, Dokdonella, Dyella, Enhydrobacter, Enterobacter Bacteria, Enterococcus, Erwinia, Escherichia, Escherichia / Shigella, Exiguobacterium, Ferroglobus, Filimonas, Finegoldia, Flavisolibacter, Flavobacterium, Frigoribacterium, Gluconacetobacter,Hafnia, Halobaculum, Halomonas, Halosimplex, Spirillum, Hymenobacter, Klebsiella, Kocuria, Oxycoperdon, Lactobacillus, Leclercia, Lentzea, Luteibacter, Luteimonas, Massilia, Mesorhizobium, Methylobacterium, Microbacterium , Micrococcus, Microvirga, Mycobacterium, Neisseria, Nocardia, Oceanibaculum, Ochrobactrum, Okibacterium, Oligotropha, Oryzihumus, Oxalophagus, Paenibacillus, Panteoa / Pantoea, Pelomonas nas), Perlucidibaca, Plantibacter, Polynucleobacter, Propionibacterium, Propioniciclava, Pseudoclavibacter, Pseudomonas, Pseudonocardia, Pseudoxanthomonas, Psychrobacter, Ralstonia, Rheinheimer Rheinheimera), Rhizobium, Rhodococcus, Rhodopseudomonas, Roseateles, Ruminococcus, Sebaldella, Sediminibacillus, Sediminibacterium, Serratia, Shigella, Shinella, Sinorhizobium, Sinosporangium, Sphingobacterium,Sphingomonas, Sphingopyxis, Sphingosinicella, Staphylococcus, 25-Sphingomonas, Sphingomonas, Streptococcus, Streptomyces, Stygiolobus, Sulfurisphaera, Tatumella, Tepidimonas, Thermomonas, Thiobacillus, Variovorax, WPS-2 incertae sedis, Xanthomonas, and Zimmermannella.

[0183] Bacteria can be obtained from any general terrestrial environment, including its soil, plants, fungi, animals (including invertebrates), and other biota, including sediments, water, and biota of lakes and rivers; from marine environments, their biota and sediments (e.g., seawater, marine mud, marine plants, marine invertebrates (e.g., sponges), marine vertebrates (e.g., fish)); terrestrial and marine lithosphere (topsoil and rocks, such as squeezed subsurface rocks, sands, and clays); cryosphere and its meltwater; atmosphere (e.g., filtered air dust, clouds, and raindrops); urban, industrial, and other man-made environments (e.g., organic matter and minerals accumulated on concrete, roadside gutters, roof surfaces, and road surfaces).

[0184] The plant from which bacterium is obtained can be a plant with one or more desired traits, such as a plant grown naturally in a specific environment or under specific conditions. As an example, a certain plant can be in sandy soil or high salinity sand, or at extreme temperatures, or grow naturally in the case of having very little water, or it can resist certain pests or diseases in the environment, and may need cash crops to grow in such conditions, especially when this is, for example, the only condition in a specific geographical location. As another example, cash crops grown in such environments, or more specifically, individual crop plants that best display the paid attention to trait from the crop grown in any specific environment collect bacteria: for example, the fastest growing plant in the crop grown in salt-restricted soil, or the plant with the lowest degree of damage in the crop exposed to severe insect damage or disease epidemic, or the plant with some metabolites of a desired amount and other compounds (including fiber content, oil content, etc.), or the plant showing the desired color, taste or smell. Bacteria can be collected from any material occurring in the plant or environment paid attention to, including fungi and other animal and plant biota, soil, water, sediment and other environmental elements as previously mentioned.

[0185] Bacteria can be separated from plant tissue. This separation can occur from any suitable tissue in the plant, including, for example, roots, stems and leaves, and plant reproductive tissue. As an example, the conventional method of separating from plants typically includes sterile excision of the plant material of interest (e.g., root or stem length, blade), surface sterilization with a suitable solution (e.g., 2% sodium hypochlorite), and then the plant material is placed on a nutrient medium to grow microorganisms. Alternatively, the surface-sterilized plant material can be crushed in a sterile liquid (usually water), and a liquid suspension (including the small pieces of the crushed plant material) is spread on the surface of one or more suitable solid agar mediums, which may or may not be selective (e.g., only contain phytic acid as a phosphorus source). This method is particularly suitable for forming isolated colonies and can be selected to plates of independent nutrient mediums, and is further purified into the bacteria of a single species by well-known methods. Alternatively, the plant root or leaf sample may not be surface sterilized, but only gently washed, thereby including surface-resident epiphytic microorganisms in the separation process, or the epiphytic microorganisms may be separated respectively by stamping and peeling off the root, stem or leaf pieces of the plant onto the agar medium surface, and then separating individual colonies as above. For example, this method is particularly suitable for bacteria. Alternatively, the root may be processed without washing off a small amount of soil attached to the root, thereby including microorganisms that colonize the rhizosphere of the plant. Otherwise, the soil adhering to the root may be removed, diluted and spread on agar of suitable selective and non-selective medium to separate individual colonies of rhizosphere bacteria.

[0186] Biologically pure cultures of Rahnella aquatica and Enterobacter sacchari were deposited with the American Type Culture Collection (ATCC; international depository) in Manassas, VA, USA on July 14, 2015, and were assigned ATCC patent deposit designation numbers PTA-122293 and PTA-122294, respectively. These deposits were made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations (Budapest Treaty).

[0187] Composition

[0188] Compositions comprising bacteria or bacterial populations produced according to the methods described herein and / or having characteristics as described herein can be in the form of liquids, foams or dry products. In some examples, compositions comprising bacterial populations can be in the form of dry powders, slurries of powders and water or flowable seed treatments.

[0189] The composition can be manufactured in bioreactors (e.g., continuous stirred tank reactors, batch reactors) and on farms. In some instances, the composition can be stored in containers such as jugs or in small bulk. In some instances, the composition can be stored in an object selected from the group consisting of: bottles, jars, ampoules, packages, vessels, bags, bags, boxes, storage boxes, encapsulations, cartons, containers, silos, shipping containers, carriages, and / or boxes.

[0190] Composition can also be used for improving plant traits. In some instances, one or more compositions can be coated on seed. In some instances, one or more compositions can be coated on seedling. In some instances, one or more compositions can be coated on the surface of seed. In some instances, one or more compositions can be coated as a layer above the seed surface. In some instances, the composition coated on the seed can be in liquid form, in dry product form, in foam form, in the slurry form of powder and water or in the form of flowable seed treatment agent. In some instances, one or more compositions can be applied to seed and / or seedling by spraying, immersing, coating, encapsulating and / or dusting with one or more compositions to seed and / or seedling. In some instances, multiple bacteria or bacterial populations can be coated on the seed and / or seedling of plant. In some examples, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more bacteria in the bacterial combination can be selected from one of the following genera: Acidovorax, Agrobacterium, Bacillus, Burkholderia, Chryseobacterium, Brevibacterium, Enterobacter, Escherichia, Methylobacterium, Paenibacillus, Pantoea, Pseudomonas, Ralstonia, Saccharibacillus, Sphingomonas and Stenotrophomonas.

[0191] In some examples, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more than ten bacteria and bacterial populations in the endophytic combination can be selected from one of the following families: Bacillaceae, Burkholderiaceae, Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae, Methylobacteriaceae, Microbacteriaceae, Paenibacillileae, Pseudomonnaceae, Rhizobiaceae, Sphingomonadaceae, Xanthomonadaceae, Cladosporiaceae, Gnomoniaceae, Incertae sedis), Lasiosphaeriaceae, Netriaceae, and Pleosporaceae.

[0192] In some examples, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more bacteria and bacterial populations in the endophytic combination can be selected from one of the following families: Bacillaceae, Burkholderiaceae, Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae, Methylobacteriaceae, Microbacteriaceae, Paenibacillusaceae, Pseudomonadaceae, Rhizobiaceae, Sphingomonasaceae, Xanthomonadaceae, Cladosporiumaceae, Gastromycetes, Undetermined, Trichosphaeriaceae, Comamonadaceae and Lachnosporaceae.

[0193] Examples of compositions may include seed coatings for commercially important crops, such as sorghum, mustard, tomatoes, strawberries, barley, rice, corn and wheat. Examples of compositions may also include seed coatings for corn, soybeans, mustard, sorghum, potatoes, rice, vegetables, cereals and oilseeds. Seeds as provided herein may be genetically modified organisms (GMOs), non-GMOs, organic or conventional. In some instances, the composition may be sprayed on the aerial parts of the plant, or applied to the roots by being inserted into furrows planted with plant seeds, watered in the soil or immersed in a suspension of the composition. In some instances, the composition may be dehydrated in a suitable manner to maintain cell viability and artificial inoculation and the ability to colonize the host plant. Bacterial species may be 108 to 10 10 The concentration between CFU / ml is present in the composition. In some instances, the composition can be supplemented with trace metal ions, such as platinum ions, iron ions, manganese ions or a combination of these ions. The ion concentration in the example of the composition as described herein can be between about 0.1mM and about 50mM. Some examples of the composition can also be prepared with a carrier, such as beta-glucan, carboxymethyl cellulose (CMC), bacterial extracellular polymeric substances (EPS), sugar, animal milk or other suitable carriers. In some instances, peat or planting materials can be used as carriers, or biopolymers can be used as carriers, wherein the composition is coated in the biopolymer.

[0194] Compositions comprising bacterial populations as described herein can be coated onto the surface of seeds. Therefore, compositions comprising seeds coated with one or more bacteria as described herein are also contemplated. Seed coating can be formed by mixing bacterial populations with porous, chemically inert granular carriers. Alternatively, the composition can be directly inserted into the furrows planted with seeds, or sprayed onto plant leaves, or applied by immersing roots in a suspension of the composition. An effective amount of the composition can be used to fill the subsoil area adjacent to the roots of plants with live bacterial growth, or to fill the blades of plants with live bacterial growth. In general, an effective amount is an amount sufficient to produce plants with improved traits (e.g., required nitrogen fixation levels).

[0195] Bacterial compositions described herein can be prepared using agriculturally acceptable carriers. The formulations applicable to these embodiments can include at least one member selected from the group consisting of: tackifiers, microbial stabilizers, fungicides, antibacterial agents, preservatives, stabilizers, surfactants, anti-complex agents, herbicides, nematicides, insecticides, plant growth regulators, fertilizers, rodenticides, desiccants, bactericides, nutrients or any combination thereof. In some instances, compositions can be storage-stable. For example, any one of the compositions described herein can include agriculturally acceptable carriers (for example, one or more of fertilizers (such as non-natural fertilizers), adhesives (such as non-natural adhesives) and pesticides (such as non-natural pesticides). The adhesives that are non-natural can be, for example, polymers, copolymers or synthetic waxes. For example, any one of the coated seeds, seedlings or plants described herein can contain such agriculturally acceptable carriers in seed coatings. In any of the compositions or methods described herein, an agriculturally acceptable carrier can be or can include a non-naturally occurring compound (e.g., a non-naturally occurring fertilizer, a non-naturally occurring binder (such as a polymer, copolymer, or synthetic wax), or a non-naturally occurring pesticide). Non-limiting examples of agriculturally acceptable carriers are described below. Other examples of agriculturally acceptable carriers are known in the art.

[0196] In some cases, the bacteria are mixed with an agriculturally acceptable carrier. The carrier can be a solid carrier or a liquid carrier, and in various forms, including microspheres, powders, emulsions, etc. The carrier can be any one or more of several carriers that impart multiple characteristics, such as improved stability, wettability or dispersibility. A wetting agent can be included in the composition, such as a natural or synthetic surfactant, which can be a nonionic or ionic surfactant, or a combination thereof. Oil-in-water emulsions can also be used to prepare compositions including isolated bacteria (see, for example, U.S. Patent No. 7,485,451). Suitable formulations that can be prepared include wettable powders, granules, gels, agar strips or granules, thickeners, etc., microencapsulated particles, etc., liquids, such as aqueous fluids, aqueous suspensions, oil-in-water emulsions, etc. The formulation can include cereals or leguminous plant products, such as ground cereals or beans, soups or powders, starch, sugars or oils derived from cereals or beans.

[0197] In certain embodiments, agricultural carrier can be soil or plant growth medium.Other agricultural carriers that can be used include water, fertilizer, plant oil, wetting agent or its combination.Or, agricultural carrier can be solid, such as diatomaceous earth, loam, silicon dioxide, alginate, clay, bentonite, vermiculite, seed shell, other plant and animal products or combination, including particle, granule or suspension.Also contemplate any mixture of the aforementioned components as carrier, such as but not limited to Pesta (pesta) (flour and kaolin), granule based on agar or flour in loam, sand or clay etc.Preparation can include food source of bacteria, such as barley, rice, or other biomaterials, such as seed, plant part, bagasse, shell or stalk from grain processing, ground plant material or wood, sawdust or small fiber from paper, fabric or wood recovery from construction site garbage.

[0198] For example, fertilizers can be used to help promote growth or provide nutrition to seeds, seedlings, or plants. Non-limiting examples of fertilizers include nitrogen, phosphorus, potassium, calcium, sulfur, magnesium, boron, chlorine, manganese, iron, zinc, copper, molybdenum, and selenium (or their salts). Other examples of fertilizers include one or more amino acids, salts, carbohydrates, vitamins, glucose, NaCl, yeast extract, NH 4 H 2 PO 4 NH 4 ) 2 SO 4In one embodiment, the preparation can include tackifiers or adhesives (referred to as adhesives) to help other active agents be attached to a substance (e.g., seed surface). Such agents are suitable for combining bacteria with a carrier (e.g., not a control agent of a biological agent) that can contain other compounds to produce a coating composition. Such compositions help produce a coating around a plant or seed to maintain contact between microorganisms and other agents and a plant or plant part. In one embodiment, the binder is selected from the group consisting of alginate, gum, starch, lecithin, formononetin, polyvinyl alcohol, alkali formononetin, hesperetin, polyvinyl acetate, cephalin, gum arabic, xanthan gum, mineral oil, polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), arabinogalactan, methylcellulose, PEG 400, chitosan, polyacrylamide, polyacrylate, polyacrylonitrile, glycerol, triethylene glycol, vinyl acetate, gellan gum, polystyrene, polyethylene, carboxymethyl cellulose, gum Ghatti, and polyoxyethylene-polyoxybutylene block copolymers.

[0199] In some embodiments, the binder can be, for example, wax, such as carnauba wax, beeswax, Chinese wax, shellac wax, spermaceti wax, candelilla wax, castor wax, ouricury wax, and rice bran wax; polysaccharides (e.g., starch, dextrin, maltodextrin, alginate, and chitosan); fats; oils; proteins (e.g., gelatin and zein); gum arabic; and shellac. The binder can be a non-naturally occurring compound, such as a polymer, a copolymer, and a wax. For example, non-limiting examples of polymers that can be used as binders include: polyvinyl acetate, polyvinyl acetate copolymers, ethylene vinyl acetate (EVA) copolymers, polyvinyl alcohol, polyvinyl alcohol copolymers, cellulose (e.g., ethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose and carboxymethyl cellulose), polyvinyl pyrrolidone, vinyl chloride, vinylidene chloride copolymers, calcium lignin sulfonate, acrylic acid copolymers, polyvinyl acrylate, polyethylene oxide, acyl amide polymers and copolymers, polyhydroxyethyl acrylate, methacrylamide monomers and polychloroprene.

[0200] In some examples, one or more of the adhesive, antifungal agent, growth regulator and pesticide (e.g., insecticide) is a non-naturally occurring compound (e.g., in any combination). Other examples of agriculturally acceptable carriers include dispersants (e.g., polyvinyl pyrrolidone / vinyl acetate PVPIVA S-630), surfactants, binders and fillers.

[0201] The formulation may also contain a surfactant. Non-limiting examples of surfactants include nitrogen surfactant blends such as Prefer 28 (Cenex), Surf-N (US), Inhance (Brandt), P-28 (Wilfarm), and Patrol (Helena); esterified seed oils include Sun-It II (AmCy), MSO (UAP), Scoil (Agsco), Hasten (Wilfarm), and Mes-100 (Drexel); and silicone surfactants include Silwet L77 (UAP), Silikin (Terra), Dyne-Amic (Helena), Kinetic (Helena), Sylgard 309 (Wilbur-Ellis), and Century (Precision). In one embodiment, the surfactant is present in a concentration between 0.01% v / v and 10% v / v. In another embodiment, the surfactant is present in a concentration between 0.1% v / v and 1% v / v.

[0202] In some cases, formulation includes microbial stabilizer. Such reagents may include desiccant, which may include any compound or mixture of compounds that may be classified as desiccant, regardless of whether one or more compounds are used in a concentration that actually has a drying effect on liquid inoculants. Such desiccant is ideally compatible with the bacterial population used, and should improve the ability of the microbial population to withstand the application on the seed and withstand drying. The example of a suitable desiccant includes one or more of trehalose, sucrose, glycerol and methylene glycol. Other suitable desiccant includes but is not limited to non-reducing sugars and sugar alcohols (e.g., mannitol or sorbitol). The amount of the desiccant introduced into the formulation may be in the range of about 5% to about 50% by weight / volume, for example, between about 10% to about 40%, between about 15% to about 35% or between about 20% to about 30%. In some cases, it is advantageous to include agents such as fungicides, antibacterial agents, herbicides, nematicides, insecticides, plant growth regulators, rodenticides, bactericides, or nutrients in the formulation. In some instances, agents may include protective agents that provide protection against pathogens that spread on the surface of the seed. In some instances, protective agents may provide a certain level of control over soil-borne pathogens. In some instances, protective agents may be effective primarily on the surface of the seed.

[0203] In some instances, fungicides can include compounds or agents that can inhibit fungal growth or kill fungi, whether chemical or biological. In some instances, fungicides can include compounds that can inhibit fungi or kill fungi. In some instances, fungicides can be protective agents, or agents that are mainly effective on seed surfaces, providing prevention of pathogens propagated on seed surfaces and providing a certain level of control of soil-borne pathogens. Non-limiting examples of protective agent fungicides include captan, maneb, thiram or fludioxonil.

[0204] In some instances, the fungicide can be a systemic fungicide that can be absorbed into the seedlings of emergence and inhibit or kill the fungi in the host plant tissue. The systemic fungicides used for seed treatment include but are not limited to the following: azoxystrobin, carboxin, mefenoxam, metalaxyl, thiabendazole, trifloxystrobin and various triazole fungicides, including difenoconazole, ipconazole, tebuconazole and triticonazole. Metalaxyl and metalaxyl are mainly used to target water molds Pythium and Phytophthora. Depending on the plant species, some fungicides are preferred compared to other fungicides because there are subtle differences in the sensitivity of pathogenic fungal species, or because there are differences in fungicide distribution or plant sensitivity. In some instances, the fungicide can be a biological control agent, such as a bacterium or a fungus. Such organisms can parasitize pathogenic fungi, or secrete toxins or other substances that can kill fungi or otherwise prevent fungal growth. Any type of fungicide, especially fungicides commonly used on plants, can be used as a control agent in the seed composition.

[0205] In some examples, the seed coating composition includes a control agent with antibacterial properties. In one embodiment, the control agent with antibacterial properties is selected from the compounds described elsewhere herein. In another embodiment, the compound is streptomycin, oxytetracycline, oxolinic acid, or gentamicin. Other examples of antibacterial compounds that can be used as part of the seed coating composition include those based on dichlorophen and benzyl alcohol hemiformal (from ICI or from Thor Chemie RS, and from Rohm & Haas MK 25) and isothiazolinone derivatives such as alkylisothiazolinone and benzisothiazolinone (from Thor Chemie MBS) compounds.

[0206] In some embodiments, the growth regulator is selected from the group consisting of abscisic acid, amidochlor, ancymidol, 6-benzylaminopurine, brassinolide, butralin, chlormequat (chlormequat chloride), choline chloride, cyclanilide, daminozide, dikegulac, dimethipin, 2,6-dimethylpurine, ethephon, flumetralin, flurprimidol, fluthiacet, forchlorfenuron, gibberellic acid, acid, inabenfide, indole-3-acetic acid, maleic hydrazide, mefluidide, mepiquat (mepiquatchloride), naphthylacetic acid, N-6-benzyladenine, paclobutrazol, prohexadione phosphorotrithioate, 2,3,5-triiodobenzoic acid, trinexapac-ethyl, and uniconazole. Other non-limiting examples of growth regulators include brassinosteroids, cytokinins (e.g., kinetin and zeatin), auxins (e.g., indoleacetic acid and indolyl acetyl aspartic acid), flavonoids and isoflavonoids (e.g., formononetin and diosmetin), phytoalexins (e.g., glyceolline), and phytoalexin-inducing oligosaccharides (e.g., pectin, chitin, chitosan, polygalacturonic acid and oligogalacturonic acid), and gibellerin. Such agents are ideally compatible with the agricultural seeds or seedlings to which the formulation is applied (e.g., they should not be harmful to the growth or health of the plant). In addition, the agent is ideally one that does not cause safety concerns for human, animal, or industrial use (e.g., there are no safety issues, or the compound is sufficiently unstable that commercial plant products derived from the plant contain negligible amounts of the compound).

[0207] Some examples of nematode antagonistic biological control agents include ARF18; 30 Arthrobotrys spp.; Chaetomium spp.; Cylindrocarpon spp.; Exophilia spp.; Fusarium spp.; Gliocladium spp.; Hirsutella spp.; Lecanicillium spp.; Monacrosporium spp.; Myrothecium spp.; Neocosmospora spp.; Paecilomyces spp.; Pochonia spp.; Stagonospora spp. spp.); vesicular-arbuscularmycorrhizal fungi, Burkholderia; Pasteurella, Brevibacillus; Pseudomonas; and rhizobacteria.Particularly preferred nematode antagonistic biological control agents include ARF18, Arthrobotrys dactyloides, Chaetomium globosum, Cylindrocarpon heteronema, Exophilia Jeanelmei, Exophilia pisciphila, Fusarium aspergilus, Fusarium solani, Gliocladium catenulatum, Gliocladium roseum, Gliocladium vixens, Hirsutellarhossiliensis, Hirsutella minnesotensis, Lecanicillium lecanii, Monacrosporium drechsleri, Monacrosporium gephyropagum, Myrotehcium verrucaria, Neocosmospora vasinfecta, Paecilomyces lilacinus, Pochonia chlamydosporia, Stagonospora heteroderae, Stagonospora phaseoli, vesicular-arbuscular mycorrhizal fungi, Burkholderia cepacia, Pasteuria punctatus, Pasteuria thornei, Pasteuria nishizawae, Pasteuria ramosa, Pasteuria usga, Brevibacillus laterosporus strain G4, Pseudomonas fluorescens, and rhizobacteria.

[0208] Some examples of nutrients can be selected from the group consisting of: nitrogen fertilizers, including but not limited to urea, ammonium nitrate, ammonium sulfate, pressureless nitrogen solution, ammonia water, anhydrous ammonia, ammonium thiosulfate, sulfur-coated urea, urea formaldehyde, IBDU, polymer-coated urea, calcium nitrate, urea formaldehyde and methylene urea; phosphate fertilizers, such as diammonium phosphate, monoammonium phosphate, ammonium polyphosphate, concentrated superphosphate and triple superphosphate; and potash fertilizers, such as potassium chloride, potassium sulfate, potassium magnesium sulfate, potassium nitrate. Such compositions can be present in the seed coating composition as free salts or ions. Alternatively, the nutrients / fertilizers can be compounded or chelated to provide sustained release over time.

[0209] Some examples of rodenticides may include a group of substances selected from the group consisting of 2-isovaleryl indan-1,3-dione, 4-(quinoxalin-2-ylamino)benzenesulfonamide, α-chlorohydrin, aluminum phosphide, antu, arsenic trioxide, barium carbonate, bisthiosemi, brodifacoum, bromadiolone, bromethalin, calcium cyanide, chloralose, chlorophacinone, cholecalciferol, coumachlor, coumafuryl, coumatetralyl, crimidine, difetoxin, chloranol ... nacoum), difethialone, diphacinone, ergocalciferol, flocoumafen, fluoroacetamide, flupropadine, flupropadine hydrochloride, hydrogen cyanide, methyl iodide, lindane, magnesium phosphide, methyl bromide, norbormide, phosacetim, phosphine, phosphorus, pindone, potassium arsenite, pyrinuron, scilliroside, sodium arsenite, sodium cyanide, sodium fluoroacetate, strychnine, thallium sulfate, warfarin, and zinc phosphide.

[0210] In liquid form, such as a solution or suspension, the bacterial population can be mixed or suspended in water or an aqueous solution. Suitable liquid diluents or carriers include water, aqueous solutions, petroleum distillates or other liquid carriers.

[0211] Solid compositions can be prepared by dispersing the bacterial population in or on a suitable divided solid carrier such as peat, wheat, bran, vermiculite, clay, talc, bentonite, diatomaceous earth, fuller's earth, pasteurized soil, etc. When such formulations are used as wettable powders, biocompatible dispersants such as nonionic, anionic, amphoteric or cationic dispersants and emulsifiers may be used.

[0212] Solid carriers used in formulation include, for example, mineral carriers such as kaolin, pyrophyllite, bentonite, montmorillonite, diatomaceous earth, acid clay, vermiculite and perlite; and inorganic salts such as ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, ammonium chloride and calcium carbonate. In addition, organic fine powders such as wheat flour, wheat bran and rice bran can be used. Liquid carriers include vegetable oils (such as soybean oil and cottonseed oil), glycerol, ethylene glycol, polyethylene glycol, propylene glycol, polypropylene glycol, etc.

[0213] Application of bacterial populations to crops

[0214] The composition of the bacteria or bacterial population described herein can be applied in furrow, in talc or as a seed treatment. The composition can be applied to the seed package in bulk, mini-bulk, bagged form or in talc.

[0215] Planters can plant treated seeds and grow crops according to conventional methods, double rows or methods that do not require tillage. Seeds can be distributed using a control hopper or a single hopper. Compressed air or manual distribution of seeds can also be used. Variable rate technology can be used to place seeds. In addition, variable rate technology can be used to apply bacteria or bacterial populations described herein. In some instances, bacteria can be applied to seeds and oilseeds of corn, soybeans, canola, sorghum, potatoes, rice, vegetables, cereals, pseudocereals. Examples of cereals can include barley, fonio, oats, palmer's grass, rye, pearl millet, sorghum, spelt, teff, triticale and wheat. Examples of pseudocereals can include breadnut, buckwheat, cattail, chia seeds, flax, grain amaranth, hanza, quinoa and sesame. In some examples, the seeds can be genetically modified organisms (GMO), non-GMO, organic, or conventional.

[0216] Crops may be treated with additives such as microfertilizers, PGRs, herbicides, insecticides, and fungicides. Examples of additives include crop protection agents such as insecticides, nematicides, fungicides; synergists such as colorants, polymers, granulators, pre-stimulants, and disinfectants; and other agents such as inoculants, PGRs, softeners, and micronutrients. PGRs may be natural or synthetic plant hormones that affect root growth, flowering, or stem elongation. PGRs may include auxins, gibberellins, cytokinins, ethylene, and abscisic acid (ABA).

[0217] The composition can be applied in the furrow in combination with a liquid fertilizer. In some examples, the liquid fertilizer can be kept in a tank. NPK fertilizer contains macronutrients of sodium, phosphorus and potassium.

[0218] Compositions can improve plant traits, such as promoting plant growth, maintaining high chlorophyll content in leaves, increasing fruit or seed number and increasing fruit or seed unit weight. The disclosed method can be used to introduce or improve one or more of a variety of desired traits. Examples of traits that can be introduced or improved include: root biomass, root length, height, bud length, leaf number, water use efficiency, total biomass, yield, fruit size, seed size, photosynthesis rate, drought tolerance, heat tolerance, salt tolerance, tolerance to low nitrogen stress, nitrogen use efficiency, resistance to nematode stress, resistance to fungal pathogens, resistance to bacterial pathogens, resistance to viral pathogens, metabolite levels, regulation of metabolite levels, proteome expression. Desired traits, including height, total biomass, root and / or shoot biomass, seed germination, seedling survival, photosynthetic efficiency, transpiration rate, seed / fruit number or quality, plant grain or fruit yield, leaf chlorophyll content, photosynthetic rate, root length, or any combination thereof, can be used to measure growth and compared with the growth rate of a reference agricultural plant (e.g., a plant without an introduced trait and / or improved trait) grown under the same conditions. In some instances, desired traits, including height, total biomass, root and / or shoot biomass, seed germination, seedling survival, photosynthetic efficiency, transpiration rate, seed / fruit number or quality, plant grain or fruit yield, leaf chlorophyll content, photosynthetic rate, root length, or any combination thereof, can be used to measure growth and compared with the growth rate of a reference agricultural plant (e.g., a plant without an introduced trait and / or improved trait) grown under similar conditions.

[0219] Agronomic traits of the host plant may include, but are not limited to, the following: altered oil content, altered protein content, altered seed carbohydrate composition, altered seed oil composition, and altered seed protein composition, chemical resistance, cold tolerance, delayed senescence, disease resistance, drought tolerance, ear weight, improved growth, enhanced health4, heat tolerance, herbicide tolerance, herbivore resistance, improved nitrogen fixation, increased nitrogen use efficiency, improved root architecture, improved water use efficiency, increased biomass, increased root length, increased seed weight, increased shoot length, increased yield, improved performance under water limited conditions, compared to isotype plants grown from seeds in the absence of the seed treatment formulation. Increased yield, kernel mass, kernel moisture content, metal tolerance, ear number, number of kernels per ear, number of pods, nutritional enhancement, pathogen resistance, pest resistance, increased photosynthetic capacity, salt tolerance, staygreen, increased vigour, increased dry weight of mature seeds, increased fresh weight of mature seeds, increased number of mature seeds per plant, increased chlorophyll content, increased number of pods per plant, increased pod length per plant, reduced number of wilted leaves per plant, reduced number of severely wilted leaves per plant, and increased number of non-wilted leaves per plant, detectable regulation at the level of metabolites, detectable regulation at the level of transcripts, and detectable regulation in the proteome.

[0220] In some cases, the plant is inoculated with the isolated bacteria or bacterial population of the same plant species as the plant component of the inoculated plant. For example, the bacteria or bacterial population found in a kind of maize (Zea mays) (corn (corn)) are associated with the plant component of another kind of maize lacking the bacteria and bacterial population under natural conditions. In one embodiment, bacteria and bacterial population are derived from the plant of the related species of the plant of the plant component of the inoculated plant. For example, the bacteria and bacterial population found in diploid perennial turfgrass (Zea diploperennis) Iltis et al. (diploid perennial class maize (diploperennial teosinte)) are applied to maize (corn), or vice versa. In some cases, the plant is inoculated with bacteria and bacterial population that are allogenic to the plant element of the inoculated plant. In one embodiment, bacteria and bacterial population are derived from the plant of another species. For example, bacteria and bacterial populations commonly found in dicots are applied to monocots (e.g., corn is inoculated with soybean-derived bacteria and bacterial populations), or vice versa. In other cases, the bacteria and bacterial populations to be inoculated on the plant are derived from species related to the plant to be inoculated. In one embodiment, the bacteria and bacterial populations are derived from related taxa, such as from related species. The plant of another species can be an agricultural plant. In another embodiment, the bacteria and bacterial populations are part of a designed composition that is inoculated into any host plant component.

[0221] In some instances, the bacteria or bacterial population is exogenous, wherein the bacteria or bacterial population is isolated from a plant different from the plant being inoculated. For example, in one embodiment, the bacteria or bacterial population can be isolated from a different plant of the same species as the plant to be inoculated. In some cases, the bacteria or bacterial population can be isolated from a species related to the inoculated plant.

[0222] In some instances, bacterium described herein and bacterial population can move to another tissue type from a type of organization. For example, the present invention has proved the ability of bacterium and bacterial population to move into the vegetative tissue of maturation plant from seed outside after coating on seed outside. Therefore, in one embodiment, bacterial population and bacterial population can move in the vegetative tissue of plant from seed outside. In one embodiment, bacterium and bacterial population coated on plant seed can be positioned in the different tissues of plant when seed germinates into vegetative state. For example, bacterium and bacterial population can be positioned in any one of the tissues in plant, including: root, adventitious root, seed root, root hair, bud, leaf, flower, bract, spike, meristem, pollen, pistil, ovary, stamen, fruit, runner, rhizome, root nodule, tuber, trichome, guard cell, water secretion hole, petal, sepal, glume, rachis, vascular cambium, phloem and xylem. In one embodiment, bacteria and bacterial populations can be located in the root and / or root hair of a plant. In another embodiment, bacteria and bacterial populations can be located in photosynthetic tissues, such as leaves and buds of a plant. In other cases, bacteria and bacterial populations are located in the vascular tissues of a plant, such as in xylem and phloem. In yet another embodiment, bacteria and bacterial populations can be located in the reproductive tissues (flowers, pollen, pistils, ovaries, stamens, fruits) of a plant. In another embodiment, bacteria and bacterial populations can be located in the roots, buds, leaves and reproductive tissues of a plant. In yet another embodiment, bacteria and bacterial populations colonize the fruit or seed tissue of a plant. In yet another embodiment, bacteria and bacterial populations can colonize plants so that they are present in the surface of the plant (that is, their presence can be detected and presented in the outside of the plant or the surface layer of the plant). In other embodiments, bacteria and bacterial populations can be located in substantially all or all tissues of a plant. In certain embodiments, bacteria and bacterial populations are not located in the root of a plant. In other cases, bacteria and bacterial populations are not located in the photosynthetic tissue of a plant.

[0223] The effectiveness of the composition can also be assessed by measuring the relative maturity of the crop or the crop heat unit (CHU). For example, the bacterial population can be applied to corn, and the corn growth can be assessed based on the relative maturity of the corn kernels or the time when the corn kernels reach maximum weight. The crop heat unit (CHU) can also be used to predict the maturity of the corn crop. CHU determines the amount of heat accumulation by measuring the daily maximum temperature of the crop growth.

[0224] In an example, the bacteria can be located in any tissue in the plant, including: roots, adventitious roots, seminal roots, root hairs, buds, leaves, flowers, bracts, meristems, pollen, pistils, ovaries, stamens, fruits, runners, rhizomes, nodules, tubers, trichomes, guard cells, water secretion pores, petals, sepals, glumes, rachis, vascular cambium, phloem and xylem. In another embodiment, the bacteria or bacterial population can be located in photosynthetic tissues, such as leaves and buds of plants. In other cases, the bacteria and bacterial populations are located in the vascular tissues of plants, such as in xylem and phloem. In another embodiment, the bacteria or bacterial populations can be located in the reproductive tissues (flowers, pollen, pistils, ovaries, stamens or fruits) of plants. In another embodiment, the bacteria and bacterial populations can be located in the roots, buds, leaves and reproductive tissues of plants. In another embodiment, the bacteria or bacterial populations colonize the fruit or seed tissues of plants. In yet another embodiment, the bacteria or bacterial population is capable of colonizing a plant such that it is present on the surface of the plant. In another embodiment, the bacteria or bacterial population is capable of localizing to substantially all or all tissues of a plant. In certain embodiments, the bacteria or bacterial population is not localized to the roots of a plant. In other cases, the bacteria and bacterial population is not localized to the photosynthetic tissue of a plant.

[0225] The effectiveness of a bacterial composition applied to a crop can be assessed by measuring various characteristics of crop growth including, but not limited to, establishment rate, seeding vigor, root strength, drought tolerance, plant height, drying, and test weight.

[0226] Plant species

[0227] Methods and bacteria described herein are applicable to any one of a variety of plants, such as plants of Hordeum, Oryza, Zea and Triticeae. Other non-limiting examples of suitable plants include mosses, lichens and algae. In some cases, plants have economic, social and / or environmental value, such as plants in food crops, fiber crops, oil crops, forestry or pulp and paper industry, raw materials and / or ornamental plants for biofuel production. In some instances, plants can be used to produce products with economic value, such as cereals, flour, starch, syrup, meal, oil, film, packaging, nutritious food products, paper pulp, animal feed, fish feed, bulk materials for industrial chemicals, cereal products, processed human food, sugar, alcohol and / or protein. Non-limiting examples of crop plants include corn, rice, wheat, barley, sorghum, millet, oats, rye triticale, buckwheat, sweet corn, sugarcane, onion, tomato, strawberry and asparagus.

[0228] In some examples, plants that can be obtained or improved using the methods and compositions disclosed herein can include plants that are important or interesting for agriculture, horticulture, biomass for the production of biofuel molecules and other chemicals, and / or forestry. Some examples of these plants may include pineapple, banana, coconut, lily, grasspea, alfalfa, tomatillo, melon, chickpea, endive, clover, kale, lentil, soybean, tobacco, potato, sweet potato, radish, cabbage, rapeseed, apple tree, grape, cotton, sunflower, Arabidopsis, canola, citrus (including orange, mandarin, kumquat, lemon, lime, grapefruit, tangerine, tangelo, citron and pomelo), pepper, beans, lettuce, Panicum virgatum (switch), Sorghum bicolor (sorghum, sudan), Miscanthus giganteus (miscanthus), Saccharum (Saccharum sp. (energy cane), Populus balsamifera (poplar), Zea mays (corn), Glycine max (soybean), Brassica napus (canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugar beet), Pennisetum glaucum (pearl millet), Panicum spp., Sorghum spp., Miscanthus spp., Saccharum spp., Erianthus spp., Populus spp., Secale cereale (rye), Salix spp. (willow), Eucalyptus spp. (eucalyptus), Triticosecale spp. (Triticum-25 wheat × rye), bamboo, Carthamus tinctorius (safflower), Jatropha curcas (Jatropha), Ricinuscommunis (castor), Elaeis guineensis (oil palm), Phoenix dactylifera (date palm), Archontophoenix cunninghamiana (king palm), Syagrus romanzoffiana (queen palm), Linum usitatissimum (flax), Brassica juncea (mustard), Manihot esculenta (cassaya), Lycopersicon esculentum (tomato), Lactuca saliva (lettuce), Musaparadisiaca (banana), Solanum tuberosum (potato), Brassica oleracea (broccoli, cauliflower, Brussels sprouts), Camellia sinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa), Coffea arabica (coffee), Vitis vinifera (grapes), Ananas comosus (pineapple), Capsicum annum (hot and sweet peppers), Allium cepa (onion), Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima (squash), Cucurbita moschata (cucumber), Spinacea oleracea (spinach), Abelmoschus esculentus (okra), Beet (Abelmoschus esculentus) esculentus), Solanum melongena (eggplant), Papaversomniferum (opium poppy), Oriental poppy (Papaverorientale), Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabis saliva, Camptotheca acuminate, Catharanthus roseus, Vincarosea, Cinchona officinalis, Coichicum autumnale, Veratrum californica, Digitalis lanata, Digitalis purpurea, 5 species of Dioscorea, Andrographis paniculata, Atropa belladonna, Datura stomonium, Berberis spp., Cephalotaxus spp., Ephedra sinica, Ephedra spp.), Erythroxylum coca, Galanthus wornorii, Scopolia spp., Lycopodium serratum (Huperzia serrata), Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp., Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis, Chrysanthemum parthenium, Coleus forskohlii, Tanacetum parthenium, Parthenium argentatum (guayule), Hevea spp. (rubber), Menthaspicata (mint), Mentha piperita (mint), Bixa orellana (rosewood), Alstroemeria spp., Rosa spp. (rose), Dianthuscaryophyllus (carnation), Petunia spp. (petunia), Poinsettia pulcherrima (poinsettia), Nicotiana tabacum (tobacco), Lupinus albus (lupin), Uniola paniculata (oat), Hordeum vulgare (barley), and Lolium spp. (rye).

[0229] In some examples, monocots can be used. Monocots belong to the following orders: Alismatales, Arales, Arecales, Bromeliales, Commelinales, Cyclanthales, Cyperales, Eriocaulales, Hydrocharitales, Juncales, Lilliales, Najadales, Orchidales, Pandanales, Poales, Restionales, Triuridales, Typhales, and Zingiberales. Plants belonging to the class of gymnosperms are Cycadales, Ginkgoales, Gnetales, and Pinales. In some examples, the monocotyledonous plant can be selected from the group consisting of corn, rice, wheat, barley, and sugarcane.

[0230] In some examples, dicots can be used, including plants belonging to the following orders: Aristochiales, Asterales, Batales, Campanulales, Capparales, Caryophyllales, Casuarinales, Celastrales, Cornales, Diapensales, Dilleniales, Dipsacales, Ebenales, Ericales, Eucomiales, Euphorbiales, Fabales, Fagus Fagales, Gentianales, Geraniales, Haloragales, Hamamelidales, Middles, Juglandales, Lamiales, Laurales, Lecythidales, Leitneriales, Magniolales, Malvales, Myricales, Myrtales, Nymphaeales, Papeverales, Piperales, Plantaginales, Plumbales aginales, Podostemales, Polemoniales, Polygalales, Polygonales, Primulales, Proteales, Rafflesiales, Ranunculales, Rhamnales, Rosales, Rubiales, Salicales, Santales, Sapindales, Sarraceniaceae, Scrophulariales, Theales, Trochodendrales, Umbellales, Urticales, and Violates.In some examples, the dicotyledonous plant can be selected from the group consisting of cotton, soybean, pepper, and tomato.

[0231] In some cases, the plant to be improved is not easy to be suitable for experimental conditions. For example, crop plants may take too long to grow to be enough to actually evaluate the improved traits continuously in multiple iterations. Therefore, the first plant from which the bacteria are initially isolated and / or a plurality of plants to which the genetically manipulated bacteria are applied can be model plants, such as plants that are more suitable for evaluation under the desired conditions. Non-limiting examples of model plants include Setaria, Brachypodium and Arabidopsis. The ability of the bacteria isolated using the model plant according to the method disclosed herein can then be applied to another type of plant (e.g., crop plants) to confirm the granting of improved traits.

[0232] Traits that can be improved by the methods disclosed herein include any observable characteristics of a plant, including, for example, changes in growth rate, height, weight, color, taste, smell, production of one or more compounds of the plant, including, for example, metabolites, proteins, drugs, carbohydrates, oils, and any other compounds. It is also contemplated to select plants based on genotypic information (e.g., including plant gene expression patterns in response to bacteria, or identifying the presence of genetic markers such as markers associated with increased nitrogen fixation). Plants can also be selected based on the absence, suppression, or inhibition of a certain feature or trait (e.g., an undesirable feature or trait) as opposed to the presence of a certain feature or trait (e.g., a desired feature or trait).

[0233] Examples

[0234] The examples provided herein describe methods for bacterial isolation, bacterial and plant analysis, and plant trait improvement. The examples are for illustrative purposes only and are not to be construed as limiting in any way.

[0235] Example 1: Isolation of microorganisms from plant tissues

[0236] Topsoil was obtained from various agricultural areas in central California. Twenty soils with different textural characteristics were collected, including heavy clay, peaty clay loam, silty clay, and sandy loam. As shown in Table 1, seeds of various non-sweet corn (field corn), sweet corn, conventional corn, and tomatoes were planted in each soil.

[0237]

[0238] Table 1: Crop types and varieties grown in soils with different characteristics

[0239] Plants were uprooted after 2-4 weeks of growth, and excess soil on the root surface was removed with deionized water. After removing the soil, the plants were surface sterilized with bleach and rinsed vigorously in sterile water. Clean 1 cm root segments were cut from the plants and placed in a phosphate buffered saline solution containing 3 mm steel balls. Slurries were generated by violently shaking the solution with Qiagen TissueLyserII.

[0240] The root and saline slurries were diluted and inoculated onto various types of growth media to isolate rhizosphere microorganisms, endophytic microorganisms, epiphytic microorganisms, and other plant-associated microorganisms. Single colonies were obtained using R2A and Nfb agar media, and nitrogen-fixing bacterial populations were obtained using semi-solid Nfb medium slants. After 2-4 weeks of incubation in semi-solid Nfb medium slants, the microbial populations were collected and streaked to obtain single colonies on R2A agar, as Figure 1A - shown in B. Single colonies were resuspended in a mixture of R2A and glycerol, subjected to PCR analysis, and frozen at -80°C for subsequent analysis. Approximately 1,000 single colonies were obtained and named "isolated microorganisms".

[0241] The isolates were then screened by colony PCR to detect the presence of the nifH gene in order to identify diazotrophs. Each isolate was probed for the presence of the nif cluster using the previously described primer set Ueda19F / 388R, which has been shown to detect over 90% of diazotrophs in a screen (Ueda et al. 1995; J. Bacteriol. 177:1414-1417). Figure 2 As shown in , a single colony of the purified isolate was picked, resuspended in PBS, and used as a template for colony PCR. Colonies of isolates that produced positive PCR bands were re-streaked, and the PCR and re-streaking process was repeated twice to prevent false positive identification of nitrogen-fixing organisms. The purified isolate was then named "candidate microorganism".

[0242] Example 2: Characterization of isolated microorganisms

[0243] Sequencing, analysis, and phylogenetic identification

[0244] Sequencing of 16S rDNA with the 515f-806r primer set was used to generate preliminary phylogenetic signatures for isolated and candidate microorganisms (see, e.g., Vernon et al.; BMC Microbiol. 2002 Dec 23;2:39.). The microorganisms included various genera including: Enterobacter, Burkholderia, Klebsiella, Brachyrhizobium, Rahnella, Xanthomonas, Raoultella, Pantoea, Pseudomonas, Brevundimonas, Agrobacterium, and Paenibacillus, as shown in Table 2.

[0245]

[0246] Table 2: Diversity of microorganisms isolated from tomato plants as determined by deep 16S rDNA sequencing.

[0247] Subsequently, the genomes of 39 candidate microorganisms were sequenced using the Illumina Miseq platform. Genomic DNA was extracted from pure cultures using the QIAmp DNA micro kit (Qiagen), and a total DNA library for sequencing was prepared by a third-party supplier (SeqMatic, Hayward). Genome assembly was then performed using the A5 pipeline (Tritt et al. 2012; PLoS One 7(9): e42304). Genes were identified and annotated, and genes related to nitrogen fixation regulation and expression were indicated as targets for mutagenesis.

[0248] Transcriptome profiling of candidate microorganisms

[0249] The transcriptome profile analysis of strain CI010 was carried out to identify promoters with activity in the presence of environmental nitrogen. The CI010 strain was cultivated in a definite nitrogen-free medium supplemented with 10mM glutamine. Total RNA (QIAGEN RNeasy test kit) was extracted from these cultures, and RNAseq sequencing was performed by HiSeq (SeqMatic, Fremont CA, CA) of Enlightenment. Geneious was used to map sequencing reads to CI010 genome data, and to identify highly expressed genes under the control of proximal transcription promoters. Table 3A-C lists genes and their relative expression levels as measured by RNASeq sequencing of total RNA. The sequence of the proximal promoter was recorded for mutagenesis nif path, nitrogen utilization-related path or other genes with required expression levels.

[0250] Genetic tractability assessment

[0251] Candidate microorganisms are characterized based on transformability and genetic tractability. First, optimal carbon source utilization is determined by growth on a small group of related culture media and growth curves in nitrogen-free and nitrogen-rich media. Second, the natural antibiotic resistance of each strain is determined by spot plate inoculation and growth in liquid culture, and the liquid culture contains a group of antibiotics used as mutagenic selective markers. Third, the transformability of each strain is tested by electroporation of the plasmid collection. The plasmid collection contains seven replication origins, i.e., p15a, pSC101, CloDF, colA, RK2, pBBR1 and pRO1600, and four antibiotic resistance markers, i.e., CmR, KmR, SpecR and TetR combinatorial expansion. This systematic evaluation of origin and resistance marker compatibility is used to identify plasmid-based mutagenic vectors in candidate microorganisms.

[0252] Example 3: Mutagenesis of candidate microorganisms

[0253] Lambda-Red-mediated knockout

[0254] Several mutants of candidate microorganisms were generated using plasmid pKD46 or derivatives containing a kanamycin resistance marker (Datsenko et al. 2000; PNAS 97(12):6640-6645). Knockout cassettes were designed with 250 bp of homology flanking the target gene and generated by overlap extension PCR. Candidate microorganisms were transformed with pKD46, cultured in the presence of arabinose to induce Lambda-Red mechanistic expression, prepared for electroporation, and transformed with knockout cassettes to generate candidate mutant strains. As shown in Table 4, four candidate microorganisms and a laboratory strain of Klebsiella oxytoca M5A1 were used to generate candidate mutants of thirteen nitrogen fixation regulatory genes nifL, glnB, and amtB.

[0255]

[0256] Table 4: List of single knockout mutants generated by Lambda-red mutagenesis Oligonucleotide-directed mutagenesis accompanied by Cas9 selection

[0257] Oligonucleotide directed mutagenesis was used to target genomic changes in the rpoB gene in Escherichia coli (E. coli) DH10B, and mutants were selected using the CRISPR-Cas system. The mutagenic oligonucleotide (ss1283: "G*T*T*G*ATCAGACCGATGTTCGGACCTTCcaagGTTTCGATCGGACATACGCGACCGTAGTGGGTCGGGTGTACGTCTCGAACTTCAAAGCC", where * represents a phosphorothioate bond) was designed to confer resistance to rifampicin by a 4-bp mutation of rpoB. Cells containing a plasmid encoding Cas9 were induced to express Cas9, prepared for electroporation, and then electroporated with both the mutagenic oligonucleotide and a plasmid encoding a constitutively expressed guide RNA (gRNA) for Cas9 cleavage targeting the WT rpoB sequence. The electroporated cells were recovered overnight in a non-selective medium to allow the resulting mutant chromosomes to be fully separated. After selection of the gRNA-encoding plasmid, two of the ten colonies screened were shown to contain the desired mutation, while the rest were shown to be escape mutants generated by either protospacer mutations in the gRNA plasmid or deletions in the Cas9 plasmid.

[0258] Lambda-Red mutagenesis accompanied by Cas9 selection

[0259] Mutants of candidate microorganisms CI006 and CI010 were generated by lambda-red mutagenesis accompanied by selection by CRISPR-Cas. The knockout box contains an endogenous promoter identified by transcriptional profiling (as described in Example 2 and depicted in Table 3) and about 250bp homology regions flanking the missing target. CI006 and CI010 were transformed with plasmids encoding the Lambda-red recombination system (exo, beta, gam genes) under the control of an arabinose-inducible promoter, and Cas9 was transformed under the control of an IPTG-inducible promoter. Red recombination and Cas9 systems were induced in the resulting transformants, and strains were prepared for electroporation. The knockout box and plasmid-encoded selection gRNA were subsequently transformed into competent cells. After inoculation on an antibiotic that was selective for both the Cas9 plasmid and the gRNA plasmid, 7 of the 10 colonies screened showed the expected knockout mutations, such as Figure 3 as shown in .

[0260] Example 4: In vitro phenotyping of candidate molecules

[0261] The effect of exogenous nitrogen on nitrogenase biosynthesis and activity in various mutants was assessed. The acetylene reduction assay (ARA) (Temme et al. 2012; 109(18): 7085-7090) was used to measure nitrogenase activity in pure culture conditions. The strains were grown in gas-tight tubes and the reduction of acetylene to ethylene was quantified using an Agilent 6890 gas chromatograph. Figure 4A -B and Fig. 10A -C shows the ARA activities of candidate microorganisms and corresponding candidate mutants grown in nitrogen-fixing medium supplemented with 0 to 10 mM glutamine.

[0262] Under anaerobic culture conditions, a range of glutamine and ammonia concentrations were tested to quantify the effect on nitrogen fixation activity. In wild-type cells, activity decreased rapidly with increasing glutamine concentrations. However, in an initial series of knockout mutations, one class of mutations was validated that enabled expression of nitrogen fixation genes at glutamine concentrations that would otherwise shut down activity in the wild type. Figure 4C This profile was generated in four different nitrogen-fixing organisms species. In addition, by rewiring the regulatory network using the identified genetic parts, the nitrogen fixation activity level could be modulated predictably. This was seen in Figure 4B , which shows strains CM023, CM021, CM015 and CI006. Strain CM023 is a low evolutionary strain; strain CM021 is a high evolutionary strain; strain CM015 is a medium evolutionary strain; strain CI006 is wild type (strain 2). Ammonia excreted into the culture supernatant was tested using an enzyme-based assay (MEGAZYME). The assay measures the amount of NADPH consumed in absorbance at 340nm. The assay was performed on bacterial cultures grown in a nitrogen-free, anaerobic environment at a starting density of 1E9 CFU / ml. As Figure 4D As seen in a panel of six evolved strains, one strain excreted up to 100 μM ammonia over a 48-hour period. Fig.11 As seen in Figure 2, the double mutant exhibited higher ammonia excretion than the single mutant from which it was derived. This demonstrates the ability of the microorganism to produce ammonia in excess of its physiological needs.

[0263] Transcriptional profiling of pure cultures

[0264] The transcriptional activity of CI006 was measured using the Nanostring Elements platform. Cells were grown in nitrogen-free medium and after 4 hours of incubation, 10E8 cells were collected. Total RNA was extracted using the Qiagen RNeasy kit. The purified RNA was submitted to Core Diagnostics in Palo Alto, CA for probe hybridization and digital analyzer analysis, such as Figure 5 as shown in .

[0265] Example 5: In planta phenotyping of candidate microorganisms

[0266] Colonization of plants by candidate microorganisms

[0267] Short-term plant growth experiments were performed to quantify the colonization of the desired host plant by candidate microorganisms. Corn was inoculated with strains expressing RFP from a plasmid or from a Tn5-integrated RFP expression cassette. Plants were grown in sterile sand and non-sterile peat media, and inoculation was performed by pipetting 1 mL of cell culture directly on newly emerged plant coleoptiles three days after germination. Plasmids were maintained by watering the plants with a solution containing the appropriate antibiotic. After three weeks, plant roots were collected, rinsed three times in sterile water to remove visible soil, and split into two samples. One root sample was analyzed by fluorescence microscopy to identify the localization pattern of the candidate microorganism. Figure 6 As shown in , a 10 mm length of the thinnest intact plant root was examined microscopically.

[0268] A second quantitative method for assessing colonization was developed. Quantitative PCR analysis was performed on a full DNA preparation from plant roots inoculated with endophytes. Corn seeds (Dekalb DKC-66-40) were germinated in previously autoclaved sand in a 2.5-inch by 2.5-inch by 10-inch pot. One day after planting, 1 ml of an overnight culture of endophytes (SOB medium) was infiltrated at the exact location where the seeds were located. 1 mL of this overnight culture is roughly equivalent to 10^9 cfu, varying within 3 times of each other, depending on the strain used. Each seedling was fertilized 3 times per week with 50 mL of a modified Hoagland's solution supplemented with 2.5 mM or 0.25 mM ammonium nitrate. Four weeks after planting, root samples were collected for DNA extraction. Soil residues were rinsed off using a pressurized water jet. These tissue samples were then homogenized using a QIAmp DNA micro kit (Qiagen) according to the recommended protocol. These DNA extracts were subjected to qPCR analysis using Stratagene Mx3005P RT-PCR using primers designed (using NCBI's Primer BLAST) to be specific to each locus in the endophyte genome. The presence of genomic copies of the endophyte was quantified. To further confirm the identity of the endophyte, the PCR amplification products were sequenced and confirmed to have the correct sequence. An overview of the colonization profiles of strains CI006 and CI008 from candidate microorganisms is presented in Table 5. Colonization rates as high as 10^7× colony forming units / gram root fresh weight were exhibited in strain CI008.

[0269] strain Colonization rate (colony forming unit / g fresh weight) CI006 1.45×10^5 CI008 1.24×10^7

[0270] Table 5: Corn colonization as measured by qPCR

[0271] RNA profiling in plants

[0272] The biosynthesis of nif pathway components in plants was estimated by measuring the transcription of nif genes.Total RNA was obtained from the root plant tissue of CI006 inoculated plants (planting method as previously described). According to the recommended scheme (Qiagen), RNA extraction was performed using the RNEasy micro kit. Then using the Nanostring Elements kit (NanoString Technologies, Inc.), the total RNA from these plant tissues was analyzed using a probe specific to the nif gene in the CI006 genome of the strain. The data of nif gene expression in plants are summarized in Table 6. The expression of the nifH gene was detected in the plants inoculated by the CM013 strain, and nifH expression was undetectable in the plants inoculated by CI006. The strain CM013 is a derivative of the strain CI006, in which the nifL gene has been knocked out.

[0273] Under fertilization conditions, the highly expressed genes of CM011 were measured in plants, and the genes were sorted by transcripts per kilobase million (TPM) per million reads. The promoters controlling the expression of some of these highly expressed genes were used as templates for homologous recombination into targeted nitrogen fixation and assimilation loci. RNA samples from greenhouse-grown CM011 inoculated plants were extracted, rRNA was removed using the Ribo-Zero kit, sequenced using the Truseq platform of Enlightenment, and mapped back to the genome of CM011. The highly expressed genes from CM011 are listed in Table 7.

[0274] strain Relative transcript expression CI006 9.4 CM013 103.25

[0275] Table 6: Expression of nifH in plants

[0276]

[0277]

[0278] Table 7

[0279] 15 N Analysis

[0280] The main method to demonstrate fixation uses the nitrogen isotope 15N, which is found in the atmosphere at a ratio relative to 14N fixation. Fixation can be observed directly from the amount of fixed 15N increased in the atmosphere supplemented with 15N2 gas by supplementing fertilizers or the atmosphere with nitrogen enriched with 15N (Yoshida 1980), or conversely, fixation can be observed by dilution of enriched fertilizers in plant tissues by atmospheric N2 gas (Iniguez 2004). The dilution method allows the observation of cumulative fixed nitrogen during plant growth, while 15N 2 The gas method is limited to measuring fixation (rate measurement) over short time intervals that can occur in plants grown in a closed atmosphere. Therefore, the gas method has superior specificity (because any elevated 15N in the plant above atmospheric rates 2 levels can all be clearly attributed to fixation), but cannot show cumulative activity.

[0281] Two types of assays have been performed to measure fixation activity of the modified strains relative to wild-type and uninoculated maize, and elevated fixation rates were observed in plants for several modified strains ( Fig.12 , Fig.14A and Fig. 14B ). These analyses served to demonstrate that the activity of the strains observed in vitro translated into in vivo results. In addition, these analyses allowed for the measurement of the effects of fertilizers on the activity of the strains, indicating suitable functionality in an agricultural setting. Similar results were observed when Setaria plants were inoculated with wild-type and modified strains ( Fig.13 ). Figures 14A-14C The in-plant fixation activity shown in is further supported by transcriptome data. The evolved strains exhibited elevated nifH transcript levels relative to their wild-type counterparts. Furthermore, on a plant-by-plant basis, microbial nitrogen levels in plants also correlated with colonization levels. These results ( Fig.12 , Fig.13 , Figures 14A-14C , Fig.15A as well as Fig. 15B ) support the hypothesis that microbial regulation, through the modified nif gene cluster, is the likely cause of the increase in atmospheric nitrogen found in plant tissues. In addition to direct measurements of fixation, the effect of inoculating plants with the modified strains in a plant biomass assay under nitrogen stress was also measured. Although plant biomass may be associated with many possible microbial-plant interactions, one would expect that the addition of fixed nitrogen would affect the phenotype of plants when nitrogen is limited. Inoculated plants were grown in the complete absence of nitrogen, and significant increases in leaf area, shoot fresh and dry weight, and root fresh and dry weight were observed in inoculated plants relative to untreated controls ( Fig. 14C). Although these differences cannot be attributed exclusively to nitrogen fixation, they support the conclusion that the modified strain is actively providing nitrogen to the plants. Corn and Setaria plants were grown and inoculated as described above. 15 N fertilizer is regularly supplied to plants. 15 N levels were used to quantify nitrogen fixation by microorganisms. The fourth leaf tissue was collected 4 weeks after planting and dried. The dried leaf samples were homogenized using beads (Qiagen Tissuelyzer) and then aliquoted into tin capsules for IRMS (MBL Stable Isotope Laboratory, The Ecosystems Center, Woods Hole, MA). Nitrogen from the atmosphere (NDFA) was calculated, and Figure 7 Nitrogen produced by CI050 and CM002 is shown in FIG.

[0282] Analysis of plant hormone production

[0283] The effect of indole-3-acetic acid on fruit ripening was previously investigated using the dwarf tomato (Solanum lycopersicum) cultivar 'Micro-Tom' by in vitro assays (Cohen 1996; J Am Soc Hortic Sci 121:520-524). To evaluate phytohormone production and secretion by candidate microorganisms, a plate-based screening assay using immature Micro-Tom fruit was developed. Twelve-well tissue culture test plates were prepared by filling the wells with agar medium, allowing it to solidify, and spotting 10 uL of an overnight microbial culture onto the agar surface, as described in Example 1. Figure 8 Wells containing increasing amounts of gibberellic acid (GA) in the agar but without bacterial culture were used as positive controls and standards. Flowers of growing Micro-Tom plants were excised one day after flowering and inserted stem first into the agar at the bacterial spot culture site. The flowers were monitored for 2-3 weeks, after which the fruits were harvested and weighed. Fig. 9 As shown in , the increase in plant fruit mass in several replicates indicates that the inoculant microorganisms produce phytohormones.

[0284] Example 6: Cyclic host-microbe evolution

[0285] Maize plants were inoculated with CM013 and grown for 4 weeks to approximately the V5 growth stage. 15Plants with improved nitrogen accumulation from microbial sources were uprooted and washed with pressurized water to remove non-rhizosphere soil. 0.25 g of root segments were cut and rinsed in PBS solution to remove fine soil particles and non-adherent microorganisms. 3 mm steel balls were used to homogenize tissue samples in QIAGEN TissueLyser II. The homogenate was diluted and inoculated on SOB agar medium. Single colonies were resuspended in liquid medium and subjected to 16s rDNA PCR analysis and mutations unique to the inoculated strain. The process of microbial differentiation, mutagenesis, inoculation and re-isolation can be repeated iteratively to improve the colonization ability of microbial traits, plant traits and microorganisms.

[0286] Example 7: Cross-geographic compatibility

[0287] The ability of improved microorganisms to colonize inoculated plants is critical to the success of plants under field conditions. Although the isolation methods described are designed to select from soil microorganisms that may have a close relationship with crop plants (such as corn), many strains may not effectively colonize under a range of plant genotypes, environments, soil types or inoculation conditions. Since colonization is a complex process that requires a series of interactions between the microbial strain and the host plant, screening for colonization ability has become a central method for selecting priority strains for further development. Early efforts to assess colonization used fluorescent markers of strains, which were effective but time-consuming and could not be expanded on a per-strain basis. Since colonization activity is not suitable for direct improvement, it is necessary to select potential product candidates from strains that are natural colonizers.

[0288] An assay was designed to test for robust colonization of wild-type strains in any given host plant using qPCR and primers designed to be strain-specific in community samples. This assay was designed to rapidly measure microbial colonization rates in maize tissue samples. Initial testing with strains that were scored as likely colonizers using fluorescence microscopy and culture plate-based techniques demonstrated that the qPCR approach was both quantitative and scalable.

[0289] A typical analysis was performed as follows: Plants, primarily maize and wheat varieties, were grown in peat potting mix in a greenhouse with six replicates per strain. Four or five days after planting, 1 mL of an early stationary phase culture of the bacteria diluted to an OD590 of 0.6-1.0 (roughly 5E+08 CFU / mL) was pipetted onto newly emerged coleoptiles. Plants were watered with tap water only and allowed to grow for four weeks before sampling, at which point the plants were uprooted and the roots were thoroughly washed to remove most of the peat residue. Clean root samples were cut and homogenized to produce a slurry of plant cell debris and associated bacterial cells. We developed a high-throughput DNA extraction protocol that efficiently produces a mixture of plant and bacterial DNA to use as a template for qPCR. Based on bacterial cell incorporation experiments, this DNA extraction process provides quantitative bacterial DNA samples relative to the fresh weight of the root. Each strain was assessed using strain-specific primers designed with Primer BLAST (Ye 2012) and compared to background amplification from uninoculated plants. Since some primers exhibit off-target amplification in non-inoculated plants, colonization can be determined by the presence of amplification or elevated amplification of the correct product compared to background levels.

[0290] This analysis is used to measure the compatibility of microbial products across different soil geographies. Field soil quality and field conditions can have a huge impact on the effectiveness of microbial products. Soil pH, water retention capacity, and competing microorganisms are just a few examples of factors in the soil that can affect the survival and colonization ability of the inoculum. Colonization analysis was performed using three different soil types sampled from agricultural fields in California as plant growth media ( Fig.16A ). A moderate inoculation density was used to approximate actual agricultural conditions. Within 3 weeks, strain 5 colonized all plants with 1E+06 to 1E+07 CFU / g fresh weight. After 7 weeks of plant growth, the evolved form of strain 1 exhibited high colonization rates (1E+06 CFU / g fresh weight) in all soil types. ( Fig. 16B ).

[0291] In addition, in order to assess colonization under the complexity of field conditions, a 1-acre field trial was conducted in San Luis Obispo in June 2015 to assess the impact and colonization of seven wild-type strains in two non-sweet corn varieties. The agronomic design and execution of the experiment were conducted by Pacific Ag Research, a contract field research organization. For inoculation, the same peat culture seed coating technology tested in the inoculation method experiment was adopted. During the growing season, plant samples were collected to assess colonization inside the roots and stems. Samples were collected from three replicate plots of each treatment at four weeks and eight weeks after planting, and samples were collected from all six replicates of each treatment shortly before harvesting at 16 weeks. At the 12th week, additional samples were collected from all six replicate treatment plots inoculated with strains 1 and 2 and untreated controls. As with other colonization analyses using qPCR and strain-specific primers, the number of cells per gram of fresh weight of washed roots was assessed. Two strains, strain 1 and strain 2, showed consistent and extensive root colonization, peaking at 12 weeks and then declining abruptly ( Fig. 16C ). Although strain 2 appeared to be present in an order of magnitude less numbers than strain 1, it was found to be more consistent between plants. No strain appeared to effectively colonize the interior of the stem. Supporting the qPCR colonization data, two strains were successfully reisolated from root samples using plating and 16S sequencing to identify isolates with matching sequences.

[0292] Examples of microbial breeding can be summarized in Fig.17 and Fig.18 in the schematic diagram. Fig.17 Microbial breeding is described, where the composition of the microbiome can first be measured and the species of interest identified. The metabolism of the microbiome can be mapped and linked to genetics. Afterwards, targeted gene variation can be introduced using methods including but not limited to conjugation and recombination, chemical mutagenesis, adaptive evolution, and gene editing. Derived microorganisms are used to inoculate crops. In some instances, crops with the best phenotypes are selected.

[0293] like Fig.17 As provided in the literature, the composition of the microbiome can be first measured and the species of interest identified. The metabolism of the microbiome can be mapped and linked to genetics. Nitrogen metabolism may involve the transport of ammonia (NH 4 +) from the rhizosphere into the cytosol of the bacteria. Glutamine synthetase and ATP catalyze ammonia and L-glutamate (L-Glu) into glutamine. Glutamine can cause biomass formation (plant growth), and it can also inhibit the expression of the nif operon. Afterwards, targeted gene variation can be introduced using methods including but not limited to conjugation and recombination, chemical mutagenesis, adaptive evolution, and gene editing. The derived microorganisms are used to inoculate crops. Crops with the best phenotype are selected.

[0294] Unless otherwise indicated herein or clearly contradictory to the content, in the case of describing the present invention (especially in the case of the appended claims), the use of the terms "a / an" and "the" and similar indicators should be understood to cover the singular and the plural. Unless otherwise indicated, the terms "comprising", "having", "including" and "containing" should be interpreted as open terms (i.e., meaning "including but not limited to") unless otherwise indicated herein. Unless otherwise indicated herein, the description of the value range herein is intended only to serve as a shorthand method of individually referring to each individual value belonging to the range, and each individual value is incorporated into this specification as if it were individually described herein. For example, if a range of 10-15 is disclosed, 11, 12, 13 and 14 are also disclosed. Unless otherwise indicated herein or clearly contradictory to the content, all methods described herein can be performed in any suitable order. Unless otherwise claimed, any and all examples or exemplary language (e.g., "such as") provided herein are intended only to better illustrate the present invention and are not intended to limit the scope of the present invention. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0295] Example 8 Promoter Verification

[0296] For each selected promoter, a strain was generated in which the nifL gene was deleted and replaced with a promoter inserted upstream of the nifA gene, a transcriptional activator of nitrogenase expression in the absence of nifL. Each of these mutants was tested in an acetylene reduction assay, in which anaerobic cultures were exposed to acetylene gas, which nitrogenase reduced to ethylene gas, which can be detected by gas chromatography. The acetylene reduction rate corresponds to the amount of nitrogenase present in the sample, and therefore serves as a readout of nifA transcription. In some cases, RNA extraction was performed on samples of the acetylene reduction assay, and transcription of nifA and nitrogenase genes was measured by qPCR. The results are shown in Table 6.

[0297] Further results are shown in Figures 19-31C. Fig.19A , 19BIn 24A and 24B, the NifL gene has been deleted and replaced with a heterologous promoter, as indicated in the figure. No DNA sequences foreign to the host cell are introduced into the genome by this process. Fig.19A , 20A , 21 and 22, when nifA is operably linked to any one of SEQ ID NO.: 1, 5, 9, 11-24, 26, 27, 30, 33-37 and 40, nifA expression is increased compared to the wild-type level. Fig.19B , 20B , 23, 24A, 24B, 25A, 25B, 26, 27, 28 and 29, when nifA is operably linked to any one of SEQ ID NO.: 1, 2, 5-7, 9-12, 26-28, 32-38, 40, 42, 45, 50-51, 61-63, 67 and 70, the nitrogenase activity is increased compared to the wild-type level. Elimination Fig.24A , 24B , 25B, and the genetically engineered strains depicted in 26-29 to remove all non-native promoter sequences. Figure 19A-23 , 25A, 30, and 31 were not cleared prior to analysis.

[0298] Figure 30-31C It is shown that the heterologous promoters disclosed herein can also be used to upregulate genes other than NifA. Figure 30-31C In the example, the native promoter sequence of bcsI, otsB, CysZ or treZ is replaced with the heterologous sequence disclosed herein. Fig.30 As shown, when the gene is operably linked to SEQ ID NO.: 2, the expression of bcsI is increased. Fig.31A As shown, when the gene is operably linked to SEQ ID NO.: 1, the expression of CysZ is increased. Fig.31B As shown, when the gene is operably linked to SEQ ID NO.: 1 or SEQ ID NO.: 2, the expression of otsB is increased. Fig.31C As shown, when the gene is operably linked to SEQ ID NO.: 2, the expression of treZ is increased.

[0299] Although preferred embodiments of the present invention have been shown and described herein, it will be clear to those skilled in the art that such embodiments are provided only as examples. Those skilled in the art will now appreciate that a large number of modifications, variations and substitutions will be appreciated without departing from the present invention. It should be understood that each alternative to the embodiments of the present invention described herein can be used to practice the present invention. The appended claims are intended to define the scope of the present invention, and thus cover methods and structures within the scope of these claims and their equivalents.

[0300] Table 3A

[0301] name Minimum Maximum length direction Murine lipoprotein CDS 2,929,898 2,930,134 237 Positive Membrane Protein CDS 5,217,517 5,217,843 327 Positive Zinc / cadmium binding protein CDS 3,479,979 3,480,626 648 Positive Acyl carrier protein CDS 4,563,344 4,563,580 237 Reverse ompX CDS 4,251,002 4,251,514 513 Positive DNA binding protein HU-βCDS 375,156 375,428 273 Positive sspA CDS 629,998 630,636 639 Reverse tatE CDS 3,199,435 3,199,638 204 Reverse LexA repressor CDS 1,850,457 1,851,065 609 Positive hisS CDS <3999979 4,001,223 >1245 Positive

[0302] Table 3B

[0303]

[0304] Table 4, strain list

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311] Strain Table (Continued)

[0312]

[0313] Table 8: Promoter sequences

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326] Table 9: Promoter activity in vitro and in plants

[0327]

[0328]

[0329]

[0330] Table 10: Species origin of the promoter sequences disclosed herein

[0331] SEQ ID NO: Species 1-10 Ostreella sucrose 11-40 Klebsiella mutans 41-59、63-66 Rahnella aquatica 60 Kluyvera intermedia 61、70-72 Ostreella pseudosucrose 62 Enterobacter 67-69 Klebsiella .

Claims

1. A genetically engineered bacterium for fixing nitrogen, wherein the genetically engineered bacterium belongs to the species Microceras sucrose and comprises an inserted Microceras promoter sequence consisting of the sequence shown in SEQ ID No. 5, wherein the inserted Microceras promoter sequence replaces the natural promoter sequence.

2. A genetically engineered bacterium that fixes nitrogen, wherein the genetically engineered bacterium belongs to the species Oxytropis sucrose and comprises a nitrogen fixation or nitrogen assimilation coding sequence operably linked to an inserted Oxytropis sucrose promoter sequence, wherein the inserted sequence consists of the sequence shown in SEQ ID No.

5.

3. The genetically engineered bacterium according to claim 2, wherein the nitrogen fixation or nitrogen assimilation coding sequence is selected from the group consisting of nifA, nifL, ntrB, ntrC, a polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, draT, amtB, a polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ and genes related to the biosynthesis of nitrogenase.

4. The genetically engineered bacterium according to claim 1 or 2, wherein the genetically engineered bacterium fixes atmospheric nitrogen under non-nitrogen limiting conditions.

5. The genetically engineered bacterium according to claim 1 or 2, wherein the genetically engineered bacterium fixes more atmospheric nitrogen than a non-engineered bacterium of the same species.

6. The genetically engineered bacterium according to claim 1 or 2, wherein the inserted promoter sequence is a natural sequence inserted in a non-natural background.

7. A composition comprising the genetically engineered bacteria according to claim 1 or 2.

8. A method for increasing bacterial gene expression in a bacterium of the species Microceras sucrosei, said method being carried out by replacing the natural promoter sequence of said bacterial gene with a Microceras promoter sequence, said Microceras promoter sequence consisting of the sequence shown in SEQ ID No.

5.

9. The method of claim 8, wherein increasing expression of the bacterial gene increases ammonium excretion by the bacteria.

10. The method of claim 8, wherein increasing expression of the bacterial gene increases nitrogen fixation by the bacteria.

11. The method of claim 8, wherein increasing expression of the bacterial gene increases colonization of a plant by the bacteria.

12. A polynucleotide comprising a coding sequence of a protein related to nitrogen fixation or nitrogen assimilation and an inserted promoter sequence of a protein related to nitrogen fixation or nitrogen assimilation.

13. The polynucleotide of claim 12, wherein the coding sequence for a protein associated with nitrogen fixation or nitrogen assimilation is selected from the group consisting of nifA, nifL, ntrB, ntrC, a polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, draT, amtB, a polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, and genes associated with the biosynthesis of nitrogenase.

14. A method for increasing the amount of atmospheric nitrogen in a corn plant, comprising contacting the corn plant with a genetically engineered bacterium belonging to the species Osterholmia sucrose, wherein the genetically engineered bacterium comprises a nitrogen fixation coding sequence operably linked to an inserted Osterholmia promoter, wherein the inserted Osterholmia promoter consists of the sequence shown in SEQ ID No.

5.

15. A method for reducing the amount of nitrogen fertilizer required between planting and harvesting of a corn crop, the method comprising inoculating the corn crop with the genetically engineered bacteria according to claim 1 or 2.

Images