methods and compositions for improving modified microbes

专利摘要:
The present description refers to the bacterial composition comprising: at least one genetically modified bacterial strain that fixes atmospheric nitrogen in an agricultural system, in which the bacterial strain comprises a modification in one or more genes selected from the group consisting of bcsll, bcsIII, yjbE, fhaB, pehA, glgA, otsB, treZ and cysZ. The present description further relates to a bacterial composition and method for increasing the colonization of a plant growth-promoting bacterial strain on a plant, wherein the plant growth-promoting bacterial strain on a plant has been remodeled to increase said colonization plant. In a further aspect, the present description relates to methods of increasing nitrogen and nitrogen fixation suitable for a plant.
公开号:BR112020002654A2
申请号:R112020002654-9
申请日:2018-08-09
公开日:2020-08-18
发明作者:Sarah BLOCH；Karsten Temme；Alvin Tamsir
申请人:Pivot Bio, Inc.；
IPC主号:

专利说明:

[0001] [0001] This patent application claims its priority benefit to Provisional U.S. Patent Application No. 62/543 288, filed on August 9, 2017, the content of which is incorporated herein by reference in this patent application. Background of the invention
[0002] [0002] By 2050, the United Nations Food and Agriculture Organization projects that total food production needs to increase 70% to meet the needs of the growing population, a challenge that is exacerbated by numerous factors, including the decreased drinking water resources, growing competition for arable land, rising energy prices, rising input costs and the likely need for crops that adapt to the pressures of drier, hotter and more extreme climatic conditions.
[0003] [0003] One area of interest is in improving nitrogen fixation. Gaseous nitrogen (N2) is a major component of the Earth's atmosphere. In addition, the element nitrogen (N) is an important component of many chemical compounds that make up living organisms. However, many organisms are unable to use N2 directly to synthesize the chemicals used in physiological processes, such as growth and reproduction. In order to be used, N2 needs to be combined with hydrogen. The combination of hydrogen and N2 is referred to as nitrogen fixation. Nitrogen fixation, whether performed chemically or biologically, requires investing large amounts of energy. In biological systems, an enzyme known as nitrogenase catalyzes the reaction that results in nitrogen fixation. An important objective of research on nitrogen fixation is the extension of this phenotype to non-leguminous plants, especially grasses of agronomic value such as wheat, rice and corn. Despite enormous advances in the understanding of the development of symbiosis for nitrogen fixation between rhizobia and legumes, the way to use this knowledge to induce nitrogen fixing nodes in non-leguminous cultures is still unclear. In the meantime, the challenge of supplying sufficient supplemental sources of nitrogen, such as fertilizers, will continue to increase with the growing need for greater food production. Summary of the invention
[0004] [0004] In one aspect, the present description provides a bacterial composition comprising: at least one genetically modified bacterial strain that fixes atmospheric nitrogen in an agricultural system, wherein the bacterial strain comprises a modification in one or more genes selected from the group consisting of bcsll, bcsIII, yjbE, fhaB, pehA, glgA, otsB, treZ and cysZ.
[0005] [0005] In another aspect, the present description provides a bacterial composition comprising: a bacterial strain that promotes plant growth, in which said strain has been remodeled to increase the colonization of said bacterial strain that promotes plant growth in a plant . In some cases, said colonization of said bacterial strain that promotes plant growth occurs in a root of said plant. In some cases, said bacterial strain comprises a genetic modification in an enzyme or pathway involved in the production of exopolysaccharides. In some cases, said genetic modification is in a gene selected from the group consisting of bcsII, bcsIII and yjbE. In some cases, said bacterial strain comprises a genetic modification in an enzyme or pathway involved in the production of a filamentous hemagglutinin. In some cases, said genetic modification is in a fhaB gene. In some cases, the so-called bacterial strain comprises a genetic modification in an enzyme or pathway involved in the production of an endo-polygalaturonase. In some cases, said genetic modification is in a pehA gene. In some cases, said bacterial strain comprises a genetic modification in an enzyme or pathway involved in the production of trehalose. In some cases, said genetic modification is in a gene selected from the group consisting of: otsB and treZ. In some cases, said bacterial composition is formulated for application to a field. In some cases, said plant growth promoting bacteria provides a nutrient for said plant. In some cases, said plant growth-promoting bacteria provides fixed nitrogen for said plant. In some cases, said plant is selected from the group consisting of corn, barley, wheat, sorghum, soy and rice.
[0006] [0006] In a further aspect, the present description provides a method to increase the colonization of a bacterial strain promoting plant growth in a plant, said method comprising: introducing a modification in said bacterial strain promoting plant growth genetics in a gene involved in a pathway selected from the group consisting of: production of exopolysaccharides, production of endo-polygalaturonase and production of trehalose.
[0007] [0007] In a further aspect, the present description provides a method to increase the nitrogen available to a plant, said method comprising: applying a plurality of remodeled bacteria on a plant, said plurality of remodeled bacteria exhibiting decreased expression of glgA.
[0008] [0008] In some cases, said remodeled bacteria have a lower degree of nitrogen assimilation within said remodeled bacteria when compared to a degree of assimilation of non-remodeled bacteria of the same species as said remodeled bacteria.
[0009] [0009] In a further aspect, the present description provides a method for increasing the fixation of the nitrogen available to a plant, said method comprising: applying a plurality of remodeled bacteria on a plant, said plurality of remodeled bacteria exhibiting increased expression of at least one nitrogenase cofactor. In some cases, said increased nitrogenous cofactor is sulfur. In some cases, said remodeled bacteria show greater expression of cysZ. In some cases, cysZ is a sulfur transporter. Incorporation by reference
[0010] [0010] All publications, patents and patent applications mentioned in this specification are incorporated by reference to the same extent that each individual publication, patent or patent application would have been specifically and individually indicated. to be incorporated by reference. Brief description of the drawings
[0011] [0011] The new features of the invention are presented with particularity in the attached claims. A better understanding of the characteristics and advantages of the present invention will be obtained by reference to the detailed description below which presents illustrative modalities, in which the principles of the invention are used, and the accompanying drawings, of which:
[0012] [0012] Figure 1A represents a soil texture map of various field soils tested for colonization. Soils that were originally the source of few microbes are referred to as stars.
[0013] [0013] Figure 1B represents the colonization rate of Cepa 1 and Cepa 5 which are tested in four different types of soil (circles). The two strains showed a relatively robust colonization profile in different types of soil.
[0014] [0014] Figure 1C represents the colonization of Cepa 1 as tested in a field trial over the period of a growing season. The strain 1 persists in the corn tissue until 12 weeks after planting, and its colonization begins to show a decline after that time.
[0015] [0015] Figure 2A represents a schematic drawing of the microbial genetic improvement according to modalities.
[0016] [0016] Figure 2B represents an enlarged view of the measurement of the composition of the microbiome as shown in Figure 2A.
[0017] [0017] Figure 2C represents the sampling of roots used in Example 7.
[0018] [0018] Figures 3A-3C illustrate derived microbes that fix and excrete nitrogen in vitro under conditions similar to agricultural soils with a high nitrate content. Figure 3A illustrates a regulatory network that controls nitrogen fixation and assimilation in PBC6.1, including the main NifL, NifA, GS, GlnE nodes represented as the enzyme with two domains ATase-AR and AmtB. Figure 3B illustrates the genome of the Kosakonia sacchari isolate PBC6.1. The three bands delimiting the genome transmit the transcription data from PBC6.1, PBC6.38, and the differential expression between the strains respectively. Figure 3C illustrates the gene cluster for fixing the nitrogen gene and the transcription data is expanded for more detailed reproduction.
[0019] [0019] Figure 4 illustrates the colonization of PBC6.1 up to almost 21% abundance of the root-associated microbiota in corn roots. The abundance data are based on the sequencing of 16S amplicon from the rhizosphere and endosphere of corn plants inoculated with PBC6.1 and grown under greenhouse conditions.
[0020] [0020] Figure 5 illustrates the increased expression of three genes of interest in remodeled K. sacchari strains, shown by qPCR. The strains were grown in medium containing 5 mM glutamine.
[0021] [0021] Figure 6 illustrates the nitrogen reduction activity of several strains remodeled in the presence or absence of glutamine.
[0022] [0022] Figure 7 illustrates the results of an in vitro adhesion assay with several strains described here.
[0023] [0023] Figure 8A illustrates the results of an in vitro adhesion assay with a remodeled strain showing greater expression of fhaB as described here. This strain showed a significantly increased relative recovery (fold recovery) when compared to the parental strain, 6-848 (2.3x, p = 0.013).
[0024] [0024] Figure 8B illustrates the results of an in vitro adhesion assay with several strains described here.
[0025] [0025] Figure 9 illustrates the greater adhesion by strains with the positively regulated yjbE2 gene and bcsIII operon. Two remodeled strains showed a slight increase compared to 6-848: 6-112 (6-848 + yjbE2-prm1): 1.9x (p = 0.07), and 6-1126 (6-848 + bcsIII-prm2 ): 1.7x (p = 0.06). Strain 6-1127 (6-848 + bcsIII-prm9) showed a significant 2.5x increase over 6-848 (p = 0.0005)
[0026] [0026] Figure 10 illustrates the results of an in vitro adhesion assay. 0 is no microbe added to water, 462 is DH10B E. coli (a control not associated with plants) added to water, 6-848 and 6-881 are parental strains of other strains used in the present assays (positive controls). Uninoculated controls e. coli showed some background, however, strain 6 of the wild type and parental controls 6-848 and 6-881 showed significant adherence in relation to the negative controls (p <0.05).
[0027] [0027] Figure 11 illustrates greater adhesion of strains with modifications in the pehA gene, in the yjbE2 gene and in the bcsII operon.
[0028] [0028] Figure 12 illustrates the overexpression of modified genes in strains remodeled in the presence and absence of glutamine.
[0029] [0029] Figure 13A illustrates the overexpression of modified genes in strains remodeled in the presence and absence of glutamine.
[0030] [0030] Figure 13B illustrates the overexpression of modified genes in strains remodeled in the presence and absence of glutamine.
[0031] [0031] Figure 13C illustrates the overexpression of modified genes in strains remodeled in the presence and absence of glutamine.
[0032] [0032] Figure 14 illustrates the excretion of ammonium under anaerobic conditions for the three strains described here.
[0033] [0033] Figure 15 illustrates the excretion of ammonium for several strains described here.
[0034] [0034] Figure 16 illustrates the excretion of ammonium for other strains described here.
[0035] [0035] Figure 17 illustrates the excretion of ammonium for other strains described here.
[0036] [0036] Figure 18 illustrates the nitrogen reduction activity for several strains described here.
[0037] [0037] Figure 19 illustrates the excretion of ammonium for other strains described here.
[0038] [0038] Figure 20 illustrates the nitrogen reduction activity for several strains described here.
[0039] [0039] Figure 21 illustrates the excretion of ammonium for other strains described here.
[0040] [0040] Figure 22A illustrates the nitrogen reduction activity for several strains described here in the presence or absence of nitrogen.
[0041] [0041] Figure 22B illustrates the nitrogen reduction activity for several strains described here in the presence or absence of nitrogen.
[0042] [0042] Figure 23A illustrates the excretion of ammonium for strains described here.
[0043] [0043] Figure 23B illustrates the excretion of ammonium for strains described here.
[0044] [0044] Figure 24 illustrates the nitrogen reduction activity for several strains described here in the presence or absence of nitrogen.
[0045] [0045] Figure 25 illustrates the excretion of ammonium for strains described here.
[0046] [0046] Figure 26A illustrates the nitrogen reduction activity for several strains described here in the presence or absence of nitrogen.
[0047] [0047] Figure 26B illustrates the nitrogen reduction activity for several strains described here in the presence or absence of nitrogen.
[0048] [0048] Figure 27A illustrates the excretion of ammonium for strains described here.
[0049] [0049] Figure 27B illustrates the excretion of ammonium for strains described here.
[0050] [0050] Figure 28 illustrates the colonization of strains described here in a greenhouse trial. The greenhouse test for colonization had no statistical power to distinguish differences with p <0.05.
[0051] [0051] Figure 29 illustrates the colonization of other strains described here in a greenhouse trial.
[0052] [0052] Figure 30 illustrates the colonization of strains described here in a greenhouse trial.
[0053] [0053] Figure 31 illustrates the colonization of strains described here in a greenhouse trial.
[0054] [0054] Figure 32 illustrates the colonization of strains described here in a greenhouse trial.
[0055] [0055] Figure 33 illustrates the excretion of ammonium for strains described here.
[0056] [0056] Figure 34 illustrates the nitrogen reduction activity for several strains described here in the presence or absence of nitrogen.
[0057] [0057] Figure 35 illustrates the excretion of ammonium for strains described here.
[0058] [0058] Figure 36 illustrates nitrogen reduction activity for several strains described here in the presence or absence of nitrogen.
[0059] [0059] Figure 37 illustrates the colonization of strains described here.
[0060] [0060] Figure 38 illustrates the colonization of strains described here. Detailed description of the invention
[0061] [0061] Although various modalities of the invention have been shown and described, it will be obvious to those skilled in the art that such modalities are provided by way of example only. Numerous variations, changes and substitutions can occur to those skilled in the art without deviating from the invention. It is to be understood that various alternatives to the embodiments of the invention described herein can be employed.
[0062] [0062] The increased use of fertilizers raises environmental concerns and is also probably not possible for many economically stressed regions of the globe. In addition, many industry operators in the microbial arena are focused on creating intergeneric microbes. However, there is a heavy regulatory burden placed on modified microbes that are characterized / classified as intergeneric. These intergeneric microbes face not only a greater regulatory burden, which makes their widespread adoption and implementation more difficult, but also very subject to public scrutiny.
[0063] [0063] Currently, there are no modified microbes on the market that are non-intergeneric and that are capable of increasing nitrogen fixation in non-leguminous cultures. This scarcity of such a microbe is a missing element in helping to enter a truly sustainable and environmentally friendly 21st century agricultural system.
[0064] [0064] The present description solves the problems mentioned above and provides a non-intergeneric microbe that has been modified to quickly fix nitrogen in cultures. These microbes are characterized / classified as intergeneric microbes and, therefore, will not face the accentuated regulatory burdens of such. In addition, the non-intergeneric microbes taught will serve to help 21st century farmers to rely less on using ever-increasing amounts of fertilizer as an exogenous source of nitrogen.
[0065] [0065] Guided microbial remodeling is a state-of-the-art technology process that identifies, characterizes and adjusts microbes so that they reach the full potential to colonize crop roots and increase nutrient absorption. In some cases, guided microbial remodeling may make use of the genetic material naturally present in each to increase nitrogen uptake by the culture. Better nutrient availability improves crop quality and yield potential.
[0066] [0066] There is a complex of enzymes called nitrogenases in microbes that are responsible for reducing atmospheric nitrogen to ammonia, and both microbes and plants convert ammonia into glutamine through glutamine synthase (GS) and, consequently, in others amino acids that are essential for the growth and development of microbes and plants. Nif genes are genes that encode the nitrogenase enzyme complex. In addition to the nitrogenase enzymes, the nif genes also encode several regulatory proteins involved in nitrogen fixation. The expression of the nif genes is induced in response to low levels of fixed nitrogen and oxygen concentrations in the rhizosphere. In most nitrogen-fixing bacteria, the transcription of the nif genes is regulated by the nitrogen-sensitive NifA protein. When there is not enough fixed nitrogen available for the body to use, NifA expression is activated, and NifA activates the rest of the nif genes. If there is a sufficient amount of reduced nitrogen or oxygen present in the rhizosphere, another protein is activated, NifL. NifL inhibits NifA activity resulting in the inhibition of nitrogenase formation.
[0067] [0067] Examples of aspects of microbial nitrogen fixation that can be changed to increase nitrogen availability for cereal crops include: a) reinforcing the interaction between the microbe and the roots of the crop in the rhizosphere. b) reinforce the microbial nitrogen fixation by removing the negative regulation of the NifA protein. For example, deletion of the nifL gene abolishes inhibition of NifA activity and improves nif expression in the presence of both oxygen and exogenous fixed nitrogen. In addition, the expression of nifA under the control of a nitrogen-independent promoter can dissociate nitrogenase biosynthesis from the detection of environmental nitrogen and oxygen. c) reinforcing the availability of ammonia to the plant by inhibiting the assimilation of ammonia to glutamine in the microbe and increasing the excretion of ammonia by the microbe. For example, the rapid assimilation of the nitrogen fixed by the microbe to glutamine by GS is reversibly regulated by the enzyme with two domains adenyltransferase (ATase), GlnE, through the adenylation and deladenylation of GS to attenuate and restore activity, respectively. Truncating the GlnE protein to delete its adenyiyi4emoving [sic] (AR) domain leads to constitutively adenylated glutamine synthetase, limiting the microbe's assimilation of ammonia and increasing intracellular and extracellular ammonia. Finally, the deletion of the amtB gene, which encodes the transporter responsible for the absorption of ammonia by the microbe, leads to greater excretion of ammonia.
[0068] [0068] In some cases, the final microbes produced by the methods described here do not contain foreign genetic material (transgenic), and may be suitable for release in a field. Genetic changes in the microbe can consist of sequence deletions or small rearrangements of the sequence within the organism's own genome, such as creating a copy of a promoter sequence within the microbe's genome and inserting the copy in front of a different gene. Therefore, any genetic elements introduced into the microbe's genome can be derived from the same parental microbe. In addition, the genetic elements introduced into the microbial genome can be non-coding genetic elements. The resulting mutations can be “markerless”, meaning that they do not contain introduced marker genes for antibiotic resistance or other selectable genes that are often used to generate mutations in bacteria. In some cases, the guided microbial remodeling process may involve introducing some helper (helper) DNA molecules into bacterial cells to facilitate the generation of non-transgenic mutations. However, all of the DNA helper can be completely removed from the strains. For example, in some cases, all foreign DNA can be removed from a strain before the strain is considered for release in the field. The removal of the entire DNA helper can be confirmed, for example, by sequencing Illumina, a sensitive method that can detect even unique molecules from DNA helper sequences. Thus, microbes generated through guided microbial remodeling may not contain transgenes or foreign DNA, and the genetic changes in the strain consist only of deletions of sequences and rearrangements of promoters, the occurrence of which has been demonstrated in nature. Definitions
[0069] [0069] The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or the like. Polynucleotides can have any three-dimensional structure, and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), RNA ribosomal (rRNA), small interfering RNA (siRNA), small RNA in hairpin (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, DNA isolated from any sequence, RNA isolated from any sequence, nucleic acid probes and primers (primers). A polynucleotide can comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be transmitted before or after assembly of the polymer. The nucleotide sequence can be interrupted by non-nucleotide components. A polynucleotide can further be modified after polymerization, such as by conjugation with a labeling component.
[0070] [0070] In this specification, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (such as and transcribed from mRNA or other RNA) and / or the process by which an mRNA is transcribed it is subsequently translated into peptides, polypeptides or proteins. Transcripts and encoded polypeptides can be collectively referred to as a “gene product”. If the polynucleotide is derived from genomic DNA, the expression may include splicing (processing) the mRNA in a eukaryotic cell.
[0071] [0071] The terms "polypeptide", "peptide" and "protein" are used interchangeably in the present to refer to polymers of amino acids of any length. The polymer can be linear or branched, can comprise modified amino acids and can be disrupted by non-amino acids. The terms also cover a polymer of amino acids that has been modified; for example, formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation, such as conjugation with a marking component. In this specification, the term “amino acid” includes natural and / or unnatural or synthetic amino acids, including glycine and optical isomers D or L, as well as amino acid and peptidomimetic analogues.
[0072] [0072] In this specification, the term “about” is used as a synonym for the term “approximately”. Illustratively, the use of the term "about" in relation to a quantity indicates values slightly outside the quoted values, p. eg, more or less 0.1% to 10%.
[0073] [0073] Microbes in and around food cultures can influence the attributes of these cultures. Plant attributes that can be influenced by microbes include: productivity (eg, grain production, biomass generation, fruit development, flowering); nutrition (eg, acquisition of nitrogen, phosphorus, potassium, iron, micronutrients); management of abiotic stress (eg, drought tolerance, salinity tolerance, heat tolerance); and management of biotic stress (eg, pest, weeds, insects, fungi and bacteria). Strategies for changing crop attributes include: increasing concentrations of essential metabolic compounds; changing the time dynamics of microbial influence on essential metabolites; link the production / degradation of microbial metabolites to new environmental stimuli; reduce negative metabolites; and improving the balance of underlying metabolites or proteins.
[0074] [0074] In some embodiments, native or endogenous control sequences of genes of the present invention are replaced by one or more intragenic control sequences.
[0075] [0075] In this specification, “introduced” refers to introduction through modern biotechnology and not a natural introduction.
[0076] [0076] In some embodiments, the bacteria of the present invention have been modified in such a way that they are not natural bacteria.
[0077] [0077] In some embodiments, the bacteria of the present invention are present in the plant in an amount of at least 103 cfu, 104 cfu, 105 cfu, 106 cfu, 107 cfu, 108 cfu, 109 cfu, 1010 cfu, 1011 cfu or 1012 cfu per gram of fresh or dry mass of the plant. In some embodiments, the bacteria of the present invention are present in the plant in an amount of at least approximately 103 cfu, approximately 104 cfu, approximately 105 cfu, approximately 106 cfu, approximately 107 cfu, approximately 108 cfu, approximately 109 cfu, approximately 1010 cfu, approximately 1011 cfu, or approximately 1012 cfu per gram of fresh or dried plant mass. In some embodiments, the bacteria of the present invention are present in the plant in an amount of at least 103 to 109, 103 to 107, 103 to 105, 105 to 109, 105 to 107, 106 to 1010, 106 to 107 cfu per gram of fresh or dry mass of the plant.
[0078] [0078] The fertilizers and exogenous nitrogen of the present invention can comprise the following nitrogen-containing molecules: ammonium, nitrate, nitrite, ammonia, glutamine etc. The nitrogen sources of the present invention can include anhydrous ammonia, ammonium sulfate, urea, diamonium phosphate, urea form, monoammonium phosphate, ammonium nitrate, nitrogenous solutions, calcium nitrate, potassium nitrate, sodium nitrate etc.
[0079] [0079] In this specification, “exogenous nitrogen” refers to non-atmospheric nitrogen readily available in the soil, field or growth medium that is present under conditions without nitrogen limitations, including ammonia, ammonium, nitrate, nitrite, urea, acid uric acid, ammonium acids etc.
[0080] [0080] In this specification, “conditions without nitrogen limitations” refers to the non-atmospheric nitrogen available in the soil, field, media in nitrogen concentrations greater than approximately 4 mM, as described by Kant at al. (2010. J. Exp. Biol. 62 (4): 1499-1509), incorporated herein by reference.
[0081] [0081] In this specification, “intergeneric microorganism” is a microorganism that is formed by the intentional combination of genetic material originally isolated from organisms of different taxonomic genera. “Intergeneric mutant” can be used alternately with “intergeneric microorganism”. An exemplary “intergenerational microorganism” includes a microorganism containing a mobile genetic element that was initially identified in a microorganism of a genus other than the recipient microorganism.
[0082] [0082] In this specification, “intragenic microorganism” is a microorganism and is formed by the intentional combination of genetic material originally isolated from organisms of the same taxonomic genera. "Intragenic mutant" can be used interchangeably with "intragenic microorganism".
[0083] [0083] In this specification, “introduced genetic material” means genetic material that is added and remains as a component of the recipient's genome.
[0084] [0084] In some embodiments, a network of genetic regulation of nitrogen fixation and assimilation comprises polynucleotides that encode non-coding genes and sequences that direct, modulate and / or regulate the microbial fixation and / or assimilation of nitrogen and that may comprise polynucleotide sequences of the nif cluster (eg, nifA, nifB, nifC, …… .nifZ), polynucleotides encoding nitrogen regulatory protein C, polynucleotides encoding nitrogen regulatory protein B, polynucleotide sequences from the gln cluster (p eg 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 a subset of NifB, NifH, NifD, NifK, NifE, NifN, NifX, hesa and NifV.
[0085] [0085] In some embodiments, the fertilizer of the present invention 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% nitrogen in large scale.
[0086] [0086] In some embodiments, the fertilizer of the present invention comprises at least approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17%, approximately 18%, approximately 19%, approximately 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35% , approximately 36%, approximately
[0087] [0087] In some embodiments, the fertilizer of the present invention comprises between approximately 5% and 50%, between approximately 5% and 75%, between approximately 10% and 50%, between approximately 10% and 75% , between approximately 15% and 50%, between approximately 15% and 75%, between approximately 20% and 50%, between approximately 20% and 75%, between approximately 25% and 50%, between approximately 25% and 75%, between approximately 30%
[0088] [0088] In some modalities, the increase in nitrogen fixation and / or the nitrogen production in the plant are measured in relation to control plants, which were not exposed to the bacteria of the present invention. All increases or decreases in bacteria are measured against control bacteria. All increases or decreases in plants are measured relative to control plants.
[0089] [0089] In this specification, “constitutive promoter” is a promoter that is active under most conditions and / or during most stages of development. There are several advantages to the use of constitutive promoters in expression vectors used in biotechnology, such as: high level of production of proteins used to select transgenic cells or organisms; high level of expression of reporter proteins or punctuated markers, allowing easy detection and quantification; high level of production of a transcription factor that is part of a regulatory system for transcription; production of compounds that require ubiquitous activity in the body; and production of compounds that are needed at all stages of development. Exemplary constitutive promoters include the CaMV 35S promoter, opine promoters, ubiquitin promoter, alcohol dehydrogenase promoter, etc.
[0090] [0090] In this specification, "non-constitutive promoter" is a promoter that is active under certain conditions, in certain types of cells and / or during certain stages of development. For example, tissue-specific promoters, preferred tissues, specific cell types and preferred cell types, inducible promoters, and promoters under development control are non-constitutive promoters. Examples of promoters under development control include promoters that preferentially initiate transcription in certain tissues.
[0091] [0091] In this specification, an “inducible” or “repressible” promoter is a promoter who is under control of chemical or environmental factors. Examples of environmental conditions that can affect transcription by inducible promoters under anaerobic conditions, certain chemicals, the presence of light, acidic or basic conditions etc.
[0092] [0092] In this specification, a "tissue-specific" promoter is a promoter that initiates transcription only in certain tissues. Unlike the expression of constitutive genes, tissue-specific expression results from several interacting levels of gene regulation. Thus, in the art, it is sometimes preferable to use promoters of homologous or closely related species to achieve efficient and reliable expression of transgenes in certain tissues. This is one of the main reasons for the large number of tissue-specific promoters isolated from certain tissues found in the scientific and patent literature.
[0093] [0093] In this specification, the term "operably linked" refers to the association of the nucleic acid sequence into a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operationally linked to a coding sequence when it is able to regulate the expression of that coding sequence (that is, that coding sequence is under the transcriptional control of the promoter). The coding sequences can be operationally linked to regulatory sequences in sense or antisense orientation. In another example, the complementary RNA regions of the invention can be operationally linked, either directly or indirectly, 5 ′ to the target mRNA, or 3 ′ to the target mRNA or within the target mRNA, or a first complementary region is 5 ′ and its complement is 3 ′ to the target mRNA.
[0094] [0094] In aspects, "applying to the plant a plurality of non-intergeneric bacteria" includes any means by which the plant (including parts of the plant such as a seed, root, stem, tissue, etc.) is made to come into contact (or exposed) with said bacteria at any stage of the plant's life cycle. Consequently, “applying to the plant a plurality of non-intergeneric bacteria” includes any of the following means to expose a plant (including parts of the plant such as a seed, root, stem, tissue, etc.) to said bacteria: spray on the plant, drip onto the plant, apply as a seed cover, apply to a field that will be planted with seeds, apply to a field already planted with seeds, apply to a field with adult plants, etc.
[0095] [0095] The term mmol is an abbreviation for millimol, which is one thousandth (10−3) of a mole, here abbreviated as mole.
[0096] [0096] In this specification the terms "microorganism" or "microbe" should be generalized. These terms, used interchangeably, include, among others, the two prokaryotic domains, Bacteria and Archaea. The term can also include fungi and eukaryotic protists.
[0097] [0097] The term “microbial consortia” or “microbial consortium” refers to a subset of a microbial community of individual microbial species, or strains of a species, which can be described as performing a common function, or can be described as participating in or leading to or correlating with a recognizable parameter, such as a phenotypic attribute of interest.
[0098] [0098] The term "microbial community" means a group of microbes that comprises two or more species or strains. Unlike microbial consortia, a microbial community does not need to perform a common function or does not need to participate in or lead to or correlate with a recognizable parameter, such as a phenotypic attribute of interest.
[0099] [0099] In this specification, “isolate”, “isolated”, “isolated microbe” and similar terms are intended to mean that one or more of the microorganisms has been separated from at least one of the materials with which it is associated in a particular environment (eg soil, water, plant tissue, etc.). Thus, an “isolated microbe” does not exist in its natural environment; instead, it is through the various techniques described here that the microbe has been removed from its natural configuration and placed in an unnatural state of existence. In this way, the isolated strain or the isolated microbe can exist as, for example, a biologically pure culture, or as spores (or other forms of the strain). In aspects, the isolated microbe may be associated with an acceptable carrier, which may be an agriculturally acceptable carrier.
[0100] [0100] In certain aspects of the invention, isolated microbes exist as "isolated and biologically pure cultures". It will be appreciated by the person skilled in the art, that an isolated and biologically pure culture of a particular microbe indicates that said culture is substantially free of other living organisms and that it contains only the individual microbe in question. The culture may contain varying concentrations of said microbe. The present invention points out that isolated and biologically pure microbes often "differ necessarily from less pure or impure materials". See, p. eg, In re Bergstrom, 427 F.2d 1394, (CCPA 1970) (discussion of purified prostaglandins), see also, In re Bergy, 596 F.2d 952 (CCPA 1979) (discussion of purified microbes), see also, Parke-Davis & Co. v. H.K.
[0101] [0101] In this specification, by “individual isolates”, a composition, or culture, must be understood that comprises a predominance of a single genus, species or strain of microorganism, after the separation of one or more other microorganisms.
[0102] [0102] The microbes of the present invention can include spores and / or vegetative cells. In some embodiments, the microbes of the present invention include microbes in a viable, but not cultured (VBNC) state. In this specification, "spore" or "spores" refer to structures produced by bacteria and fungi that are adapted for survival and dispersion. Spores are generally characterized as dormant structures; however, spores are capable of differentiation through the germination process. Germination is the differentiation of spores in vegetative cells that are capable of metabolic activity, growth and reproduction. The germination of a single spore results in a single vegetative fungal or bacterial cell. Fungal spores are units of asexual reproduction and, in some cases, are necessary structures in the life cycle of fungi. Bacterial spores are structures for survival conditions that may not normally lead to the survival or growth of vegetative cells.
[0103] [0103] In this specification, "microbial composition" refers to a composition that comprises one or more microbes of the present invention. In some embodiments, a microbial composition is administered to plants (including various parts of the plant) and / or in agricultural fields.
[0104] [0104] In this specification, "carrier", "acceptable carrier", or "agriculturally acceptable carrier" refers to a diluent, adjuvant, excipient or vehicle with which the microbe can be administered, which does not adversely affect the microbe.
[0105] [0105] The use of the terms "one", "one", "o" and "a" "one" and similar referents in the context of describing the invention (especially in the context of the following claims) should be interpreted to include the singular and the plural, unless otherwise indicated in the present or clearly denied by the context. The terms "comprising", "having", "including" and "containing" shall be interpreted as open terms (that is, meaning "including, among others",) unless otherwise noted. The recitation of ranges of values in the present is simply intended to serve as an abbreviated method for referring individually to each separate value within the range, unless otherwise indicated in the present, and each separate value is incorporated into the specification. as it would be if it were recited individually in the present. For example, if the range 10-15 is described, then 11, 12, 13 and 14 are also described. All of the methods described herein may be performed in any appropriate order unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples, or exemplary language (eg, "as"), in this specification is simply intended to further clarify the invention and does not impose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be interpreted as indicating any element not claimed as essential to the practice of the invention. Bacterial colonization
[0106] [0106] In some modalities, microbes can be modified to increase nitrogen delivery to plants. For example, microbes can be modified to increase colonization of plant roots or to increase cofactors necessary for nitrogen fixation.
[0107] [0107] In some cases, exopolysaccharides may be involved in bacterial colonization of plants. In some cases, plant-colonizing microbes can produce a biofilm. In some cases, plant-colonizing microbes secrete molecules that can assist in plant adherence, or in evading an immune response from the plant. In some cases, plant-colonizing microbes can excrete signaling molecules that alter the response of plants to microbes. In some cases, plant-colonizing microbes can secrete molecules that alter the local microenvironment. In some cases, a plant colonizing microbe can alter gene expression to adapt to a plant to which the said microbe is close. In some cases, a plant colonizing microbe can detect the presence of a plant in the local environment and can change the expression of genes in response. Regulation of nitrogen fixation
[0108] [0108] In some cases, the nitrogen fixation pathway can act as a target for genetic engineering and optimization. One attribute that can serve as a target for regulation by the methods described here is nitrogen fixation. Nitrogen fertilizers represent the largest operating expense on a farm, and the biggest driver for higher crop yields in rows like corn and wheat. The present invention describes microbial products that can provide new renewable forms of nitrogen in non-leguminous crops. Although some endophytes have the genetics necessary to fix nitrogen in pure crops, the fundamental technical challenge is that wild type endophytes of cereals and grasses stop fixing nitrogen in fertilized fields. The application of chemical fertilizers and residual levels of nitrogen in soil in the field signal the microbe to end the biochemical pathway for nitrogen fixation.
[0109] [0109] Changes to the transcriptional and post-translational levels of components of the nitrogen fixing regulation network may be beneficial for the development of a microbe capable of fixing and transferring nitrogen to corn in the presence of fertilizer. To this end, the Host-Microbe Evolution (HoME) technology is described here for the precise evolution of the regulation and induction of new phenotypes. Also described here are unique libraries owned exclusively by nitrogen-fixing endophytes isolated from corn, paired with extensive omic data involving the interaction of microbes and host plant under different environmental conditions such as stress and excess nitrogen. In some modalities, this technology allows the precise evolution of the genetic regulation network of endophytes to produce microbes that actively fix nitrogen even in the presence of fertilizer in the field. Assessments of the technical potential of developing microbes that colonize root tissues of corn and produce nitrogen for fertilized plants are also described, and assessments of endophyte compatibility with standard formulation practices and various soils to determine the feasibility of integrating microbes into modern strategies for nitrogen management.
[0110] [0110] For the element nitrogen (N) to be used in chemical synthesis, life forms combine nitrogen gas (N2) available in the atmosphere with hydrogen in a process known as nitrogen fixation. Because of the highly energetic nature of biological nitrogen fixation, diazotrophs (Bacteria and Archaea that fix atmospheric nitrogen gas) have developed a sophisticated and rigid regulation of the cluster of nif genes in response to environmental oxygen and available nitrogen. The Nif genes encode enzymes involved in nitrogen fixation (such as the nitrogenase complex) and proteins that regulate nitrogen fixation. Shamseldin (2013. Global J. Biotechnol. Biochem. 8 (4): 84-94) provides detailed descriptions of the nif genes and their products, which is incorporated by reference. The present invention describes methods of producing a plant with an improved attribute, which comprise isolating bacteria from a first plant, introducing a genetic variation into a gene of the isolated bacteria to increase nitrogen fixation, exposing a second plant to variant bacteria, isolate bacteria from the second plant having an improved attribute compared to the first plant and repeat the steps with bacteria isolated from the second plant.
[0111] [0111] In Proteobacteria, the regulation of nitrogen fixation centers around the σ54-dependent enhancer-binding protein (NifA), the positive transcriptional regulator of the nif cluster. The intracellular levels of active NifA are controlled by two essential factors: transcription of the nifLA operon and inhibition of NifA activity by protein-protein interaction with NifL. These two processes respond to intracellular levels of glutamine through the signaling cascade of the PII protein. This cascade is mediated by GlnD, which directly perceives the level of glutamine and catalyzes the uridylation or desuridylation of the two PII regulatory proteins - GlnB and GlnK - in response to the absence or presence, respectively, of bound glutamine. Under conditions with excess nitrogen,
[0112] [0112] NifA is transcribed from the nifLA operon, whose promoter is activated by phosphorylated NtrC, another σ54-dependent regulator. The phosphorylation state of NtrC is mediated by histidine kinase NtrB, which interacts with desuridylated GlnB, but not with uridylated GlnB. Under conditions of excess nitrogen, a high intracellular level of glutamine leads to GlurB deuridation, which then interacts with NtrB to deactivate its phosphorylation activity and activate its phosphatase activity, resulting in NtrC dephosphorylation and deactivation of the nifLA promoter. However, under nitrogen-limited conditions, a low level of intracellular glutamine results in GlnB uridylation, which inhibits its interaction with NtrB and allows NtrC phosphorylation and transcription of the nifLA operon. In this way, nifLA expression is tightly controlled in response to environmental nitrogen through the signaling cascade of the PII protein. nifA, ntrB, ntrC and glnB, are all genes that can mutate in the methods described here. These processes can also respond to intracellular or extracellular levels of ammonia, urea or nitrates.
[0113] [0113] NifA activity is also regulated after translation in response to environmental nitrogen, most typically through NifL-mediated inhibition of NifA activity. In general, the interaction of NifL and NifA is influenced by the signaling cascade of the PII protein through GlnK, although the nature of the interactions between GlnK and NifL / NifA varies significantly between diazotrophs. 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 within the cell. Under conditions with excess nitrogen, desuridylated GlnK interacts with the AmtB ammonium transporter, which has the function of blocking ammonium absorption by AmtB and sequestering GlnK to the membrane, allowing the inhibition of NifA by NifL. On the other hand, in Azotobacter vinelandii, the interaction with desuridylated GlnK is necessary for NifL / NifA interaction and NifA inhibition, while GlnK uridylation inhibits its interaction with NifL. In diazotrophs lacking the nifL gene, there is evidence that NifA activity is directly inhibited by the interaction with the desuridylated forms of GlnK and GlnB under conditions with excess nitrogen. In some bacteria, the Nif cluster can be regulated by glnR and, even in some cases, it can comprise negative regulation. Regardless of the mechanism, post-translational inhibition of NifA is an important regulator of the nif cluster in most known diazotrophs. In addition, nifL, amtB, glnK and glnR are genes that can mutate in the methods described here.
[0114] [0114] In addition to regulating the transcription of the cluster of nif genes, many diazotrophs have developed a mechanism for direct post-translational modification and inhibition of the nitrogenase enzyme itself, known as nitrogenase shutdown. This is mediated by ADP-ribosylation of the Fe protein (NifH) under conditions with excess nitrogen, which disrupts its interaction with the MoFe protein complex (NifDK) and abolishes the nitrogenase activity. DraT catalyzes ADP-ribosylation of the Fe protein and nitrogenase shutdown, while DraG catalyzes the removal of ADP-ribose and reactivation of nitrogenase. As with nifLA transcription and NifA inhibition, nitrogenase shutdown is also regulated via the PII protein signaling cascade.
[0115] [0115] Although some endophytes are able to fix nitrogen in vitro, genetics are often silenced in the field by high levels of exogenous chemical fertilizers. It is possible to dissociate the detection of exogenous nitrogen from the expression of the nitrogenase enzyme to facilitate field-based nitrogen fixation. Improving the global activity of nitrogenase over time also has the function of increasing the production of nitrogen for use by the culture. Specific targets for genetic variation to facilitate field-based nitrogen fixation using the methods described here 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.
[0116] [0116] An additional target for genetic variation to facilitate field-based nitrogen fixation using the methods described here is the NifA protein. The NifA protein is typically the activator for expression of nitrogen fixing genes. Increasing NifA production (either constitutively or during a condition with a high level of ammonia) bypasses the native ammonia detection pathway. In addition, reducing the production of NifL proteins, a known inhibitor of NifA, also leads to a higher level of freely active NifA. Furthermore, increasing the level of transcription of the nifAL operon (either constitutively or during a condition with a high level of ammonia) also leads to a higher overall level of NifA proteins. Raising the level of nifAL expression is achieved by altering the promoter itself or reducing the expression of NtrB (part of the signaling cascade of ntrB and ntrC that originally would result in the shutdown of the nifAL operon during a high nitrogen condition). The high level of NifA achieved by these or any other methods described here increases the nitrogen fixing activity of endophytes.
[0117] [0117] Another target for genetic variation to facilitate field-based nitrogen fixation using the methods described here is the GlnD / GlnB / GlnK PII signaling cascade. The intracellular level of glutamine is detected through the GlnD / GlnB / GlnK PII signaling cascade. Active GlnD site mutations that abolish GlnD's uridyl removing activity disrupt the nitrogen detection cascade. In addition, reducing the GlnB concentration acts as a short circuit in the glutamine detection cascade. These mutations “trick” cells into detecting a state with limited nitrogen, thereby increasing the activity of the nitrogen fixation level. These processes can also respond to intracellular or extracellular levels of ammonia, urea or nitrates.
[0118] [0118] The amtB protein is also a target of genetic variation to facilitate field-based nitrogen fixation using the methods described here. The ammonia absorption of the environment can be reduced by decreasing the expression level of the amtB protein. Without intracellular ammonia, the endophyte is unable to detect the high level of ammonia, preventing negative regulation of nitrogen fixing genes. Any ammonia that can penetrate the intracellular compartment is converted to glutamine. The intracellular level of glutamine is the main currency of nitrogen detection. Lowering the intracellular level of glutamine prevents cells from detecting high levels of ammonium in the environment. This effect can be achieved by increasing the level of expression of glutaminase, an enzyme that converts glutamine to glutamate. In addition, intracellular glutamine can also be reduced by lowering glutamine synthase (an enzyme that converts ammonia to glutamine). In diazotrophs, the fixed ammonia is quickly assimilated into glutamine and glutamate to be used for cellular processes. Disturbances to the assimilation of ammonia can allow the diversion of fixed nitrogen to be exported from the cell as ammonia. Fixed ammonia is predominantly assimilated into glutamine by glutamine synthetase (GS), encoded by glnA, and subsequently in glutamine by glutamine oxoglutarate aminotransferase (GOGAT). In some examples, glnS encodes a glutamine synthetase. GS is post-translationally regulated by GS adenyl transferase (GlnE), a bifunctional enzyme encoded by glnE that catalyzes GS adenylation and deadenylation through the activity of its adenyl transferase (AT) and adenyl (AR) domains, respectively . Under nitrogen-limited conditions, glnA is expressed, and the AR domain of GlnE deactivates GS, allowing it to be active. Under conditions of excess nitrogen, the expression of glnA is turned off, and the AT domain of GlnE is activated allosterically by glutamine, causing adenylation and deactivation of GS.
[0119] [0119] Furthermore, the draT gene can also be a target of genetic variation to facilitate field-based nitrogen fixation using the methods described here. Once nitrogen-fixing enzymes are produced by the cell, shutdown of nitrogenase represents another level at which the cell negatively regulates fixation activity under conditions of high nitrogen level. This shutdown could be removed by lowering the DraT expression level.
[0120] [0120] Methods for transmitting new microbial phenotypes can be performed at the transcriptional, translational and post-translational level. The transcription level includes changes in the promoter (such as changing affinity for the sigma factor or transcription factor binding sites, including deletion of all or part of the promoter) or changing terminators and attenuators of transcription. The translational one includes changes in the ribosome binding sites and changes in mRNA degradation signals. The post-translational level includes mutation at the active site of an enzyme and changes in protein-protein interactions. These changes can be achieved in multiple ways. The reduction in the level of expression (or complete abolition) can be achieved by exchanging the native ribosome binding site (RBS) or promoter for another with less strength / efficiency. Departure sites with ATG can be exchanged for a departure codon GTG, TTG or CTG, resulting in a reduction in the translation activity of the coding region. The complete abolition of expression can be carried out by knocking out (deletion) the coding region of a gene. Alteration in the open reading phase (ORF) (frameshifting) will probably result in a premature stop codon along the ORF, thus creating a non-functional truncated product. Inserting stop codons in frame (without changing the matrix) will also create a non-functional truncated product. The addition of a degradation tag at the N or C terminal can also be performed to reduce the effective concentration of a particular gene.
[0121] [0121] On the other hand, the level of expression of the genes described here can be achieved using a stronger promoter. In order to ensure high promoter activity during a high nitrogen condition (or any other condition), a transcription profile of the entire genome in a high nitrogen condition could be obtained and active promoters with a desired level of transcription can be chosen from that data set to replace the weak promoter. Weak start codons can be swapped out with an ATG start codon for better efficiency at the start of translation. Weak ribosome binding sites (RBS) can also be disabled with a different RBS of higher efficiency at the beginning of the translation. In addition, site-specific mutagenesis can also be performed to alter the activity of an enzyme.
[0122] [0122] In addition to the regulation of nitrogenase genes and nitrogen assimilation, the levels of excreted nitrogen can also be affected by the availability of cofactors involved in fixation and / or assimilation. For example, sulfur is a component of some nitrogenases. Sulfur generally needs to be transferred across cell membranes. In L. japonicus, the sulfate transporter LjSST1 is essential for nitrogen fixation; knockout mutants (disabled) are unable to develop functional nodes. In some modalities, nitrogenase activity can be increased by positively regulating transporters that increase the availability of cofactors such as sulfur. For example, the expression or activity of the sulfur transporter, cysZ, can be increased.
[0123] [0123] Increasing the level of nitrogen fixation that occurs in a plant can lead to a reduction in the amount of chemical fertilizer needed to produce the crop and reduce greenhouse gas emissions (eg nitrous oxide). Generation of bacterial populations Isolation of bacteria
[0124] [0124] Microbes useful in the methods and compositions disclosed herein can be obtained by extracting microbes from surfaces or tissues of native plants. Microbes can be obtained by grinding seeds to isolate microbes. Microbes can be obtained by planting seeds in various soil samples and recovering tissue microbes. In addition, microbes can be obtained by inoculating plants with exogenous microbes and determining which microbes appear in plant tissues. Non-limiting examples of plant tissues may include a seed, seedling, leaf, cut, plant, bulb or tuber.
[0125] [0125] One method of obtaining microbes can be by isolating bacteria from soils. Bacteria can be collected from various types of soil. In some examples, the soil can be characterized by attributes such as high or low fertility, moisture levels, mineral levels and various cultivation practices. For example, the soil may be involved in crop rotation when different crops are planted on 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. The bacteria can be isolated from growing plants in the selected soils. Young plants can be harvested after 2-6 weeks of growth. For example, at least 400 isolates can be collected in a harvest cycle. The types of soil and plant reveal the phenotype of the plant as well as the conditions, which allow enrichment downstream of certain phenotypes.
[0126] [0126] Microbes can be isolated from plant tissues to assess microbial attributes. The parameters for processing tissue samples can be varied to isolate different types of associative microbes, such as rhizophrenic bacteria, epiphytes or endophytes. The isolates can be grown in a nitrogen-free medium to enrich bacteria that perform nitrogen fixation. Alternatively, microbes can be obtained from worldwide strain banks.
[0127] [0127] In-plant analytics are performed to assess microbial attributes. In some embodiments, plant tissue can be processed for screening by high-throughput DNA and RNA processing. In addition, non-invasive measurements can be used to assess plant characteristics, such as colonization. Measurements on wild microbes can be obtained from plant to plant. Measurements on wild microbes can also be obtained in the field using medium yield methods. Measurements can be carried out successively over time. Plant model system can be used, including, among others, Setaria.
[0128] [0128] Microbes in a plant system can be screened by determining the transcription profile of a plant system. Examples of screening through the transcription profile are methods that use quantitative polymerase chain reaction (qPCR), molecular barcodes for transcript detection, Next Generation Sequence and microbial tagging with fluorescent markers. Impact factors can be measured to assess colonization in the greenhouse including, among others, microbiome, abiotic factors, soil conditions, oxygen, humidity, temperature, inoculum conditions and root location. Nitrogen fixation can be assessed in bacteria by measuring 15N gas / fertilizer (dilution) with IRMS or NanoSIMS as described here. NanoSIMS is high resolution secondary ion mass spectrometry. The NanoSIMS technique is a way to investigate the chemical activity of biological samples. The reduction catalysis of oxidation reactions that direct the metabolism of microorganisms can be investigated at the cellular, subcellular, molecular and elementary levels. NanoSIMS can offer a special high resolution greater than 0.1 µm. NanoSIMS can detect the use of isotopic tracers such as 13C, 15N and 18O. Therefore, NanoSIMS can be used for nitrogen with chemical activity in the cell.
[0129] [0129] Automated greenhouses can be used for plant analytics. The metric of plants in response to microbial exposure includes, among others, biomass, chloroplasty analysis, CCD chamber, volumetric measurements by tomography.
[0130] [0130] One way to enrich a population of microbes is according to the genotype. For example, a polymerase chain reaction (PCR) assay with a targeted primer or specific primer. Primers designed for the nifH gene can be used to identify diazotrophs because diazotrophs express the nifH gene in the nitrogen fixation process. A microbial population can also be enriched through culture-independent single cell approaches and chemotaxis-guided isolation approaches. Alternatively, the isolation of microbes can be directed by culturing the microbes in selection media. Premeditated approaches to enrich microbial populations for desired attributes can be guided by bioinformatics data and are described below. Domestication of microbes
[0131] [0131] Microbes isolated from nature can undergo a domestication process in which the microbes are converted into a form that is genetically traceable and identifiable. One way to domesticate a microbe is to modify it with resistance to antibiotics. The antibiotic resistance modification process can begin by determining antibiotic sensitivity in the wild type microbial strain. If bacteria are sensitive to the antibiotic, then the antibiotic can be a good candidate for antibiotic resistance modification. Subsequently, an antibiotic resistance gene or a suicide negative selection vector (counterselectable) can be incorporated into the genome of a microbe using recombination methods. A suicide negative selection vector may consist of a deletion of the gene of interest, a selectable marker and the sacB negative selection marker. Negative selection can be used to exchange native microbial DNA sequences for antibiotic resistance genes. A medium yield method can be used to evaluate multiple microbes simultaneously, allowing parallel domestication. Alternative methods of domestication include using homed nucleases (from English, homing) to prevent sequences of the suicide vector from escaping from the loop or obtaining sequences that intervene with the vector.
[0132] [0132] DNA vectors can be introduced into bacteria using several methods, including electroporation and chemical transformations. A standard vector library can be used for the transformations. An example of a gene editing method is CRISPR preceded by tests with Cas9 to ensure Cas9 activity in microbes.
[0133] [0133] The remodeling process may include transiently transfecting certain plasmids in microbes. These plasmids can then be cured of microbes to remove them. Various methods involving chemical and physical agents have been developed to eliminate plasmids. Protocols for curing plasmids often consist of exposing a culture to subinhibitory concentrations of some chemical agents, e.g. eg, acridine orange, acriflavine and sodium dodecyl sulfate, or at a temperature above the optimum followed by the selection of cured derivatives.
[0134] [0134] In cases where the plasmid is stable or the loss of property difficult to determine, the bacteria can be treated with curing agents. These include chemical and physical agents, some of which can mutate DNA, specifically interfere with its replication, or affect particular structural components or enzymes of the bacterial cell. Protocols for curing plasmids often consist of exposing a culture to subinhibitory concentrations of some chemical agents, e.g. eg, acridine orange, acriflavin and sodium dodecyl sulfate, or at an optimum temperature followed by selection of cured derivatives. DNA intercalating agents, such as acridine orange and ethidium bromide, are the most commonly used because they have been shown to be effective against plasmids in a wide variety of genera. Although all of these agents have been used to enhance the recovery of plasmid less derived from various bacteria, they are individually effective only against some plasmids and their likely response is unpredictable. The efficiency of the cure can also vary widely depending on the plasmid of the particular bacterial host that carries it. In some cases, a plasmid difficult to cure can be removed by introducing an additional plasmid, easier to cure or a system capable of reaching and degrading the plasmid difficult to cure. For example, a plasmid difficult to cure can be cured by introducing a plasmid encoding a CRIPSR system directed against the plasmid difficult to cure. Once a bacterial strain has been cured, samples can be sequenced to ensure complete removal of the plasmid sequence. Non-GMO modification of microbes
[0135] [0135] A microbial population with favorable attributes can be obtained through targeted evolution. Targeted evolution is an approach in which the natural selection process is imitated for the development of proteins or nucleic acids towards a user-defined goal. An example of direct evolution is when random mutations are introduced into a microbial population, the microbes with the most favorable attributes are selected and the growth of the selected microbes is continued. The most favorable attributes in growth-promoting rhizobacteria (PGPRs) can be nitrogen fixation. The method of directed evolution can be iterative and adaptive based on the selection process after each iteration.
[0136] [0136] Plant growth promoting rhizobacteria (PGPRs)
[0137] [0137] Intragenic genes can be transferred to the host microbe. In addition, reporter systems can also be transferred to the microbe. Reporter systems characterize promoters, determine the success of the transformation, track mutants and act as negative screening tools.
[0138] [0138] The microbes that carry the mutation can be cultured through serial passages. A bacterial colony contains a single variant of the microbe. Microbial colonies are tracked with the help of an automatic colony collector and liquid handler. Mutants with duplication of genes and a higher number of copies express a genotype with more desired attributes. Selection of plant growth promoting microbes based on nitrogen fixation
[0139] [0139] Microbial colonies can be screened using various assays to assess nitrogen fixation. One way to measure nitrogen fixation is through a simple fermentation test, which measures nitrogen excretion. An alternative method is the acetylene reduction test (ARA) with in-line sampling over time. ARA can be performed on high-performance microtube array plates. ARA can be performed with live plants and plant tissues. The formulation of the medium and the concentration of oxygen in the medium can be varied in ARA assays. Another method for tracking microbial variants is using biosensors. The use of NanoSIMS and Raman microspectroscopy can be used to investigate the activity of microbes. In some cases, the bacteria can also be grown and expanded with fermentation methods in bioreactors. Bioreactors are designed to improve the robustness of bacterial growth and to decrease the sensitivity of bacteria to oxygen. Microfermenters based on medium to high PT plates are used to assess oxygen sensitivity, nutritional needs, nitrogen fixation and nitrogen excretion. Bacteria can also be co-cultivated with competitive or beneficial microbes to elucidate cryptic pathways. Flow cytometry can be used to track bacteria that produce high levels of nitrogen using chemical, colorimetric or fluorescent indicators. Bacteria can be grown in the presence or absence of a nitrogen source. For example, bacteria can be grown with glutamine, ammonia, urea or nitrates.
[0140] [0140] Microbial genetic improvement is a method to systematically identify and improve the function of species within the culture microbiome. The method comprises three stages: 1) selection of candidate species by mapping plant-microbe interactions and predicting regulation networks linked to a particular phenotype, 2) pragmatic and predictable improvement of microbial phenotypes through interspecies regulation networks and clusters of genes and 3) screening and selection of new microbial genotypes that produce the desired phenotypes for cultures. To systematically assess the improvement of strains, a model is created that links the colonization dynamics of the microbial community to genetic activity by key species. The model is used to predict the improvement of genetic targets and improves the frequency of selected improvements in attributes encoded in the agronomically relevant microbiome. See, Figure 2A for a graphical representation of a process modality. Specifically, Figure 2A represents a schematic drawing of microbial genetic improvement, according to modalities. As illustrated in Figure 2A, the rational improvement of the crop microbiome can be used to increase soil biodiversity, adjust the impact of key species and / or change the timing and expression of important metabolic pathways. To this end, the inventors have developed a microbial genetic improvement pipeline to identify and improve the role of strains within the crop microbiome. The method comprises three stages: 1) selection of candidate species by mapping plant-microbe interactions and predicting regulation networks linked to a particular phenotype, 2) pragmatic and predictable improvement of microbial phenotypes through the intragenomic crossing of regulation networks and clusters of genes and 3) screening and selection of new microbial genotypes that produce desired phenotypes for cultures. To systematically assess strain improvement, the inventors employ a model that links the colonization dynamics of the microbial community to genetic activity by key species. This process represents a methodology for genetic improvement and selection of improvements in attributes encoded in the agronomically relevant microbiome.
[0141] [0141] The production of bacteria to improve plant attributes (eg nitrogen fixation) can be achieved through serial passage. The production of this bacterium can be performed by selecting plants, which have a specific improved attribute that is influenced by microbial flora, in addition to identifying bacteria and / or compositions that are capable of transmitting one or more improved attributes to one or more plants. A method of producing bacteria to improve a plant attribute includes the steps of: (a) isolating bacteria from the tissue or soil of a first plant; (b) introducing a genetic variation in one or more of the bacteria to produce one or more variant bacteria; (c) exposing a plurality of plants to variant bacteria; (d) isolating bacteria from the tissue or soil of one of the plurality of plants, in which the plant from which the bacteria are isolated has an improved attribute compared to other plants in the plurality of plants; and (e) repeating steps (b) to (d) with bacteria isolated from the plant with an improved attribute (step (d)). Steps (b) to (d) can be repeated several times (eg, once, two, three, four, five, ten or more times) until the improved attribute in a plant reaches a desired level. In addition, 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.
[0142] [0142] In addition to obtaining a plant with an improved attribute, a bacterial population is obtained that comprises bacteria including one or more genetic variations introduced in one or more genes (p.
[0143] [0143] The genetic 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, nifQ, bcsII, bcsIII, yjbE, fhaB, pehA, glgA, otsB, treZ and cysZ. The genetic variation can be a variation in a gene that encodes a protein with functionality selected from the group consisting of: glutamine synthetase, glutaminase, glutamine synthetase adenyltransferase, transcriptional activator, anti-transcriptional activator, flavodoxin oxidoreductase, flavodoxin, NAD + - aDP-D-ribosyltransferase dinitrogen-reductase, exopolysaccharide production, filamentous hemagglutinin, glycogen synthase, trehalose synthesis or a sulfate transporter. The genetic variation can be a mutation that results in one or more of: greater expression or activity of NifA glutaminase, bcsII, bcsIII, yjbE, fhaB, pehA, otsB, treZ or CysZ; lesser expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB, or glgA; decreased GlenE adenyl removal activity; or decreased urnyl removal activity from GlnD. Introducing a genetic variation may comprise the insertion and / or deletion of one or more nucleotides at a target site, such as 1, 2, 3, 4, 5, 10, 25, 50, 100, 250, 500 or more nucleotides. The genetic variation introduced in one or more bacteria of the methods disclosed herein may be a disabling mutation (eg, deletion of a promoter, insertion or deletion to produce a premature stop codon, deletion of an entire gene), or it may be the elimination or abolition of the activity of a protein domain (eg, point mutation affecting an active site or deletion of a part of a gene that encodes the relevant part of the protein product), or it can alter or abolish a regulatory sequence of a target gene. One or more regulatory sequences can also be inserted, including heterologous regulatory sequences and regulatory sequences found in a genome of a bacterial species or genus corresponding to the bacteria into which the genetic variation is introduced.
[0144] [0144] In general, the term "genetic variation" refers to any change introduced in a polynucleotide sequence in relation to a reference polynucleotide, such as a reference genome or part of it, or a reference gene or part of it.
[0145] [0145] The genetic variations introduced in microbes can be classified as transgenic, cisgenic, intragenomic, intragenetic, intergeneric, synthetic, evolved, rearranged or SNPs.
[0146] [0146] Genetic variation can be introduced in numerous metabolic pathways within microbes to induce improvements in the attributes described above. Representative pathways include sulfur absorption pathways, glycogen biosynthesis, glutamine regulation pathway, molybdenum absorption pathway, nitrogen fixation pathway, ammonia assimilation, ammonia excretion or secretion, nNitrogen absorption - nio, glutamine biosynthesis, annamox, phosphate solubilization, organic acid transport, organic acid production, agglutinin production, reactive oxygen radical scavenger genes, indole acetic acid biosynthesis, trehalose biosynthesis, pathways or plant cell wall degradation enzymes, genes for root adhesion, exopolysaccharide secretion, glutamate synthase pathway, iron absorption pathways, siderophores pathway, chitinase pathway, ACC deaminase, glutathione biosynthesis, phosphorus signaling genes, the hiona biosynthesis pathway, phosphorus signaling genes, quorum quenching pathways (communication block), cytochrome pathways, the hemoglobin pathway, the hemoglobi pathway in the bacterial type, small rsmZ RNA, rhizobitoxin biosynthesis, lapA adhesion protein, the AHL quorum sensing pathway (detection of communication signals), phenazine biosynthesis, cyclic lipopeptide biosynthesis and production of antibiotics.
[0147] [0147] CRISPR / Cas9 (Clustered regularly interspaced short palindromic repeats) / CRISPR-associated (Cas) systems can be used to introduce desired mutations. CRISPR / Cas9 provides bacteria and archeas with adaptive immunity against viruses and plasmids using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. The Cas9 protein (or functional equivalent and / or variant thereof, that is, Type Cas9 protein) naturally contains endonuclease DNA activity that depends on the association of the protein with two natural or synthetic DNA molecules called crRNA and tracrRNA (also called guide RNAs) . In some cases, the two molecules are covalently linked and form a single molecule (also called a single guide RNA
[0148] [0148] As a cyclic amplification technique, polymerase chain reaction (PCR) mutagenesis uses mutagenic primers to introduce desired mutations. PCR is performed by cycles of denaturation, annealing and extension. After PCR amplification, selection of mutated DNA and removal of DNA from a parent plasmid can be performed by: 1) replacement of dCTP with hydroxymethylated dCTP during PCR, followed by digestion with restriction enzymes to remove only DNA non-hydroxymethylated parent; 2) simultaneous mutagenesis of an antibiotic resistance gene and the studied gene changing the plasmid to a different antibiotic resistance, the new antibiotic resistance facilitating the selection of the desired mutation later; 3) after a desired mutation has been introduced, digestion of the parental methylated template DNA by the restriction enzyme Dpnl that cleaves only methylated DNA, whereby mutagenized non-methylated strands are recovered; or 4) circularization of the mutated PCR products in an additional ligation reaction to increase the efficiency of mutated DNA transformation. Further description of exemplary methods can be found at, p. US7132265, US6713285, US6673610, US6391548, US5789166, US5780270, US5354670, US5071743 and US20100267147.
[0149] [0149] Oligonucleotide-directed mutagenesis, also called site-directed mutagenesis, typically uses a synthetic DNA primer. This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so that it can hybridize with the DNA in the gene of interest. The mutation can be a single base change (point mutation), multiple base changes, deletion, or insertion, or a combination of these. The single strand primer is then amplified using a DNA polymerase, which copies the rest of the gene. The gene thus copied contains the mutated site and can then be introduced into a host cell as a vector and cloned. Finally, mutants can be selected by DNA sequencing to verify that they contain the desired mutation.
[0150] [0150] Genetic variations can be introduced by error-prone PCR. In this technique, the gene of interest is amplified using a DNA polymerase under conditions with impaired sequence replication fidelity. The result is that the amplification products contain at least one error in the sequence. When a gene is amplified and the product (s) resulting from the reaction contain / contain one or more changes in the sequence when compared to the template molecule, the resulting products are mutagenized when compared to the template. Another way to introduce random mutations is to expose the cells to a chemical mutagen, such as nitrosoguanidine or ethyl methanesulfonate (Nestmann, Mutat Res 1975 June; 28 (3): 323-30), and the vector containing the gene is then isolated host.
[0151] [0151] Saturation mutagenesis is another form of random mutagenesis, in which one tries to generate all or practically all possible mutations in a specific site, or a narrow region of a gene. In general, saturation mutagenesis encompasses the mutagenesis of a complete set of mutagenic cassettes (where each cassette contains, for example, 1-500 bases in length) in a polynucleotide sequence defined to undergo mutagenesis (where the sequence mutagenesis contains, for example, 15 to 100,000 bases in length). Therefore, a group of mutations (eg, ranging from 1 to 100 mutations) is introduced in each cassette to undergo mutagenesis. A cluster of mutations to be introduced in a cassette can be different or equal to a second cluster of mutations to be introduced in a second cassette during the application of a saturation mutagenesis cycle. Such groupings are exemplified by deletions, additions, groupings of codons in particular and groupings of nucleotide cassettes in particular.
[0152] [0152] Mutagenesis by shuffling fragments, also called DNA shuffling, is a way to quickly propagate beneficial mutations. In an example of a scrambling process, DNAse is used to fragment a set of original genes into pieces of, p. , about 50-100 bp in length. This is followed by a polymerase chain reaction (PCR) without primers - DNA fragments with sufficient homologous sequences overlapping will ring with each other and are then extended by DNA polymerase. Several cycles of this extension can occur by PCR, after some of the DNA molecules reach the size of the original genes. These genes can then be amplified with another PCR, this time with the addition of primers that are designed to complement the ends of the strands. Primers may have additional sequences at their 5 'ends, such as sequences for recognition enzyme sites for ligation into a cloning vector. Other examples of shuffling techniques are provided in US20050266541.
[0153] [0153] Mutagenesis by homologous recombination involves the recombination between an exogenous DNA fragment and the target polynucleotide sequence. After a double strand break occurs, sections of DNA around the 5 'ends of the break are cut out in a process called resection. In the following strip invasion step, a 3 'protruding end of the broken DNA molecule then "invades" a similar or identical DNA molecule that is not broken. The method can be used to delete a gene, remove exons, add a gene and introduce point mutations. Mutagenesis by homologous recombination can be permanent or conditioned. Typically, a recombination mold is also provided. A recombination template can be a component of another vector, contained in a separate vector or supplied as a separate polynucleotide. In some embodiments, a recombination template is created to serve as a template for homologous recombination, such as within or near a nicked target sequence (cleaved) or cleaved by a site-specific nuclease. A template polynucleotide can be of any suitable length, such as approximately or more than 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000 or more nucleotides in length. In some embodiments, the template polynucleotide is complementary to a part of a polynucleotide that comprises the target sequence. When optimally aligned, one or more nucleotides of a target sequence may overlap with a template polynucleotide (eg, approximately or more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 , 50, 60, 70, 80, 90, 100 or more nucleotides). In some embodiments, when a template sequence and a polynucleotide comprising a target sequence are optimally aligned, the nucleotide closest to the template polynucleotide is within approximately 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10,000 or more nucleotides of the target sequence. Non-limiting examples of site-directed nucleases useful in homologous recombination methods include zinc finger nucleases, CRISPR nucleases, TALE nucleases and mega-nuclease. For a more detailed description of the use of such nucleases, see, p. US8795965 and US20140301990.
[0154] [0154] Mutagens that create primarily punctual mutations and short deletions, insertions, transversions and / or transitions, including chemical mutagens or radiation, can be used to create genetic variations. Mutagens include, but are not limited to, ethyl methanesulfonate, methylmethane sulfonate, N-ethyl-N-nitrosurea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melamine, mustard , vincristine, dimethylnitrosamine, N-methyl-N'-nitro-Nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12-dimethyl-benz (a) anthracene, ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxialtanes and diepoxialtanes and diepoxialtanes and diepoxoctanides , 2-methoxy-6-chloro-9 [3- (ethyl-2-chloro-ethyl) aminopropylamino] acridine dihydrochloride and formaldehyde.
[0155] [0155] The introduction of a genetic variation can be an incomplete process, such that some bacteria in a population of treated bacteria carry a desired mutation while others are not. In some cases, it is convenient to apply a selection pressure in order to enrich bacteria that carry a desired genetic variation. Traditionally, the selection of successful genetic variants has involved selection for or against some functionality transmitted or abolished by genetic variation, such as in the case of inserting an antibiotic resistance gene or abolishing a metabolic activity capable of converting a non-lethal compound into a lethal metabolite. It is also possible to apply a selection pressure based on the polynucleotide sequence itself, such that only a desired genetic variation needs to be introduced (eg, without also requiring a selectable marker). In this case, the selection pressure may comprise cleaving genomes devoid of the genetic variation introduced in a target site, in such a way that the selection is effectively directed against the reference sequence in which the genetic variation is sought to be introduced. Typically, cleavage occurs at up to 100 nucleotides at the target site (e.g., at up to 75, 50, 25, 10 or less nucleotides at the target site, including cleavage at or within the target site). Cleavage can be driven by a site-specific nuclease selected from the group consisting of zinc finger nuclease, CRISPR nuclease, TALE (TALEN) nuclease or meganuclease. Such a process is similar to processes that enhance homologous recombination at a target site, except that no template for homologous recombination is provided. As a result, bacteria deprived of the desired genetic variation are more likely to suffer cleavage which, left unrepaired, results in cell death. Bacteria that survive selection can then be isolated for use in plant exposure to assess the attribution of an improved attribute.
[0156] [0156] A CRISPR nuclease can be used as the site-specific nuclease to direct cleavage to a target site. It is possible to obtain an improved selection of mutated microbes using Cas9 to eliminate cells without mutation. The plants are then inoculated with the mutated microbes to reconfirm symbiosis and create evolutionary pressure and select efficient symbionts. The microbes can then be re-isolated from plant tissues. CRISPR nuclease systems used for selection against non-variants may employ elements similar to those described above in relation to the introduction of genetic variation, except that no template for homologous recombination is provided. Cleavage directed to the target site thus reinforces the death of affected cells.
[0157] [0157] Other options are available to specifically induce cleavage at a target site, such as zinc finger nucleases, systems with TALE nuclease (TALEN) nuclease and meganuclease. Zinc finger nucleases (ZFNs) are artificial DNA endonucleases generated by fusing a zinc finger binding domain to DNA with a DNA cleavage domain. ZFNs can be designed to target desired DNA sequences and this allows zinc finger nucleases to cleave unique target sequences. When introduced into a cell, ZFNs can be used to edit a target DNA in the cell (eg, in the cell's genome) by inducing double strand breaks. Nucleases with transcriptional activator-like effector nucleases, TALENs, are artificial DNA endonucleases generated by the fusion of a TAL effector binding domain (of the transcriptional activator type) to DNA with a DNA cleavage domain . TALENS can be quickly designed to bind to almost any desired DNA sequence and, when introduced into a cell, TALENs can be used to edit a target DNA in the cell (eg, in the cell's genome) by inducing breaks double tapes. Meganucleases (endonuclease homing) are endodesoxyribonucleases characterized by a large recognition site (double stranded DNA sequences with 12 to 40 base pairs). Meganucleases can be used to replace, eliminate or modify sequences in a highly targeted manner. With the modification of its recognition sequence through protein engineering, the target sequence can be changed. Meganucleases can be used to modify all types of genomes, whether bacterial, plant or animal, and are commonly grouped into four families: the LAGLIDADG family (SEQ ID NO: 1), the GIY-YIG family, the His-Cyst family box 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. Genetic variation - Identification methods
[0158] [0158] The microbes of the present invention can be identified by one or more modifications or genetic alterations, which have been introduced in said microbe. One method by which said genetic modification or alteration can be identified is by reference to a SEQ ID NO which contains a part of the microbe's genomic sequence which is sufficient to identify the genetic modification or alteration.
[0159] [0159] In addition, in the case of microbes that have not had a genetic modification or alteration (eg, wild type, WT) introduced in their genomes, the invention can use the 16S nucleic acid sequence to identify the said microbes. A 16S nucleic acid sequence is an example of a "molecular marker" or "genetic marker", which refers to an indicator that is used in methods to visualize differences in nucleic acid sequence characteristics. Examples of other such indicators are restriction fragment length polymorphism markers (RFLP), amplified fragment length polymorphism markers (AFLP), single nucleotide polymorphisms (SNPs), insertion mutations, microsatellite markers (SSRs) , characterized sequence amplified regions (SCARs), cleaved amplified polymorphic sequence markers (CAPS) isozyme markers or combinations of the markers described here that define a specific and chromosomal genetic location. The markers also include polynucleotide sequences encoding 16S or 18S rRNA, and transcribed internal spacer (ITS) sequences, which are sequences found between small subunit and large subunit rRNA genes that have proven to be especially useful in elucidating relationships or distinctions when compared against each other. In addition, the invention uses unique sequences found in genes of interest (e.g., nif H, D, K, L, A, glnE, amtB etc.) to identify the microbes disclosed herein.
[0160] [0160] The primary structure of the important 16S rRNA subunit comprises a special combination of conserved, variable and hypervariable regions that evolve at different rates and enable the resolution of very old strains, such as domains, and more modern strains such as genera. The secondary structure of the 16S subunit includes approximately 50 helices that result in base pairing of approximately 67% of the residues. These highly conserved secondary characteristics are of great functional importance and can be used to ensure positional homology in multiple sequence alignments and in phylogenetic analysis. Over the past few decades, the 16S rRNA gene has become the most sequenced taxonomic marker and is the cornerstone for the current systematic classification of Bacteria and Archaea (Yarza at al. 2014. Nature Rev. Micro. 12: 635-45 ). Genetic variation - Detection methods: Primers, probes and assays
[0161] [0161] The present invention teaches primers, probes and assays that are useful for detecting the microbes now taught. In some respects, the invention provides methods of detecting parental WT strains. In other respects, the invention provides methods for detecting modified non-intergeneric microbes derived from the WT strains. In aspects, the present invention provides methods of identifying non-intergenerational genetic changes in a microbe.
[0162] [0162] In some respects, the genomic engineering methods of this invention lead to the creation of unnatural “junction” sequences of nucleotides in non-intergeneric derived microbes. These unnatural nucleotide junctions can be used as a type of diagnosis that is indicative of the presence of a particular genetic alteration in a microbe now taught.
[0163] [0163] The present techniques are able to detect these unnatural nucleotide junctions through the use of specialized quantitative PCR methods, including uniquely designed primers and probes. In some respects, the probes of the invention bind to sequences with unnatural nucleotide junctions. In some ways, traditional PCR is used. In other respects, real-time PCR is used. In some aspects, quantitative PCR (qPCR) is used.
[0164] [0164] Thus, the invention can cover the use of two common methods for the detection of PCR products in real time: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA and (2) Sequence-specific DNA consisting of oligonucleotides that are labeled with a fluorescent reporter that allows detection only after hybridization of the probe to its complementary sequence. In some aspects, only the junction of unnatural nucleotides will be amplified through the primers taught and, consequently, can be detected by means of a non-specific dye or by using a specific hybridization probe. In other respects, the primers of the invention are chosen in such a way that the primers flank either side of a splice sequence, such that if an amplification reaction occurs, then said splice sequence is present.
[0165] [0165] Aspects of the invention involve molecules of the sequence with unnatural nucleotide junction per se, together with other nucleotide molecules which are capable of binding to said sequences with unnatural nucleotide junctions under mild to stringent hybridization conditions. In some respects, nucleotide molecules that are able to bind to said sequences with unnatural nucleotide junctions under light to stringent hybridization conditions are called "nucleotide probes".
[0166] [0166] In some respects, genomic DNA can be extracted from samples and used to quantify the presence of microbes of the invention using qPCR. The primers used in the qPCR reaction can be primers designed by Primer Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) to amplify regions unique to the wild-type genome or regions unique to mutant strains modified non-intergenerational. The qPCR reaction can be performed with the SYBR GreenER qPCR SuperMix Universal kit (Thermo Fisher, P / N 11762100), using only forward and reverse amplification primers; alternatively, the Kapa Sonda Force kit (Kapa Biosystems, P / N KK4301) can be used with amplification primers and a TaqMan probe containing a FAM dye marking on the 5 'end, an internal ZEN quencher (signal suppressor) and a ligand narrow groove (minor groove binder) and fluorescent quencher at the 3 'end (Integrated DNA Technologies).
[0167] [0167] The efficiency of the qPCR reaction can be measured by a standard curve generated from a known amount of gDNA from the target genome. The data can be normalized for copies of the genome per g of fresh mass using tissue mass and extraction volume.
[0168] [0168] The quantitative polymerase chain reaction (qPCR) is a method to quantify, in real time, the amplification of one or more nucleic acid sequences. The real-time quantification of the PCR assay allows to determine the amount of nucleic acids generated by the PCR amplification steps by comparing the amplified nucleic acids of interest and an appropriate control nucleic acid sequence, which can act as a calibration standard.
[0169] [0169] TaqMan probes are often used in qPCR assays that require greater specificity to quantify a target nucleic acid sequence. TaqMan probes comprise an oligonucleotide probe with a fluorophore attached to the 5 'end and a quencher attached to the 3' end of the probe. When the TaqMan probes remain as they are, with the 5 'and 3' ends of the probe in close contact with each other, the quencher prevents transmission of the fluorescent signal by the fluorophore. TaqMan probes are designed to ring within a region of nucleic acid amplified by a specific set of primers. As Taq polymerase extends the primer and synthesizes the nascent ribbon, the 5 'to 3' exonclease activity of Taq polymerase degrades the probe that has ringed the mold. This degradation of the probe releases the fluorophore, thus breaking the close proximity to the quencher and allowing the fluorophore to fluoresce. The fluorescence detected in the qPCR assay is directly proportional to the fluorophore released and the amount of the DNA template present in the reaction.
[0170] [0170] The characteristics of qPCR allow the user to eliminate the laborious post-amplification step of preparing gel electrophoresis, which is generally necessary to observe the amplified products of traditional PCR assays. The benefits of qPCR over conventional PCR are considerable, and include greater speed, ease of use, reproducibility and quantitative capacity. Attribute improvement
[0171] [0171] The methods of the present invention can be employed to introduce or improve one or more of a variety of desirable attributes. The attributes to be improved can be attributes of the bacterium, or the bacteria can be modified to improve an attribute on an associated plant. Examples of attributes that can be introduced or improved include: root biomass, root length, height, length of branches, number of leaves, water use efficiency, global biomass, yield, fruit size, grain size, photosynthesis rate , drought tolerance, heat tolerance, salinity tolerance, resistance to nematode stress, resistance to a fungal pathogen, resistance to a bacterial pathogen, resistance to a viral pathogen, level of a metabolite and expression of the proteome. Desirable attributes, including height, global biomass, root and / or aerial branch biomass, seed germination, seedling survival, photosynthetic efficiency, transpiration rate, number or mass of seeds / fruits, yield of grades or fruits of the plant, leaf chlorophyll content, photosynthetic rate, root length, or any combination thereof, can be used to measure growth, and compared to the growth rate of reference agricultural plants (eg, plants without the attributes (improved) grown under identical conditions.
[0172] [0172] Additional examples of attributes that can be improved include the ability of a bacterium to adhere and colonize a plant. For example, a bacterium can be modified to better adhere to a plant's root, or to produce or secrete a beneficial compound for colonization.
[0173] [0173] A preferred attribute to be introduced or improved is nitrogen fixation, as described here. In some cases, a plant resulting from the methods described here exhibits a difference in attribute that is at least approximately 5% greater, for example, at least approximately 5%, at least approximately 8%, at least approximately 10%, at least approximately 15 %, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 75%, at least approximately 80%, at least approximately 80%, at least approximately 90%, or at least 100%, at least approximately 200%, at least approximately 300%, at least approximately 400% or more than an agricultural plant reference crops grown under the same soil conditions. In additional examples, a plant resulting from the methods described here exhibits a difference in attribute that is at least approximately 5% greater, for example, at least approximately 5%, at least approximately 8%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 75 %, at least approximately 80%, at least approximately 80%, at least approximately 90%, or at least 100%, at least approximately 200%, at least approximately 300%, at least approximately 400% or more than an agricultural plant reference crops grown under similar soil conditions.
[0174] [0174] The attribute to be improved can be evaluated under conditions that include the application of one or more biotic or abiotic stressors. Examples of stressors include abiotic stresses (such as heat stress, salinity stress, drought stress, cold stress and low nutrient stress) and biotic stresses (such as nematode stress, herbivorous insect stress, fungal pathogen stress, stress by bacterial pathogen and stress by viral pathogen).
[0175] [0175] The attribute improved by methods and compositions of the present invention can be nitrogen fixation, including in a plant not previously capable of nitrogen fixation. In some cases, bacteria isolated according to a method described here produce 1% or more (eg, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% , 15%, 20% or more) of a plant's nitrogen, which can represent an increase in nitrogen fixing capacity of at least 2 times (eg, 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) when compared to bacteria isolated from the first plant before any genetic variation was introduced. In some cases, bacteria produce 5% or more of a plant's nitrogen. The desired level of nitrogen fixation can be achieved after repeating the steps of introducing genetic variation, exposure to a plurality of plants and isolation of plant bacteria with an improved attribute one or more times (eg, 1 , 2, 3, 4, 5, 10, 15, 25 or more times). In some cases, enhanced levels of nitrogen fixation are achieved in the presence of fertilizer supplemented with glutamine, ammonia or another chemical source of nitrogen. Methods for assessing the degree of nitrogen fixation are known, examples of which are described here.
[0176] [0176] Microbial genetic improvement is a method to systematically identify and improve the role of species within the culture microbiome. The method comprises three stages: 1) selection of candidate species by mapping plant-microbe interactions and predicting regulation networks linked to a particular phenotype, 2) pragmatic and predictable improvement of microbial phenotypes through intra-species crossing regulatory networks and gene clusters; and 3) screening and selection of new microbial genotypes that produce desired phenotypes for cultures. In order to systematically assess the improvement of strains, a model is created that links the colonization dynamics of the microbial community to genetic activity by major species. The model is used to predict genetic targets for breeding and improves the frequency of selected improvements in attributes encoded in the agronomically relevant microbiome. Measurement of nitrogen delivered in a field of agricultural relevance
[0177] [0177] In the field, the amount of nitrogen delivered can be determined by the colonization function multiplied by the activity. Nitrogen delivered Colonization x activity Time and space
[0178] [0178] The above equation requires (1) the average colonization per unit of plant tissue and (2) the activity either as the amount of nitrogen fixed or the amount of ammonia excreted by each microbial cell. To convert pounds of nitrogen per area, the physiology of corn growth is tracked over time, p. eg, plant size and associated root system throughout the stages of maturity.
[0179] [0179] The pounds of nitrogen delivered to a crop per area-season can be calculated using the following equation: Nitrogen delivered Plant tissue (t) x Colonization (t) x activity (t) dt
[0180] [0180] Vegetable tissue (t) is the fresh mass of tissue from the corn plant over time growing (t). The values for reasonably performing the calculations are described in detail in the publication entitled Roots, Growth and Nutrient Uptake (Mengel. Department of Agronomy, Publication in AGRY-95-08 (Rev. May 95., p. 1-8.).
[0181] [0181] Colonization (t) is the amount of microbes of interest found in plant tissue per gram of fresh mass of plant tissue, at any specific time, t, during the growing season. In case there is only one moment available, the only moment is normalized as the maximum colonization rate over the season, and the colonization rate for the other moments is adjusted accordingly.
[0182] [0182] Activity (t) is the rate at which N is fixed by the microbes of interest per unit of time, at any specific time, t, during the growing season. In the modalities disclosed here, this activity rate is approximated by the in vitro acetylene reduction test (ARA) in ARA medium in the presence of 5 mM glutamine or in the ammonium excretion test in medium ARA medium in the presence of 5 mM ammonium ions.
[0183] [0183] The amount of nitrogen delivered is then calculated by numerically integrating the above function. In cases where the values of the variables described above are measured distinctly at defined moments, the intermediate values between those moments are approximated by linear interpolation. Nitrogen fixation
[0184] [0184] The present invention describes methods to increase nitrogen fixation in a plant, which include exposing the plant to bacteria including one or more genetic variations introduced in one or more genes that regulate nitrogen fixation, in which the bacteria produce 1% or more nitrogen in the plant (eg, 2%, 5%, 10% or more), which can represent a nitrogen fixation capacity of at least 2 times compared to the plant in the absence of bacteria. The bacteria can produce nitrogen in the presence of fertilizer supplemented with glutamine, urea, nitrates or ammonia. The genetic variations can be any genetic variation described herein, including the examples provided above, in any number and any combination. The genetic variation can be introduced in a gene selected from the group consisting of nifA,
[0185] [0185] The amount of nitrogen fixation that occurs in the plants described here can be measured in several ways, for example, by an acetylene reduction (AR) test. An acetylene reduction assay can be performed in vitro or in vivo. The evidence that a given bacterium is supplying nitrogen fixed to a plant may include: 1) the total N of the plant increases significantly upon inoculation, preferably with a concomitant increase in the concentration of N in the plant; 2) the symptoms of nitrogen deficiency are alleviated under N-limited conditions at inoculation (which should include an increase in dry matter); 3) N2 fixation is documented using an N approach (which can be isotope dilution experiments, 15N2 reduction assays or N abundance assays); 4) N fixed is incorporated into a protein or metabolite of the plant; and 5) all of these effects are not seen in uninoculated plants or in plants inoculated with a mutant of the inoculated strain.
[0186] [0186] The regulating cascade of nitrogen fixation of the wild type can be represented as a digital logic circuit where the O2 and NH4 + inputs pass through a NOR gate, whose output enters an AND gate in addition to ATP. In some modalities, the methods disclosed here interrupt the influence of NH4 + in this circuit, at multiple points in the regulatory cascade, so that microbes can produce nitrogen even in fertilized fields. However, the methods disclosed here also envisage changing the impact of ATP or O2 on the circuit, or replacing the circuit with other regulatory cascades in the cell, or altering genetic circuits in addition to nitrogen fixation. Gene clusters can be modified again to generate functional products under the control of a heterologous regulatory system. By eliminating native regulatory elements outside and inside encoding sequences of gene clusters, and replacing them with alternative regulatory systems, the functional products of complex genetic operons and other gene clusters can be controlled and / or moved to heterologous cells , including cells of a species different from the species from which the native genes were derived. Once modified back, synthetic gene clusters can be controlled by genetic circuits or other inducible regulatory systems, thereby controlling the expression of products as desired. Expression cassettes can be created to act as logic gates, pulse generators, oscillators, switches or control devices. The controller expression cassette can be connected to a promoter in such a way that the expression cassette functions as an environmental sensor, such as an oxygen, temperature, touch, osmotic stress, membrane stress or redox sensor.
[0187] [0187] As an example, the nifL, nifA, nifT and nifX genes can be deleted from the nif gene cluster. Synthetic genes can be designed by randomizing the codons of the DNA that encodes each sequence of amino acids. Codon selection is performed, specifying the use of the codons as divergent as possible from the use of codons in the native gene. The proposed sequences are examined for any undesired characteristics, such as restriction enzyme recognition sites, transposon recognition sites, repetitive sequences, sigma 54 and sigma 70 promoters, cryptic ribosomal binding sites and rho independent terminators. Synthetic ribosomal binding sites are chosen to match the strength of each corresponding native ribosomal binding site, such as by constructing a fluorescent reporter plasmid in which the 150 bp around a gene start codon (from −60 to + 90) are fused to a fluorescent gene. This chimera can be expressed under the control of the Ptac promoter, and the fluorescence measured by flow cytometry. To generate synthetic ribosomal binding sites, a library of reporter plasmids is generated using 150 bp (−60 to +90) of a synthetic expression cassette. Briefly, a synthetic expression cassette can consist of a random DNA spacer, a degenerate sequence that encodes a RBS library and the coding sequence for each synthetic gene. Multiple clones are screened to identify the synthetic ribosomal binding site that best matches the native ribosomal binding site. Synthetic operons that consist of the same genes as native operons are thus constructed and tested for functional complementation. An additional exemplary description of synthetic operons is provided in US20140329326. Bacterial species
[0188] [0188] Microbes useful in the methods and compositions disclosed herein can be obtained from any source. In some cases, microbes can be bacteria, archaea, protozoa or fungi. The microbes of this invention can be nitrogen-fixing microbes, for example, nitrogen-fixing bacteria, nitrogen-fixing arches, nitrogen-fixing fungi, nitrogen-fixing yeasts or nitrogen-fixing protozoa. The microbes useful in the methods and compositions disclosed herein can be spore-forming microbes, for example, spore-forming bacteria. In some cases, the bacteria useful in the methods and compositions disclosed herein may be Gram positive bacteria or Gram negative bacteria. In some cases, the bacteria may be endospore-forming bacteria of the phylum Firmicute. In some cases, the bacteria can be diazotrophic. In some cases, the bacteria may not be diazotrophic. Klebsiella variicola
[0189] [0189] Klebsiella variicola is a nitrogen-fixing bacteria that lives freely in the soil, and has been isolated from the rhizospheres of banana, rice, sugar cane and corn. Strain 137 was originally isolated from a soil sample collected from the corn root rhizosphere of a field in Missouri in St. Charles County. The same strain was also found in fields in California and Puerto Rico, as confirmed by alignments between the 16S rRNA of strain 137 and the 16S rRNA of natural K. variicola organisms in the soils of California and Puerto Rico. Klebsiella variicola is not known to exhibit plant pest characteristics; although a research group has reported that a strain of K. variicola can cause soft rot1 in bananas in China. No virulence factors were found in the genome of strain 137 of K. variicola. Kosakonia sacchari
[0190] [0190] Kosakonia sacchari is a new species within the new genus Kosakonia, which was included in the genus Enterobacter2. K sacchari is a free-living nitrogen-fixing bacteria, known for its association with sugar cane (Saccharum officinarum L.). K sacchari bacteria are gram-negative, aerobic rods, not spore-forming, and are capable of colonizing and fixing nitrogen in with sugar cane plants, thus promoting plant growth. Strain CI006 was isolated from a soil sample taken from San Joaquin County, California. Kosakonia sacchari is not known for exhibiting characteristics of plant pests.
[0191] [0191] The identities of the two wild-type microbes described above (strains 137 and CI006) have been confirmed by sequence analysis of the 16S rRNA gene, an established method for phylogenetic studies of prokaryotes. Biological deposit information for these two strains is included in this patent application.
[0192] [0192] The methods and compositions of that invention can be used with an arch, such as, for example, Methoutromobacter thermoautotrophicus.
[0193] [0193] In some cases, bacteria that may be useful include, but are not limited to, Agrobacterium radiobacter, Bacillus acidocaldarius, Bacillus acidoterrestris, Bacillus agri, Bacillus aizawai, Bacillus albolactis, Bacillus alcalophilus, Bacillus alvei, Bacillus aminoglucosillususus, Bacillus aminoglucosillusus, Bacillus (also known as Paenibacillus amylolyticus) Bacillus amyloliquefaciens, Bacillus aneurinolyticus, Bacillus atrophaeus, Bacillus azotoformans, Bacillus badius, Bacillus cereus (synonyms: Bacillus endorhythmos, Bacillus medusa), Bacillus chitinosporus, Bacillus circulans, Bacillus cirillus, Bacillus circulans , Bacillus kurstaki, Bacillus lacticola, Bacillus lactimorbus, Bacillus lactis, Bacillus laterosporus (also known as Brevibacillus laterosporus), Bacillus lautus, Bacillus lentimorbus,
[0194] [0194] In some cases, the bacterium may be a species of Clostridium, for example, Clostridium pasteurianum, Clostridium beijerinckii, Clostridium perfringens, Clostridium tetani, Clostridium acetobutylicum.
[0195] [0195] In some cases, the bacteria used with the methods and compositions of the present invention may be cyanobacteria. Examples of cyanobacterial genera include Anabaena (for example, Anagaena sp. PCC7120), Nostoc (for example, Nostoc punctiforme) or Synechocystis (for example, Synechocystis sp. PCC6803).
[0196] [0196] In some cases, the bacteria used with the methods and compositions of the present invention may belong to the phylum Clorobi, for example, Clorobium tepidum.
[0197] [0197] In some cases, microbes used with the methods and compositions of the present invention may comprise a gene homologous to a known NifH gene. Known NifH gene sequences can be found in, for example, the Zehr lab NifH database, (https://wwwzehr.pmc.ucsc.edu/nifH_Database_Public/, April 4, 2014), or the database Buckley lab NifH (http://www.css.milhoell.edu/faculty/buckley/nifh.htm, and in 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, the microbes used with the methods and compositions of the present invention may comprise a sequence that encodes a polypeptide with at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 96% , 98%, 99% or more than 99% sequence identity with a sequence from the Zehr lab NifH database, (https://wwwzehr.pmc.ucsc.edu/nifH_Database_Public/, April 4, 2014).
[0198] [0198] The microbes useful in the methods and compositions discussed here can be obtained by extracting microbes from surfaces or tissues of native plants; grinding seeds to isolate microbes; planting seeds in different soil samples and recovering tissue microbes; or by inoculating plants with exogenous microbes and determining which microbes appear in plant tissues. Non-limiting examples of plant tissues include seed, seedling, leaf, cut, plant, bulb or tuber. In some cases, the bacteria are isolated from a seed. The parameters for sample processing can be varied to isolate different types of associative microbes, such as rhizospheric, epiphytes or endophytes. Bacteria can also be acquired in a repository, such as collections of environmental strains, instead of initially isolating from a first plant. Microbes can be genotyped and phenotyped, by sequencing the genomes of isolated microbes; determine the profile of the composition of communities in planta; characterization of the transcriptome functionality of isolated communities or microbes; or tracking microbial characteristics using selective or phenotypic means (eg, nitrogen fixation phenotypes or phosphate solubilization). The selected strains or candidate populations can be obtained by data on sequence, data on phenotype, data on plants (eg, data on genome, phenotype and / or yield); soil data
[0199] [0199] The bacteria and bacterial production methods described here can be applied to bacteria capable of efficient self-propagation on the leaf surface, root surface or internal plant tissues, without inducing a harmful defense reaction for the plant, or the bacteria are resistant to the plant's defense responses. The bacteria described here can be isolated by culturing an extract of plant tissue or washing the leaf surface in a medium without added nitrogen. However, bacteria may be non-cultivable, that is, not known to be cultivable or difficult to cultivate using standard methods known in the art. The bacteria described here can be an endophyte or an epiphyte or a bacterium that inhabits the plant rhizosphere (rhizospheric bacteria). The bacteria obtained, after repeating the steps of introducing genetic variation, exposure to a plurality of plants and isolation of bacteria from plants with an improved attribute one or more times (eg, 1, 2, 3, 4 , 5, 10, 15, 25 or more), can be endophytic, epiphytic or rhizospheric. Endophytes are organisms that penetrate the interior of plants without causing symptoms of disease or causing the formation of symbiotic structures, and are of agronomic interest because they are able to reinforce plant growth and improve plant nutrition (eg, by fixing nitrogen). The bacteria can be a seed endophyte. Seed endophytes include bacteria associated with or derived from the seed of a grass or plant, such as bacterial endophytes in seed found in ripe, dry, undamaged seeds (eg, without cracking, visible or prematurely germinated fungal infection ). The seed bacterial endophyte for being associated with or derived from the seed surface; alternatively or in addition to, it can be associated with or derived from the inner seed compartment (eg, from a seed with a sterilized surface). In some cases, a seed bacterial endophyte is capable of replication within plant tissue, for example, within the seed. In addition, in some cases, the seed bacterial endophyte is able to survive drying.
[0200] [0200] Bacteria isolated according to methods of the invention, or used in methods or compositions of the invention, may comprise a plurality of different bacterial taxa in combination. For example, bacteria can include Proteobacteria (such as Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azorhizobium, Azotobacter, Duganella, Delftia, Bradorhizobiun, and Bacillus, Paenibacillus, Lactobacillus, Mycoplasma and Acetabacterium) and Actinobacteria (such as Streptomyces, Rhodacoccus, Microbacterium and Curtobacterium). The bacteria used in the methods and compositions of this invention may include bacterial nitrogen-fixing pools of two or more species. In some cases, one or more bacterial species from bacterial pools may be able to fix nitrogen. In some cases, one or more species of the bacterial pools can facilitate or enhance the ability of other bacteria to fix nitrogen. Bacteria that fix nitrogen and bacteria that enhance the ability of other bacteria to fix nitrogen can be the same or different. In some instances, a bacterial strain may be able to fix nitrogen when in combination with a different bacterial strain, or in certain bacterial pools, but it may be unable to fix nitrogen in monoculture. Examples of bacterial genera that can be found in nitrogen-fixing bacterial pools include, among others, Herbaspirillum, Azospirillum, Enterobacter and Bacillus.
[0201] [0201] The bacteria which can be produced by the methods disclosed herein include Azotobacter sp., Bradyrhizobium sp., Klebsiella sp. and Sinorhizobium sp. In some cases, the bacteria may be selected from the group consisting of: Azotobacter vinelandii, Bradyrhizobium japonicum, Klebsiella pneumoniae and Sinorhizobium meliloti. In some cases, the bacteria may be of the genus Enterobacter or Rahnella. In some cases, the bacteria may be of the genus Frankia or Clostridium. Examples of bacteria of the genus Clostridium include, but are not limited to, Clostridium acetobutilicum, Clostridium pasteurianum, Clostridium beijerinckii, Clostridium perfringens and Clostridium tetani. In some cases, the bacteria may be of the genus Paenibacillus, for example, Paenibacillus azotofixans, Paenibacillus borealis, Paenibacillus durus, Paenibacillus macerans, Paenibacillus polymyxa, Paenibacillus alvei, Paenibacillus amylolyticus, paenibacis, paenibacillus, Paenibacillus, Paenibacillus subsp. Larvae, Paenibacillus larvae subsp. Pulvifaciens, Paenibacillus lautus, Paenibacillus macerans, Paenibacillus macquariensis, Paenibacillus macquariensis, Paenibacillus pabuli, Paenibacillus peoriae or Paenibacillus polymyxa.
[0202] [0202] In some examples, the bacteria isolated according to methods of the invention may belong to one or more of the following taxa: Achromobacter, Acidithiobacillus, Acidovorax, Acidovoraz, Acinetobacter, Actinoplanes, Adlercreutzia, Aerococcus, Aeromonas, Afipia, Agromyces, Ancylobacter Arthrobacter, Atopostipes, Azospirillum, Bacillus, Bdellovibrio, Beijerinckia, Bosea, Bradyrhizobium, Brevibacillus, Brevundimonas, Burkholderia, Candidatus Haloredivivus, Caulobacter, Cellulomonas, Cellvibrio, Chryseobobium, Cryobacterium, Citrobacterium, Citrobacter, Citrobacter , Unemployment, devosia,
[0203] [0203] In some cases, bacterial species selected from at least one of the following genera are used: Enterobacter, Klebsiella, Kosakonia and Rahnella. In some cases, a combination of bacterial species of the following genera is used: Enterobacter, Klebsiella, Kosakonia and Rahnella. In some cases, the species used can be one or more of: Enterobacter sacchari, Klebsiella variicola, Kosakonia sacchari and Rahnella aquatilis.
[0204] [0204] In some cases, a Gram positive microbe may have a Molybdenum-Iron nitrogenase system comprising: nifH, nifD, nifK, nifB, nifE, nifN, nifX, hesA, nifV, nifW, nifU, nifS, nifI1 and nifI2. In some cases, a Gram positive microbe may have a vanadium nitrogenase system comprising: vnfDG, vnfK, vnfE, vnfN, vupC, vupB, vupA, vnfV, vnfR1, vnfH, vnfR2, vnfA (transcriptional regulator). In some cases, a Gram positive microbe may have an iron nitrogenase-only system comprising: anfK, anfG, anfD, anfH, anfA (transcriptional regulator). In some cases, a Gram positive microbe may have a nitrogenase system comprising glnB and glnK (nitrogen signaling proteins). Some examples of enzymes involved in nitrogen metabolism in Gram-positive microbes include glnA (glutamine synthetase), gdh (glutamate dehydrogenase), bdh (3-hydroxybutyrate dehydrogenase), glutaminase, gltAB / gltB / gltS (glutamate synthase), asnA / asnB (aspartate-ammonia ligase / asparagine synthase) and ansA / ansZ (asparaginase). Some examples of proteins involved in nitrogen transport in Gram-positive microbes include amtB (ammonium transporter), glnK (ammonium transport regulator), glnPHQ / glnQHMP (ATP-dependent glutamine / glutamate carriers), glnT / alsT / yrbD / yflA (glutamine-like proton carrier) and gltP / gltT / yhcl / nqt (glutamate-like proton carrier).
[0205] [0205] Examples of Gram-positive microbes that may be of particular interest include Paenibacillus polymixa, Paenibacillus riograndensis, Paenibacillus sp., Frankia sp., Heliobacterium sp., Heliobacterium clorum, Heliobacillus sp., Heliophilum sp., Heliorestis sp. Clostridium acetobutylicum, Clostridium sp., Mycobacterium flaum, Mycobacterium sp., Arthrobacter sp., Agromyces sp., Corynebacterium autitrophicum, Corynebacterium sp., Micromonspora sp., Propionibacteria sp., Streptomyces sp. and Microbacterium sp.
[0206] [0206] Some examples of genetic changes that can be made in Gram-positive microbes include: deleting glnR to remove negative regulation of BNF in the presence of environmental nitrogen, inserting different promoters directly upstream of the nif cluster to eliminate regulation by GlnR in response to environmental nitrogen, induce mutation in glnA to reduce the rate of ammonium assimilation via the GS-GOGAT pathway, delete amtB to reduce the absorption of ammonium from the medium, induce mutation in glnA so that it is constitutively in the inhibited state by feedback (FBI-GS) to reduce the assimilation of ammonium via the GS-GOGAT route.
[0207] [0207] In some cases, glnR is the main regulator of N metabolism and fixation in the Paenibacillus species. In some cases, the genome of a Paenibacillus species may not contain a gene to produce glnR. In some cases, the genome of a Paenibacillus species may not contain a gene to produce glnE or glnD. In some cases, the genome of a Paenibacillus species may not contain a gene to produce glnB or glnK. For example, Paenibacillus sp. WLY78 does not contain a gene for glnB, or its counterparts found in the archaea Methanococcus maripaludis, nifI1 and nifI2. In some cases, the genomes of Paenibacillus species can be variable. For example, Paenibacillus polymixa E681 is devoid of glnK and gdh, has several nitrogen-carrying compounds, but only amtB appears to be controlled by GlnR. In another example, Paenibacillus sp. JDR2 has glnK, gdh and most of the central genes for nitrogen metabolism, let alone nitrogen-carrying compounds, but has glnPHQ controlled by GlnR. Paenibacillus riograndensis SBR5 contains a standard glnRA operon, an fdx gene, a main nif operon, a secondary nif operon, and an anf operon (encoding only nitrogenase iron). Supposed glnR / tnrA sites were found upstream of each of these operons. GlnR can regulate all of the above operons, except the anf. GlnR can bind to each of these regulatory sequences as a dimer.
[0208] [0208] Paenibacillus N-fixing strains can be located in two subgroups: Subgroup I, which contains only a minimal cluster of nif genes and subgroup II, which contains a minimal cluster, plus an uncharacterized gene between nifX and hesA and, often, other clusters duplicating some of the nif genes, such as nifH, nifHDK, nifBEN or clusters encoding the vanadium nitrogenase (vnf) or iron nitrogenase (anf) genes only.
[0209] [0209] In some cases, the genome of a Paenibacillus species may not contain a gene to produce glnB or glnK. In some cases, the genome of a Paenibacillus species may contain a minimal nif cluster with 9 genes transcribed from a sigma-70 promoter. In some cases, a nif Paenibacillus cluster may be negatively regulated by nitrogen or oxygen. In some cases, the genome of a Paenibacillus species may not contain a gene to produce sigma-54. For example, Paenibacillus sp. WLY78 does not contain a gene for sigma-54. In some cases, a nif cluster can be regulated by glnR and / or TnrA. In some cases, the activity of a nif cluster can be altered by changing the activity of glnR and / or TnrA.
[0210] [0210] In Bacilli, glutamine synthetase (GS) is inhibited by feedback from high concentrations of intracellular glutamine, causing a shift in confirmation (referred to as FBI-GS). Nif clusters contain distinct binding sites for the GlnR and TnrA regulators in several Bacilli species. GlnR binds and suppresses gene expression in the presence of excess intracellular glutamine and AMP. A function of GlnR may be to prevent the influx and intracellular production of glutamine and ammonium under conditions of high nitrogen availability. TnrA can bind and / or activate (or repress) gene expression in the presence of limiting intracellular glutamine and / or in the presence of FBI-GS. In some cases, the activity of a nif Bacilli nif cluster can be altered by changing the activity of GlnR.
[0211] [0211] Feedback-inhibited glutamine synthetase (FBI-GS) can bind GlnR and stabilize GlnR binding to recognition sequences. Several bacterial species have a GlnR / TnrA binding site upstream of the nif cluster. Changing the connection of FBI-GS and GlnR can change the activity of the nif pathway. Microbe Sources
[0212] [0212] Bacteria (or any microbe according to the invention) can be obtained from any general terrestrial environment, including their soils, plants, fungi, animals (including invertebrates) and other biota, including sediment, water and biota lakes and rivers; of the marine environment, its biota and sediments (for example, seawater, marine mud, marine plants, marine invertebrates (for example, sponges), marine vertebrates (for example, fish); the terrestrial and marine geosphere (regolith and rock, for example, crushed underground rocks, sand and clays); the cryosphere and its melting water; the atmosphere (for example, filtered aerial dust, cloud and rain droplets); urban, industrial and other man-made environments (eg example, organic and mineral material accumulated in concrete, roadside gutters, roof surfaces and road surfaces).
[0213] [0213] The plants from which the bacteria (or any microbe according to the invention) are obtained may be a plant having one or more desirable attributes, for example, a plant that grows naturally in a particular environment under certain conditions of interest. For example, a certain plant can grow naturally on sandy soil or on high salinity sand, or under extreme temperatures, or with little water, or it can be resistant to certain pests or diseases present in the environment, and it may be desirable for a crop commercial cultivation under such conditions, especially if these are, for example, the only conditions available in a specific geographical location. As an additional example, bacteria can be collected from commercial crops grown in such environments or, more specifically, from plants of individual crops that best exhibit an attribute of interest among a crop grown in any specific environment: for example, fastest-growing plants among a crop grown in soils of limited salinity, or the least damaged plants in crops exposed to severe insect damage or epidermal disease, or plants with the desired amounts of certain metabolites and other compounds, including fibers, oil content and others, or plants exhibiting desirable colors, taste or smell. Bacteria can be collected from a plant of interest or any material existing in the environment of interest, including fungi and other animal and plant biota, soil, water, sediment and other elements of the environment as previously mentioned.
[0214] [0214] Bacteria (or any microbe according to the invention) can be isolated from plant tissue. Such isolation can occur from any suitable tissue woven into the plant, including for example, root, stem and leaves, and reproductive plant tissues. For example, conventional methods of isolation from plants typically include sterile excision of the plant material of interest (eg, root or stem length, leaves), sterilizing the surface with a solution (e.g. 2% sodium hypochlorite), after which the plant material is placed in a nutrient medium for microbial growth. Alternatively, plant material with a sterile surface can be crushed in a sterile liquid (usually water) and the liquid suspension, including small pieces of crushed plant material, is spread over the surface of a suitable solid agar medium, or media, which may or may not be selective (eg, contain only phytic acid as a source of phosphorus). This approach is especially useful for bacteria that form isolated colonies and can be picked up individually and transferred to separate plates of nutrient medium and further purified to a single species by well-known methods. Alternatively, samples of the plant's root or foliage may not have the surface sterilized, but only gently washed, thus including epiphytic microorganisms that live on the surface in the isolation process, or the epiphytic microbes can be isolated separately , pressing for printing and lifting pieces of roots, stems or leaves of the plant on the surface of an agar medium, and then isolating individual colonies as above. This approach is especially useful for bacteria, for example. Alternatively, the roots can be processed without washing small amounts of soil attached to the roots, thus including microbes that colonize the plant's rhizosphere. Otherwise, the soil adhered to the roots can be removed, diluted and spread on agar using suitable selective and non-selective media to isolate individual colonies of rhizospheric bacteria. Budapest Treaty on international recognition of the deposit of microorganisms for the purposes of patent procedures
[0215] [0215] The microbial deposits of the present invention were made in accordance with the provisions of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedures (Treaty of Budapest).
[0216] [0216] The Claimants declare that, according to 37 C.F.R. §
[0217] [0217] A biologically pure culture of Kosakonia sacchari (WT) was deposited on January 6, 2017 with the Bigelow National
[0218] [0218] A biologically pure culture of Klebsiella variicola (WT) was deposited on August 11, 2017 located at 60 Bigelow Drive, East Boothbay, Maine 04544, USA, and received the NCMA Patent Filing Designation number 201708001. Information applicable to the deposit are shown below in Table 1.
[0219] [0219] Table 1: Microorganisms deposited under the Budapest Treaty Main designation of the Depositor Number strain (some strains have Taxonomy Date of deposit accessing multiple designations) January 6, NCMA CI006, PBC6.1, 6 Kosakonia sacchari (WT) 201701001 2017 August 11, NCMA CI137, 137, PB137 Klebsiella variicola (WT) 201708001 2017 Isolated and biologically pure microorganisms
[0220] [0220] The present invention, in certain modalities, provides isolated and biologically pure microorganisms that have applications, among others, in agriculture. The revealed microorganisms can be used in their isolated and biologically pure states, as well as be formulated in compositions (see the section on exemplary composition descriptions below). In addition, the invention provides microbial compositions containing at least two members of the revealed microorganisms isolated and biologically pure, as well as methods of using said microbial compositions. In addition, the invention provides methods of modulating nitrogen fixation in plants through the use of the revealed microbes isolated and biologically pure.
[0221] [0221] In some respects, the isolated and biologically pure microorganisms of the invention are those in Table 1. In other respects, the isolated and biologically pure microorganisms of the invention are derived from a microorganism in Table 1. For example, strain, son, mutant or derivative of a microorganism from Table 1 are provided at present. The invention contemplates all possible combinations of microbes listed in Table 1, said combinations forming, sometimes, a microbial consortium. The microbes in Table 1, either individually or in any combination, can be combined with any plant, active compound (synthetic, organic, etc.), adjuvant, carrier, supplement or biological agent mentioned in the invention. Compositions
[0222] [0222] The compositions, comprising bacteria or bacterial populations produced according to methods described herein and / or having characteristics as described herein, can be in the form of liquid, foam or a dry product. Compositions, comprising bacteria or bacterial populations produced according to the methods described herein and / or having characteristics as described herein, can also be used to improve plant attributes. In some examples, a composition comprising bacterial populations may be in the form of dry powder, a paste of powder and water or a wettable seed treatment. Compositions comprising bacterial populations can be coated on the surface of a seed, and can be in liquid form.
[0223] [0223] The composition can be manufactured in bioreactors such as reactors of the continuous agitated tank type, batch reactors and on the farm. In some examples, the compositions can be stored in a container, such as a jug, or in mini bulk. In some examples, the compositions can be stored inside an object selected from the group consisting of vial, ampoule, packaging, vase, bag, box, crate, envelope, cardboard box,
[0224] [0224] Compositions can also be used to improve plant attributes. In some examples, one or more compositions can be coated on a seed. In some examples, one or more compositions can be coated on a seedling. In some examples, one or more compositions can be coated on a seed surface. In some examples, one or more compositions can be coated as a layer on top of a seed surface. In some examples, a composition that is coated on a seed may be in liquid form, in the form of a dry product, in the form of foam, in the form of a paste of powder and water or in a wettable seed treatment. In some examples, one or more compositions can be applied to a seed and / or seedling by spraying, dipping, coating, encapsulating and / or dusting the seed and / or seedling with one or more of the compositions. In some instances, multiple bacteria or bacterial populations can be coated on a seed and / or a plant seedling. In some instances, 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 from a bacterial combination can be selected from one of the following genera: Acidovorax, Agrobacterium, Bacillus, Burkholderia, Chryseobacterium, Curtobacterium, Enterobacter, Escherichia, Metilobacterium, Paenibacillus, Pantoea, Pseudomonas, Ralstonia, Saccharibacillus, Sphingomonas and Stenotrophomonas.
[0225] [0225] 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 of an endophytic combination are selected from one of the following families: Bacillaceae, Burkholderiaceae, Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae, Metilobacteriaceae, Microbacteriaceae, Paenibacillileae, Pseudomonneae, Rhizobiaceae, Sphingomonadeae, Ephonaceae, Ephonaceae
[0226] [0226] In some instances, 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 of an endophytic combination are selected from one of the following families: Bacillaceae, Burkholderiaceae, Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae, Metilobacteriaceae, Microbacteriaceae, Paenibacillileae, Pseudomonneae, Rhizobiaceae, Sphingomonadeae, Ephonaceae, Sphingomonadaceae,
[0227] [0227] Examples of compositions may include seed coatings for commercially important agricultural crops, for example, sorghum, canola, tomatoes, strawberries, barley, rice, corn and wheat. Examples of compositions may also include seed coatings for corn, soybeans, canola, sorghum, potatoes, rice, vegetables, cereals and oilseeds. The seeds, now provided, may be genetically modified (GMO), non-GMO, organic or conventional organisms. In some examples, the compositions can be sprayed on the branches of the plants or applied to the roots, inserting in grooves in which the plant's seeds are planted, watering the soil or immersing the roots in a suspension of the composition. In some instances, the compositions may be dehydrated in a manner that adequately maintains cell viability and the ability to artificially inoculate and colonize host plants. Bacterial species can be present in the compositions at a concentration between 108 and 1010 CFU / mL. In some instances, the compositions may be supplemented with attribute metal ions, such as molybdenum ions, iron ions, manganese ions or combinations of these ions. The ion concentration in examples of compositions as described herein can be between approximately 0.1 mM and 50 mM. Some examples of compositions can also be formulated with a carrier, such as beta-glycan, carboxymethylcellulose (CMC), bacterial extracellular polymeric substance (EPS), sugar, animal milk or other suitable carriers. In some examples, peat or planting materials can be used as carriers, or biopolymers in which a composition is trapped in the biopolymer can be used as a carrier. Compositions comprising the bacterial populations described here can improve plant attributes, such as promoting plant growth, maintaining high leaf content of chlorophyll, increasing the number of fruits or seeds and increasing the unit mass of fruits or seeds.
[0228] [0228] The compositions comprising the bacterial populations described herein can be coated on the surface of a seed. Thus, compositions comprising a seed coated with one or more bacteria described herein are also contemplated. The seed coating can be formed by mixing the bacterial population with a porous, chemically inert granular carrier. Alternatively, the compositions can be inserted directly into the grooves in which the seed is planted, or sprayed on the leaves of the plant or applied by immersing the roots in a suspension of the composition. An effective amount of the composition can be used to populate the subsoil region adjacent to the plant roots with viable bacterial growth, or to populate the plant leaves with viable bacterial growth. In general, an effective amount is an amount sufficient to result in plants with improved attributes (eg, a desired level of nitrogen fixation).
[0229] [0229] The bacterial compositions described herein can be formulated using an agriculturally acceptable carrier. The formulation useful for these modalities can include at least one element selected from the group consisting of a tackifier (adhesive), a microbial stabilizer, a fungicide, an antibacterial agent, a preservative, a stabilizer, a surfactant, an agent anti-complex, a herbicide, a nematicide, an insecticide, a plant growth regulator, a fertilizer, a rodenticide, a desiccant, a bactericide, a nutrient or any combination thereof. In some examples, the compositions can be stable at room temperature. For example, any of the compositions described herein may include an agriculturally acceptable carrier (eg, one or more of a fertilizer as an unnatural fertilizer, an adhesion agent as an unnatural adhesion agent and a pesticide as a pesticide unnatural). An unnatural adhesion agent can be, for example, a polymer, copolymer or synthetic wax. For example, any of the coated seeds, seedlings or plants described herein may contain such an agriculturally acceptable carrier in the seed coat. In any of the compositions or methods described herein, an agriculturally acceptable carrier can be or include an unnatural compound (eg, an unnatural fertilizer, an unnatural adhesion agent, such as a polymer, copolymer or synthetic wax, or an unnatural pesticide). Non-limiting examples of agriculturally acceptable carriers are described below. Additional examples of agriculturally acceptable carriers are known in the art.
[0230] [0230] In some cases, the bacteria are mixed with an agriculturally acceptable carrier. The carrier can be a solid carrier or liquid carrier, and in various forms including microspheres, powders, emulsions and the like. The carrier can be any one or more of several carriers that provide a variety of properties, such as stability, greater wettability or dispersibility. Wetting agents, such as natural or synthetic surfactants, which can be non-ionic or ionic surfactants, or a combination of these, can be included in the composition. Water-in-oil emulsions can also be used to formulate a composition that includes 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 pellets, thickeners, and the like, microencapsulated particles and the like, liquids such as aqueous flowables, aqueous suspensions, water emulsions in oil etc. The formulation can include grain or vegetable products, for example, ground grain or beans, broth or flour derived from grain or beans, starch, sugar or oil.
[0231] [0231] In some modalities, the agricultural carrier can be the soil or a plant growth medium. Other agricultural carriers that can be used include water, fertilizers, vegetable-based oils, humectants, or combinations of these. Alternatively, the agricultural carrier can be a solid, such as diatomaceous earth, clay, silica, alginate, clay, bentonite, vermiculite, seed husks, other plant and animal products, or combinations, including granules, pellets or suspensions. Mixtures of any of the ingredients mentioned above are also contemplated as carriers, such as, among others, pesta (flour and kaolin clay), agar or flour-based pellets in clay, sand or clay etc. The formulations may include food sources for bacteria, such as barley, rice or other biological materials such as seeds, parts of plants, sugarcane bagasse, husks or stems from grain processing, ground vegetable material or mulch waste works, sawdust or small fibers from the recycling of paper, fabric or wood.
[0232] [0232] For example, a fertilizer can be used as an aid to promote growth or to provide nutrients to a seed, seedling or plant. Non-limiting examples of fertilizers include nitrogen, phosphorus, potassium, calcium, sulfur, magnesium, boron, chloride, manganese, iron, zinc, copper, molybdenum and selenium (or a salt thereof). Additional examples of fertilizers include one or more amino acids, salts, carbohydrates, vitamins, glucose, NaCl, yeast extract, NH4H2PO4, (NH4) 2SO4, glycerol, valine, L-leucine, lactic acid, propionic acid, succinic acid, malic acid, citric acid, KH tartrate, xylose, lixose and lecithin. In one embodiment, the formulation may include a tackifier or adherent (referred to as an adhesive agent) to aid in the attachment of other active agents to a substance (eg, seed surface). Such agents are useful for combining bacteria with carriers that may contain other compounds (eg, non-biological control agents), to produce a coating composition. Such compositions help to create coatings around the plant or seed to maintain contact between the microbe and other agents with the plant or part of the plant. In one embodiment, the adhesives are selected from the group consisting of: alginate, gums, starches, lecithins, formononetine, polyvinyl alcohol, alkaline formononetinate, hesperetin, polyvinyl acetate, cephalins, arabic gum, xanthan gum, mineral oil , polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), Arabino-galactan, methyl cellulose, PEG 400, chitosan, polyacrylamide, polyacrylate, polyacrylonitrile, glycerol, triethylene glycol, vinyl acetate, gum gum, polystyrene, polystyrene, polystyrene, polystyrene, polystyrene, polystyrene, polystyrene, polystyrene, polystyrene, polystyrene, polystyrene, polystyrene, polystyrene, polystyrene, polystyrene, polystyrene polyoxyethylene-
[0233] [0233] In some embodiments, adhesives can be, e.g. , a wax such as carnauba wax, beeswax, Chinese wax, shellac, spermaceti wax, candelilla wax, castor oil wax, ouricuri wax and rice bran wax, a polysaccharide ( eg starch, dextrins, maltodextrins, alginate and chitosans), a fat, oil, protein (eg gelatin and zeins), arable gums and enamels. Adhesive agents can be unnatural compounds, e.g. , polymers, copolymers and waxes. For example, non-limiting examples of polymers that can be used as an adhesive include: polyvinyl acetates, polyvinyl acetate copolymers, ethylene vinyl acetate (EVA) copolymers, polyvinyl alcohols, polyvinyl alcohol copolymers, cellulose ( e.g., ethyl celluloses, methylcelluloses, hydroxymethylcelluloses, hydroxypropylcelluloses and carboxymethylcelluloses), polyvinylpyrrolidones, vinyl chloride, vinylidene chloride copolymers, calcium lignosulfonates, acrylic copolymers, polyethyls, polyvinylacids and polyethylacrylates acrylate, methylacrylamide and polychloroprene monomers.
[0234] [0234] In some instances, one or more of the adhesives, antifungal agents, growth regulating agents and pesticides (eg, insecticide) are unnatural compounds (eg, in any combination). Additional examples of agriculturally acceptable carriers include dispersants (eg, polyvinylpyrrolidone / vinyl acetate PVPIVA S-630), surfactants, binders and fillers.
[0235] [0235] The formulation may also contain a surfactant. Non-limiting examples of surfactants include mixtures of nitrogenous surfactants such as Prefer 28 (Cenex), Surf-N (US), Inhance (Brandt), P-28 (Wilfarm) and Patrol (Helena); esterified seed oils including
[0236] [0236] In certain cases, the formulation includes a microbial stabilizer. This agent can include a desiccant, which can include any compound or mixture of compounds that can be classified as desiccant regardless of whether the compound or compounds are used in such concentrations as to actually have a desiccant effect on an inoculant. liquid. Such desiccants are ideally compatible with the bacterial population used, and should promote the ability of the microbial population to survive application on seeds and to survive drying. Examples of suitable desiccants include one or more of trehalose, sucrose, glycerol and methylene glycol. Other suitable desiccants include, among others, non-reducing sugars and sugar alcohols (eg mannitol or sorbitol). The amount of desiccant introduced into the formulation can vary between approximately 5% and 50% in mass / volume, for example, between approximately 10% and 40%, between approximately 15% and 35% or between approximately 20% and 30%. In some cases, it is advantageous that the formulation contains agents such as a fungicide, antibacterial agent, herbicide, nematocide, insecticide, plant growth regulator, rodentide, bactericide or nutrient. In some instances, agents may include protectors that provide protection against pathogens on the seed surface. In some instances, protectors may provide some level of control of pathogens present in the soil.
[0237] [0237] In some examples, a fungicide may include a compound or agent, whether chemical or biological, that can inhibit the growth of a fungus or kill a fungus. In some examples, a fungicide can include compounds that can be fungistatic or fungicidal. In some examples, the fungicide may be a protector, or agents that are effective predominantly on the seed surface, providing protection against pathogens present on the seed surface and providing some level of pathogen control in the soil. Non-limiting examples of protective fungicides include captan, maneb, tiram or fludioxonil.
[0238] [0238] In some examples, the fungicide can be a systemic fungicide, which can be absorbed by the emerging seedling and inhibit or kill the fungus within the tissues of the host plant. Systemic fungicides used for seed treatment include, but are not limited to, the following: azoxystrobin, carboxine, mefenoxam, metalaxyl, thiabendazole, trifloxystrobin and various triazole fungicides, including diphenoconazole, ipconazole, tebuconazole and triticonazole. Mefenoxam and metalaxyl are used primarily to target the fungal fungi in water Pythium and Phytophthora. Some fungicides are preferred over others, depending on the plant species, either because of subtle differences in the sensitivity of the pathogenic fungal species, or because of differences in the distribution or sensitivity to the plants' fungicide. In some instances, the fungicide can be a biological control agent, such as a bacterium or fungus. Such organisms can be parasites of pathogenic fungi, or secrete toxins or other substances that can kill or otherwise prevent the growth of fungi. Any type of fungicide, especially those that are commonly used in plants, can be used as a control agent in a seed composition.
[0239] [0239] In some examples, the seed coating composition comprises a control agent with antibacterial properties. In one embodiment, the control agent with antibacterial properties is selected from among the compounds described elsewhere in the present. In another embodiment, the compound is streptomycin, oxytetracycline, oxolinic acid or gentamicin. Other examples of antibacterial compounds that can be used as part of a seed coating composition include those based on dichlorophene and hemiformal benzyl alcohol (Proxel® from ICI or Acticide® RS from Thor Chemie and Kathon® MK 25 from Rohm & Haas) and isothiazolinone derivatives such as alkyl isothiazolinones and benzisothiazolinones (Thor Chemie's Acticide® MBS).
[0240] [0240] In some examples, the growth regulator is selected from the group consisting of: abscisic acid, amidochlor, ancimidol, 6-benzylaminopurine, brassinolide, butralin, clormequate (chlormequate chloride), choline chloride, cyclanilide , daminozide, dikegulac, dimethypine, 2,6-dimethylpuridine, etefon, flumetraline, flurprimidol, flutiacet, forclorfenuron, gibberellic acid, inabenfide, indole-3-acetic acid, maleic hydrazide, mefluidide, mepiquate, mepiquate, mepiquate, chloride N-6-benzyladenine, paclobutrazol, prohexadione phosphorothioate, 2,3,5-triiodiodobenzoic acid, trinexapatethyl and uniconazole. Additional non-limiting examples of growth regulators include brassinosteroids, cytokinins (eg, kinetin and zeatin), auxins (eg, indolacetic acid and indolacetyl aspartate), flavonoids and isoflavanoids (eg, formononetine and diosmetin ), phytookines (eg, gliceolin) and phytoalexin-inducing oligosaccharides (eg, pectin, chitin, chitosan, polygalacturonic acid and oligogalacturonic acid) and gibelerins. Such agents are ideally compatible with the seed or agricultural seedling on which the formulation is applied (eg, 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 (eg, no safety issue, or the compound is sufficiently labile so that the plant product as a commodity, derived from the plant contains disposable amounts of the compound).
[0241] [0241] Some examples of nematode antagonist 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 .; vesicular-arbuscular mycorrhizal fungi, Burkholderia spp .; Pasteuria spp., Brevibacillus spp .; Pseudomonas spp .; and Rhizobacteria. Especially preferred nematode antagonist biocontrol agents include ARF18, Arthrobotrys oligospora, Arthrobotrys dactyloides, Chaetomium globosum, Cylindrocarpon heteronema, Exophilia jeanselmei, Exophilia pisciphila, Fusarium aspergilus, Fusarium solani, Gliocirsis, Glium , Lecanicillium lecanii, Monacrosporium drechsleri, Monacrosporium gephyropagum, Myrotehcium verrucaria, Neocosmospora vasinfecta, Paecilomyces lilacinus, Pochonia chlamydosporia, Stagonospora heteroderae, Stagonospora pasteur, Pasteuriseur, type of germs, , use of Pastrueia, Brevibacillus laterosporus strain G4, Pseudomonas fluorescens and Rhizobacteria.
[0242] [0242] Some examples of nutrients can be selected from the group consisting of a nitrogen fertilizer including,
[0243] [0243] Some examples of rodenticides can be selected from the group of substances consisting of 2-isovalerylindan-1,3-dione, 4- (quinoxalin-2-ylamino) benzenesulfonamide, alpha-hydrochlorine, aluminum phosphide, antu, arsenious oxide, barium carbonate, bistiosemi, brodifacoum, bromadiolone, bromethalin, calcium cyanide, chloralose, chlorofacinone, cholecalciferol, coumaclor, coumafuril, coumatetralila, crimidine, difenacoum, difetifamily, fluethaginine, flacetamone, flacetamone , hydrogen cyanide, iodomethane, lindane, magnesium phosphide, methyl bromide, norbormide, fosacetim, phosphine, phosphorus, pindone, potassium arsenite, pyrinuron, scylliroside, sodium arsenite, sodium cyanide, sodium fluoroacetate, strychnine, sulfate of thallium, warfarin and zinc phosphide.
[0244] [0244] In liquid form, for example, solutions or suspensions, bacterial populations can be mixed or suspended in water or in aqueous solutions. Suitable liquid diluents or carriers include water, aqueous solutions, petroleum distillates or other liquid carriers.
[0245] [0245] Solid compositions can be prepared by dispersing bacterial populations in and over a finely divided solid carrier, such as peat, wheat, bran, vermiculite, clay, talc, bentonite, diatomaceous earth, Fuller's soil, pasteurized soil and others. When such formulations are used as wettable powders, biologically compatible dispersing agents, such as nonionic, anionic, amphoteric or cationic dispersing and emulsifying agents, can be used.
[0246] [0246] Solid carriers when formulated include, for example, mineral carriers such as kaolin clay, pyrophyllite, bentonite, montmorillonite, diatomaceous earth, acidic white soil, vermiculite and perlite, and inorganic salts such as ammonium sulphate, phosphate ammonium, ammonium nitrate, urea, ammonium chloride and calcium carbonate. In addition, fine organic 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. Application of bacterial populations in cultures
[0247] [0247] The composition of the bacteria or bacterial population described here can be applied furrow, in talc or as seed treatment. The composition can be applied to seeds in bulk packaging, mini bulk, in a bag or in talc.
[0248] [0248] The farmer can plant the treated seed and grow the crop according to conventional, double row, or ways that do not require plowing. The seeds can be distributed using a control funnel or an individual funnel. The seeds can also be distributed using pressurized air or manually. Seed placement can be done using variable rate technologies. In addition, the application of the bacteria or bacterial population described here can be done using variable rate technologies. In some instances, the bacteria can be applied to corn, soy, canola, sorghum, potato, rice, vegetables, cereals, pseudo-cereals and oilseeds. Examples of cereals may include barley, millet, oats, Distichlis palmeri, rye, millet, sorghum, spelled, teff, triticale and wheat. Examples of pseudocereal can include breadfruit, buckwheat, cattail, chia, flaxseed, amaranth grain, hanza, quinoa and sesame. In some instances, seeds may be genetically modified (GMO), non-GMO, organic or conventional organisms.
[0249] [0249] Additives such as micro-fertilizer, PGR, herbicide, insecticide and fungicide can also be used to treat crops. Examples of additives include crop protectors such as insecticides, nematicides, fungicides, breeding agents such as dyes, polymers, pellets, preparations and disinfectants, and other agents such as inoculants, PGR, softeners (softener) and micronutrients. PGRs can be natural or synthetic plant hormones that affect root growth, flowering or stem elongation. PGRs can include auxins, gibberellins, cytokinins, ethylene and abscisic acid (ABA).
[0250] [0250] The composition can be applied to the furrow in combination with liquid fertilizer. In some instances, liquid fertilizer can be kept in tanks. NPK fertilizers contain macronutrients of sodium, phosphorus and potassium.
[0251] [0251] The composition can improve plant attributes, such as promoting plant growth, maintaining a high leaf content of chlorophyll, increasing the number of fruits or seeds and increasing the unit mass of fruits or seeds. The methods of the present invention can be employed to introduce or improve one or more of a variety of desirable attributes. Examples of attributes that can be introduced or improved include: root biomass, root length, height, branch length, number of leaves, water use efficiency, global biomass, yield, fruit size, grain size, rate of photosynthesis, drought tolerance, heat tolerance, tolerance to salinity, tolerance to low nitrogen stress, efficiency in the use of nitrogen, resistance to nematode stress, resistance to a fungal pathogen, resistance to a bacterial pathogen , resistance to a viral pathogen, metabolite level, metabolite level modulation, proteome expression. Desirable attributes, including height, global biomass, root and / or branch biomass, seed germination, seedling survival, photosynthetic efficiency, transpiration rate, number / mass of seeds / fruits, yield of grades or fruits of the plant , chlorophyll leaf content, photosynthetic rate, root length, or any combination thereof, can be used to measure growth, and compared to the growth rate of reference agricultural plants (eg, plants without introduced and / or improved attributes) grown under identical conditions. In some examples, desirable attributes, including height, global biomass, root and / or branch biomass, seed germination, seedling survival, photosynthetic efficiency, transpiration rate, number or mass of seeds / fruits, yield of degrees or fruits of the plant, leaf content of chlorophyll, photosynthetic rate, length of roots, or any combination thereof, can be used to measure growth, and compared with the growth rate of reference agricultural plants ( (eg, plants without the introduced and / or improved attributes) grown under similar conditions.
[0252] [0252] An agronomic attribute for a host plant may include, among others, the following: alteration of the oil content, alteration of the protein content, alteration of the composition of carbohydrates in seeds, alteration of the composition of oils in seeds and alteration of the composition of protein in seeds, tolerance to chemicals, tolerance to cold, retardation of senescence, resistance to diseases, tolerance to drought, weight of ears, growth improvement, health improvement, heat tolerance, tolerance to herbicides, improved nitrogen fixation resistance to herbivores, better use of nitrogen, better root architecture, better efficiency in the use of water, greater biomass, greater length of roots, greater mass of seeds, greater length of branches, greater yield, greater yield under conditions of water limitation , grain weight, grain moisture content, metal tolerance, number of ears, number of grains per ear, number of spaces, m improved nutrition, resistance to pathogens, resistance to pests, better phytosynthetic capacity, tolerance to salinity, staying green, better vigor, greater dry mass of mature seeds, greater fresh mass of mature seeds, greater number of mature seeds per plant, higher chlorophyll content, greater number of pods per plant, greater length of pods per plant, lesser number of wilted leaves per plant, lesser number of severely wilted leaves per plant and greater number of non-wilted leaves per plant, detectable modulation in the level of a metabolite, detectable modulation at the level of a transcript and detectable modulation in the proteome, compared to an isolated plant grown from a seed without the said formulation for seed treatment.
[0253] [0253] In some cases, plants are inoculated with bacteria or bacterial populations that are isolated from the same plant species as the plant element of the inoculated plant. For example, a bacterium or bacterial population that is normally found in a variety of Zea mays (corn) is associated with a plant element in a plant of another variety of Zea mays that, in its natural state, lacks said bacteria and bacterial populations. . In one embodiment, the bacteria and bacterial populations are derived from a plant of a plant-related species to the plant element of the inoculated plant. For example, a bacteria and bacterial populations that are normally found in Zea diploperennis Iltis at al., (Diploperennial teosinte) are applied to Zea mays (maize) or vice versa. In some cases, the plants are inoculated with bacteria and bacterial populations that are heterologous to the plant element of the inoculated plant. In one embodiment, bacteria and bacterial populations are derived from a plant of another species. For example, a bacterium and bacterial populations that are normally found in dicots are applied to a monocot plant (eg, inoculation of maize with a bacterium and bacterial populations derived from soybeans) or vice versa. In other cases, the bacteria and bacterial populations to be inoculated into a plant are derived from a related species of the plant being inoculated. In one embodiment, bacteria and bacterial populations are derived from a related taxon, for example, from a related species. The plant of another species can be an agricultural plant. In another fashion, bacteria and bacterial populations are part of a projected composition inoculated into any element of the host plant.
[0254] [0254] In some instances, the bacterium or bacterial population is exogenous, in which the bacterium and bacterial population are isolated from a plant other than the inoculated plant. For example, in a fashion, the bacteria or bacterial population can be isolated from a plant other than the same species as the inoculated plant. In some cases, the bacteria or bacterial population may be isolated from a species related to the inoculated plant.
[0255] [0255] In some examples, the bacteria and bacterial populations described here are able to move into one type of tissue to another.
[0256] [0256] The effectiveness of the compositions can also be evaluated by measuring the relative maturity of the culture or the thermal unit of the culture (CHU). For example, the bacterial population can be applied to corn, and corn growth can be evaluated according to the relative maturity of the corn kernels or the time until the corn kernels are at maximum mass. The thermal crop unit (CHU) can also be used to predict the maturity of the corn crop. The CHU determines the amount of heat accumulated by measuring the maximum daily temperatures during the growth of the crop.
[0257] [0257] In examples, bacteria can be located in any of the tissues in the plant, including: the root, adventitia root, seminal root, radicular, branch, leaf, flower, bud, tassel, meristem, pollen, pistil , ovaries, stamen, fruit, stolon, rhizome, nodule, tuber, trichome, guard cells, hydatidium, petal, sepal, glume, rachis, vascular camphor, phloem and xylem. In another modality, the bacterium or bacterial population is able to be located in photosynthetic tissues, for example, leaves and branches of the plant. In other cases, the bacterium and bacterial populations are located in the vascular tissues of the plant, for example, in xylem and phloem. In another modality, the bacterium or bacterial population is able to be located in reproductive tissues (flower, pollen, pistil, ovaries, stamen or fruit) of the plant. In another modality, bacteria and bacterial populations are able to be located in the root, branches, leaves and reproductive tissues of the plant. In another embodiment, the bacterium or bacterial population colonizes a tissue of fruit or seed from the plant. In yet another embodiment, the bacterium or bacterial population is able to colonize the plant such that it is present on the plant's surface. In another embodiment, the bacterium or bacterial population is able to be located in substantially all or all tissues of the plant. In certain modalities, the bacterium or bacterial population is not located at the root of a plant. In other cases, the bacteria and bacterial populations are not located in the photosynthetic tissues of the plant.
[0258] [0258] The effectiveness of bacterial compositions applied to crops can be assessed by measuring various characteristics of crop growth including, among others, planting rate, sowing vigor, root strength, drought tolerance, plant height , loss of moisture in the grains (dry down) and volumetric weight. Plant species
[0259] [0259] The methods and bacteria described here are suitable outside any one of a variety of plants, such as plants in the genera 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 food crops, fibrous crops, oil crops, plants of forest species or pulp and paper industries, raw material for the production of biofuel and / or ornamental plants. - such. In some examples, plants can be used to generate products of economic value such as a grain, a flour, a starch, a syrup, a meal, an oil, a film, a package, a nutraceutical product, a pulp, a feed for animals, a fish feed, a fish feed, a bulk material for industrial chemicals, a cereal product, a human processed food product, sugar, alcohol and / or protein. Non-limiting examples of crop plants include corn, rice, wheat, barley, sorghum, millet, oats, rye, triticale, buckwheat, sweet corn, sugar cane, onion, tomato, strawberry and asparagus.
[0260] [0260] In some examples, plants that can be obtained or improved using the methods and composition disclosed herein may 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, pea plants and grasses; and dicotyledonous plants, such as peas, alfalfa, tomato, melon, chickpeas, chicory, clover, curly kale, lentils, soybeans, tobacco, potatoes, sweet potatoes, radishes, cabbage, rapeseed, apple trees, vine, cotton , sunflower, Aradopsis thaliana, canola, citrus (including orange, mandarin, kumquat, lemon, lime, grapefruit, mandarin, tangelo, cider and pomelo), pepper, beans, lettuce, Panicum virgatum (switchgrass), Bicolor sorghum (sorghum, Sudan ), Miscanthus giganteus (miscanthus), Saccharum sp. (energy cane), Populus balsamifera (poplar), Zea mays (corn), Glycine max (soy), Brassica napus (canola), Triticum aestivum (wheat), Gossypium hirsutum (something), Oryza sativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris (beet), Pennisetum glaucum (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 - wheat X rye), bamboo, Carthamus tinctorius (saffron), Jatropha curcas (Jatropha), Ricinus communis (castor), Elaeis guineensis (oil palm), Phoenix dactylifera (date palm), Archontophoenix cunninghamiana (king's palm), Syagr romanzoffiana (queen palm), Linum usitatissimum (linseed), Brassica juncea, Manihot esculenta (madioca), Lycopersicon esculentum (tomato), Lactuca saliva (lettuce), Musa paradisiaca (banana), Solanum tuberosum (potato), Brassica oleracea (broccoli) , cauliflower, Brussels sprouts), Camellia sinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa), Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus (pineapple), Capsicum annum (pepper chilli and sweet), Allium cepa (onion), Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima (pumpkin), Cucurbita moschata (pumpkin), Spinacea oleracea (spinach), Citrullus lanatus (watermelon), Abelmoschus esculentus ( okra), Solanum melongena (eggplant), Papaver so mniferum (poppy poppy), Papaver orientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabis saliva, Camptotheca acuminate, Catharanthus roseus, Vinca rosea, Cinchona officinalis, Coichicum autumnale, Veratrum californica, Digitalis lanata, Digitaliscorpeaia, Dysporepureia, Digitaliscorpureia, Dysporepureia, Dariusporeographia paniculata, Atropa belladonna, Datura stomonium, Berberis spp., Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca, Galanthus wornorii, Scopolia spp., Lycopodium serratum (Huperzia serrata), Lycopodium sp. , Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis, Chrysanthemum parthenium, Coleus forskohlii, Tanacetum parthenium, Parthenium argentatum (guaiúle), Hevea spp. (rubber tree), Mentha spicata (mint), Mentha piperita (mint), Bixa orellana, Alstroemeria spp., Rosa spp. (pink), Dianthus caryophyllus (coal), Petunia spp. (petunia), Poinsettia pulcherrima (poinsettia), Nicotiana tabacum (tobacco), Lupinus albus (white lupine), Uniola paniculata (oats), Hordeum vulgare (barley) and Lolium spp. (rye).
[0261] [0261] In some examples, a monocot plant can be used. The monocotyledonous plants belong to the orders of Alismatales, Arales, Arecales, Bromeliales, Commelinales, Cyclanthales, Cyperales, Eriocaulales, Hydrocharitales, Juncales, Lilliales, Najadales, Orchidales, Pandanales, Poales, Restionales, Triuridales, Typhales and Zingibes. The plants that belong to the Gymnospermae class are Cycadales, Ginkgoales, Gnetales and Pinales. In some examples, the monocot plant can be selected from the group consisting of corn, rice, wheat, barley and sugar cane.
[0262] [0262] In some examples, a dicotyledonous plant may be used, including those belonging to the orders of Aristochiales, Asterales, Batales, Campanulales, Capparales, Caryophyllales, Casuarinales, Celastrales, Cornales, Diapensales, Dilleniales, Dipsacales, Ebenales, Ericales, Eucomiales Euphorbiales, Fabales, Fagales, Gentianales, Geraniales, Haloragales, Hamamelidales, Middles, Juglandales, Lamiales, Laurales, Lecythidales, Leitneriales, Magniolales, Malvales, Myricales, Myrtales, Nymphaeales, Papeverales, Piperales, Polerales, Agaisales, Plaisales , Polygonales, Primulales, Proteales, Rafflesiales, Ranunculales, Rhamnales, Rosales, Rubiales, Salicales, Santales, Sapindales, Sarraceniaceae, Scrophulariales, Theales, Trochodendrales, Umbellales, Urticales and Violates. In some examples, the dicot plant can be selected from the group consisting of cotton, soy, pepper and tomato.
[0263] [0263] In some cases, the plant to be improved is not quickly receptive to experimental conditions. For example, a crop plant may take too long to grow long enough to evaluate practically a series-enhanced attribute over multiple iterations. Thus, a first plant from which the bacteria are initially isolated and / or the plurality of plants to which genetically manipulated bacteria are applied can be a model plant, such as a plant more receptive to evaluation under desired conditions. Non-limiting examples of model plants include Setaria, Brachypodium and Arabidopsis. The ability of bacteria isolated according to a method of the invention, using a model plant, can then be applied to a plant of another type (e.g., a crop plant) to confirm the delegation of the improved attribute.
[0264] [0264] The attributes that can be improved by the methods disclosed here include any observable characteristic of the plant, including, for example, growth rate, height, mass, color, flavor, odor, variations in the production of one or more compounds by plant (including, for example, metabolites, proteins, drugs, carbohydrates, oils and any other compounds). Plant selection based on genotypic information is also contemplated (for example, including the plant's gene expression pattern in response to bacteria, or identifying the presence of genetic markers, such as those associated with greater nitrogen fixation). Plants can also be selected based on the absence, suppression or inhibition of a certain characteristic or attribute (such as an undesirable characteristic or attribute) as opposed to the presence of a certain characteristic or attribute (such as a desirable characteristic or attribute). Concentrations and application rates of agricultural compositions
[0265] [0265] As mentioned before, the agricultural compositions of the present invention, which may comprise a taught microbe, can be applied to plants in multiple ways. In two aspects in particular, the invention contemplates a furrow treatment or a seed treatment.
[0266] [0266] For seed treatment modalities, the microbes of the invention can be present in the seed in a variety of concentrations. For example, microbes can be found in a seed treatment at a cfu concentration, per seed, of: 1 × 101, 1 × 102, 1 × 103, 1 × 104, 1 × 105, 1 × 106, 1 × 107 , 1 × 108, 1 × 109, 1 × 1010 or more. In particular aspects, compositions for seed treatment comprise between approximately 1 × 104 and 1 × 108 cfu per seed. In other aspects in particular, seed treatment compositions comprise between approximately 1 × 105 and 1 × 107 cfu per seed. In other respects, compositions for seed treatment comprise between approximately 1 × 106 cfu per seed.
[0267] [0267] In the United States, about 10% of the corn area is planted with a seed density above approximately
[0268] [0268] Table 2 below uses various cfu concentrations per seed in a contemplated seed treatment modality (perpendicular lines) and various seed densities in the planting area (1st column: 15K-41K) to calculate the total amount of cfu per area, which would be used in various agricultural scenarios (ie concentration per seed × density of seeds planted per area of seed treatment). Thus, if a seed treatment with 1 × 106 cfu per seed and 30,000 plant seeds per area were used, then the total cfu content per area would be 3 × 1010 (ie 30K * 1 × 106).
[0269] [0269] Table 2: Calculation of total CFU by area for seed treatment modalities Maize population (ie seeds 1.00E + 02 1.00E + 03 1.00E + 04 1.00E + 05 1.00E + 06 1.00E + 07 1.00E + 08 1.00E + 09 by area)
[0270] [0270] For furrow modalities, the microbes of the invention can be applied at a cfu concentration per area of: 1 × 106, 3.20 × 1010, 1.60 × 1011, 3.20 × 1011, 8.0 × 1011 , 1.6 × 1012, 3.20 × 1012 or more. Therefore, in aspects, the liquid compositions in the groove can be applied at a concentration between approximately 1 × 106 and 3 × 1012 cfu per area.
[0271] [0271] In some respects, the groove compositions are contained in a liquid formulation. In the grooved liquid modes, microbes can be present at a cfu concentration per milliliter of: 1 × 101, 1 × 102, 1 × 103, 1 × 104, 1 × 105, 1 × 106, 1 × 107, 1 × 108, 1 × 109, 1 × 1010, 1 × 1011, 1 × 1012, 1 × 1013 or more. In certain respects, liquid compositions in the groove comprise microbes at a concentration between approximately 1 × 106 and 1 × 1011 cfu per milliliter. In other respects, the liquid groove compositions comprise microbes at a concentration between approximately 1 × 107 and 1 × 1010 cfu per milliliter. In other respects, the liquid compositions in the groove comprise microbes at a concentration between approximately 1 × 108 and 1 × 109 cfu per milliliter. In other respects, the liquid compositions in the groove comprise microbes at a concentration of up to approximately 1 × 1013 cfu per milliliter. Examples
[0272] [0272] The examples provided below describe methods of bacterial isolation, analysis of bacteria and plants and improvement of plant attributes. The examples are for illustrative purposes only and should not be construed as limitations in any way. Example 1: Determination of the transcriptomic profile of candidate microbes
[0273] [0273] The determination of the transcriptomic profile of strain CI010 was performed to identify promoters that are active in the presence of environmental nitrogen. The CI010 strain was grown in a defined, nitrogen-free medium, supplemented with 10 mM glutamine. Total RNA was extracted from these cultures (Kit QIAGEN RNeasy) and subjected to RNAseq sequencing using Illumina HiSeq (SeqMatic, Fremont CA). The sequencing readings were mapped against the CI010 genome data using Geneious, and high-expression genes under the control of proximal transcriptional promoters were identified.
[0274] [0274] Tables 3 and 4 list genes and their relative expression levels as measured by RNASeq sequencing of the
[0275] [0275] Several candidate microbial mutants were generated using plasmid pKD46 or a derivative containing a kanamycin resistance marker (Datsenko at al. 2000; PNAS 97 (12): 6640- 6645). The knockout cassettes (silencing) were designed with 250bp homology flanking the target gene and generated via PCR with overlap extension. Candidate microbes were transformed with pKD46, cultured in the presence of arabinose to induce the expression of Lambda-Red machinery, prepared for electroporation and transformed with knockout cassettes to produce candidate mutant strains. Four candidate microbes and a laboratory strain, Klebsiella oxytoca M5A1, were used to generate thirteen candidate mutants of the nitrogen-fixing regulatory genes nifL, glnB, and amtB, as shown in Table 5.
[0276] [0276] Table 5: List of single knockout mutants created through mutagenesis by the Lambda-red Cepa system Oligo-directed mutagenesis with selection using Cas9
[0277] [0277] Oligo-directed mutagenesis was used to target genomic changes to the rpoB gene in E. coli DH10B, and mutants were selected with a CRISPR-Cas system. A mutagenic oligo (ss1283: “G * T * T * G * ATCAGACCGATGTTCGGACCTTCcaagGTTTCGATCGGA
[0278] [0278] Mutants of candidate microbes CI006 and CI010 were generated through mutagenesis by the lambda-red system with selection by CRISPR-Cas. The knockout cassettes contained an endogenous promoter identified through the transcriptional profile (as shown in Tables 3-4) and regions with ~ 250bp homology flanking the deletion 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 under the control of an IPTG-inducible promoter. The Red and Cas9 recombination systems were induced in the resulting transformers, and strains were prepared for electroporation. Knockout cassettes and selection gRNA encoded by a plasmid were subsequently transformed into the competent cells. After plating in a selective medium with antibiotics for the Cas9 plasmid and the gRNA plasmid, 7 of the 10 screened colonies revealed the desired knockout mutation. Example 3: Phenotyping in plant of candidate microbes Colonization of plants by candidate microbes
[0279] [0279] The colonization of host plants desired by a candidate microbe was quantified through short-term plant growth experiments. Corn plants were inoculated with strains that express RFP either from a plasmid or from an RFP expression cassette integrated with Tn5. The plants were grown in a medium with sterile sand and non-sterile peat, and inoculation was performed by introducing 1 mL of cell culture with a pipette directly onto the coleoptile of the emerging plant three days after germination. The plasmids were maintained by watering the plants with a solution containing the appropriate antibiotic. After three weeks, roots of the plant were collected, rinsed three times with sterile water to remove the visible soil and divided into two samples. A root sample was analyzed by fluorescence microscopy to identify patterns of location of candidate microbes. Microscopy was performed on intact, thinner roots of the plant, 10 mm long.
[0280] [0280] A second quantitative method for assessing colonization has been developed. A quantitative PCR assay was performed on whole DNA preparations from the roots of plants inoculated with endophytes. Corn seeds (Dekalb DKC-66-40) were germinated on sand previously autoclaved in a 2.5 inch by 2.5 inch by 10 inch (6.4 cm x 6.4 cm x 25.4 cm) pot ). One day after planting, 1 mL of endophyte culture overnight (SOB medium) was squirted (drenched) right at the point where the seed was located. 1 mL of this culture overnight is almost equivalent to 109 cfu, varying within 3 times of each other, depending on which strain is being used. Each seedling was fertilized 3x with 50 mL of Hoagland's modified solution, supplemented with ammonium nitrate at either 2.5 m or 0.25 mM. Four weeks after planting, root samples were collected for DNA extraction. Soil residues were washed using pressurized water spray. These tissue samples were then homogenized with QIAGEN Tissuelyzer and the DNA was then extracted using the QIAmp DNA Mini Kit (QIAGEN) according to the recommended protocol. The qPCR assay was performed with Stratagene Mx3005P RT-PCR on these DNA extracts using primers that were designed (with the NCAST BLAST Primer) to be specific for a locus in the genome of each endophyte. The presence of copies of the endophyte genome was quantified. To further confirm the identity of the endophytes, the PCR amplification products were sequenced and confirmed to have the correct sequence. The summary of the colonization profile of strain CI006 and CI008 from candidate microbes is presented in Table
[0281] [0281] Biosynthesis of components of the nif in planta pathway was estimated by measuring the transcription of nif genes. Total RNA was obtained from the root tissue of plants inoculated with CI006 (planting methods as previously described). RNA extraction was performed using the RNEasy Mini Kit according to the recommended protocol (QIAGEN). The total RNA of these plant tissues was then analyzed with the kits
[0282] [0282] Highly expressed CM011 genes, classified by transcripts per million kilobases (TPM), were measured in planta under fertilized condition. The promoters that control the expression of some of these highly expressed genes were used as templates for homologous recombination in loci targeted for nitrogen fixation and assimilation. RNA samples were extracted from plants inoculated with CM011, grown in the greenhouse, the rRNA was removed with the Ribo-Zero kit, sequenced using the Illumina Truseq platform and mapped back against the CM011 genome. Highly expressed CM011 genes are listed in Table 8. Table 7: Expression of nifH in planta Strains Relative expression of CI006 transcripts 9.4 CM013 103.25 Table 8 Gross PMS count (Transcripts per million Gene name Location of the gene Direction of readings (kilobases) rpsH CDS 18196 - 18588 reverse 4841.5 27206.4 rplQ CDS 11650 - 12039 reverse 4333 24536.2 rpsJ CDS 25013 - 25324 reverse 3423 24229 rplV CDS 21946 - 22278 reverse 3367.5 22333 rpsN CDS 18622 - 18927 reverse 2792 20150.1 rplN CDS 19820 - 20191 reverse 3317 19691.8 rplF CDS 17649 - 18182 reverse 4504.5 18628.9 rpsD CDS 13095 - 13715 reverse 5091.5 18106.6 rpmF CDS 8326 - 8493 forward 1363.5 17923.8
[0283] [0283] The ability of improved microbes to colonize an inoculated plant is critical to the success of the plant in field conditions. Although the methods described for isolation are designed to select soil microbes that may exhibit an intimate relationship with crop plants such as corn, many strains may not effectively colonize in a number of plant genotypes, environments, soil types or inoculation conditions . Since colonization is a complex process that requires a set of interactions between a microbial strain and the host plant, screening for colonization competence has become a central method for selecting strains with priority for future development. Initially, attempts were made to assess colonization using fluorescent labeling of strains, which was effective, but time-consuming and was not scalable based on individual strains. As the colonization activity cannot be directly improved, it is imperative that the potential candidate products are selected from strains that are natural colonizers.
[0284] [0284] An assay was designed to test robust colonization of wild-type strains on any given host plant using qPCR primers designed to be strain-specific in a community sample. This test is designed to quickly measure the colonization rate of microbes from corn tissue samples. Initial tests using strains evaluated as likely colonizers by fluorescence microscopy and plate-based techniques indicated that a qPCR approach would be quantitative and scalable.
[0285] [0285] A typical test is performed as follows: Plants, mainly varieties of corn and wheat, are grown in a mixture of peat pots in the greenhouse with 6 replicates per strain. Four or five days after planting, a 1 mL drench of cultures in the initial stationary phase of bacteria diluted to OD590 of 0.6-1.0 (approximately 5E + 08 CFU / mL) is pipetted over the emerging coleoptile. The plants are watered with running water only and allowed to grow for four weeks before sampling, whereupon the plants are uprooted and the roots washed thoroughly to remove most peat residue. Clean root samples are removed and homogenized to create a paste of plant cell debris and associated bacterial cells. A high-performance DNA extraction protocol was developed that effectively produced a mixture of plant and bacterial DNA to use as a template for qPCR. Based on spike-in (peak) experiments on bacterial cells, this DNA extraction process provides a quantitative sample of bacterial DNA in relation to the fresh root mass. Each strain is evaluated using strain-specific primers designed using the BLAST Primer (Ye 2012) and compared to background amplification of uninoculated plants. Since some primers exhibit off-target off-target amplification in uninoculated plants, colonization is determined either by the presence of amplification or high amplification of the correct product compared to the background level.
[0286] [0286] This test was used to measure the compatibility of the microbial product between different geographies of the soil. The qualities of the soil in the field and the conditions of the field can have an immense influence on the effect of a microbial product. Soil pH, water holding capacity and competitive microbes are just a few examples of factors in the soil that can affect inoculum survival and colonization capacity. A colonization test was carried out using three different types of agricultural field soil in California as the plant growth medium (Figure 1A). An intermediate density of inoculation was used to approximate the real conditions of agriculture. Within 3 weeks, Cepa 5 colonized all plants with 1E + 06 to 1E + 07 CFU / g FW. After 7 weeks of plant growth, a developed version of Cepa 1 exhibited high rates of colonization (1E + 06 CFU / g FW) in all soil types (Figure 1B).
[0287] [0287] In addition, to assess colonization in the complexity of field conditions, a 1-acre field trial was initiated in San Luis Obispo in June 2015 to assess the impacts and colonization of seven of the wild-type strains in two varieties of field corn. The agronomic design and the execution of the study were carried out by an organization contracted for field research, Pacific Ag Research. For inoculation, the same technique of seed coating in peat culture was used, tested in the experiment of inoculation methods. During the growing season, samples of plants were collected to assess colonization at the root and inside the stem. Samples were collected from three plots in duplicate of each treatment in four and eight weeks after planting, and from all six replicates of each treatment a little from the harvest in 16 weeks. Additional samples were collected from all six treatment plots, in duplicate, inoculated with Cepa 1 and Cepa 2, as well as from untreated controls, in 12 weeks. The numbers of cells per gram fresh weight of washed roots were evaluated as in other colonization assays with qPCR and strain-specific primers. Two strains, Cepa 1 and Cepa 2, showed consistent and generalized root colonization that peaked in 12 weeks and then declined precipitously (Figure 1C). Although it appeared to be present in numbers in an order of magnitude lower than that of Cepa 1, Cepa 2 was found in more consistent numbers from plant to plant. No strain appeared to effectively colonize the interior of the stem. In support of colonization data by qPCR, both strains were successfully isolated again from root samples using 16S plating and sequencing to identify isolates from concordant sequences. Example 5: Microbial genetic improvement
[0288] [0288] Examples of microbial genetic improvement can be summarized in the schematic drawing in Figure 2A. Figure 2A represents the microbial genetic improvement in which the composition of the microbiome can be initially measured and a species of interest is identified. The metabolism of the microbiome can be mapped and linked to genetics. After that, a targeted genetic variation can be introduced using methods that include, among others, conjugation and recombination, chemical mutagenesis, adaptive evolution and gene editing. Derivatives of microbes are used to inoculate cultures. In some examples, cultures with the best phenotypes are selected.
[0289] [0289] As shown in Figure 2A, the composition of the microbiome can be initially measured and a species of interest is identified. Figure 2B represents an enlarged view of the step measurement in the microbiome. The metabolism of the microbiome can be mapped and linked to genetics. Nitrogen metabolism may involve the entry of ammonia (NH4 +) from the rhizosphere to the bacteria's cytosol through the AmtB transporter. Ammonia and L-glutamate (L-Glu) are catalyzed by glutamine synthetase and ATP to glutamine. Glutamine can lead to the formation of biomass (plant growth), and can also inhibit the expression of nif operon. Thereafter, a targeted genetic variation can be introduced using methods that include, but are not limited to, conjugation and recombination, chemical mutagenesis, adaptive evolution and gene editing. Microbial derivatives are used to inoculate cultures. The crops with the best phenotypes are selected.
[0290] [0290] A diversity of nitrogen-fixing bacteria can be found in nature, including in agricultural soils. However, the potential of a microbe to supply sufficient nitrogen to crops that would reduce the use of fertilizers can be restricted by the repression of nitrogenase genes in fertilized soils, as well as low abundance in close association with the roots of the crop. The identification, isolation and genetic improvement of microbes that are closely associated with important commercial crops could disrupt and improve the regulatory networks, linked to nitrogen detection and nitrogen fixation, and unlock significant nitrogen contributions by microbes associated with crops. To this end, nitrogen-fixing microbes were identified that associate with and colonize the root system of corn.
[0291] [0291] Root samples of corn plants grown in agronomically relevant soils were collected, and microbial populations extracted from the rhizosphere and endosphere. Genomic DNA was extracted from these samples, followed by sequencing of amplicon 16S to determine the profile of the community composition. A Kosakonia sacchari microbe (strain PBC6.1) was isolated and classified by sequencing the 16S rRNA and the entire genome. This is an especially interesting nitrogen fixer, capable of colonizing with up to almost 21% abundance of the root-associated microbiota (Figure 4). To assess the sensitivity to the strain's exogenous nitrogen, nitrogen fixation rates in pure culture were measured with the classic acetylene reduction test (ARA) and variable levels of glutamine supplementation. The species exhibited a high level of nitrogen fixation activity in a nitrogen-free medium, although fixed exogenous nitrogen suppressed the expression of nif genes and nitrogenase activity (Cepa PBC6.1, Figure 3C). In addition, when the released ammonia was measured in the PBC6.1 supernatant grown under nitrogen fixing conditions, very little fixed nitrogen release could be detected.
[0292] [0292] The hypothesis was raised that PBC6.1 could significantly contribute to nitrogen fixed in fertilized fields, if the regulation networks that control nitrogen metabolism were rewired to allow optimal expression of nitrogenase and the release of ammonia in the presence fixed nitrogen. There must be sufficient genetic diversity within the PBC6.1 genome to allow for broad phenotypic remodeling without the insertion of transgenes or synthetic regulatory elements. The isolated strain has a genome of at least 5.4 Mbp and a canonical cluster of nitrogen fixing genes. The pathways related to nitrogen metabolism in PBC6.1 are similar to those of the model nitrogen fixing organism, Klebsiella oxytoca m5al.
[0293] [0293] Several nodes of the regulatory gene network have been identified that can increase nitrogen fixation and subsequent transfer to a host plant, especially at high exogenous concentrations of fixed nitrogen (Figure 3A). The nifLA operon directly regulates the rest of the nif cluster through transcriptional activation by NifA and the repression of nitrogen and oxygen dependent NifA by NifL. Disruption of nifL can abolish NifA inhibition and improve expression of nif in the presence of oxygen and exogenous fixed nitrogen. Furthermore, the expression of nifA under the control of a nitrogen-independent promoter may dissociate nitrogenase biosynthesis from regulation by the NtrB / NtrC nitrogen detection complex. The assimilation of nitrogen fixed by the microbe to glutamine by glutamine synthetase (GS) is reversibly regulated by the enzyme of two domains GlenE adenyltransferase (ATase), through the adenylation and deladenylation of GS, to attenuate and restore the activity,
[0294] [0294] Table 9. List of isolated and derived strains used in this work. Prm, promoter sequence derived from the PBC6.1 genome; ΔglnEAR1 and ΔglnEAR2, different truncated versions of the glnE gene that removes the sequence from the adenyl removing domain.
[0295] [0295] Several in vitro tests have been carried out to characterize specific phenotypes of the derived strains. The ARA assay was used to assess the strain's sensitivity to exogenous nitrogen, in which PBC6.1 exhibited repression of nitrogenase activity at high glutamine concentrations. In contrast, most of the derived strains show an unrepressed phenotype with varying levels of acetylene reduction, observed in high glutamine concentrations. The rates of nifA transcription in samples analyzed by qPCR correlated well with the rates of acetylene reduction, supporting the hypothesis that nifL disruption and the insertion of a nitrogen-independent promoter to drive nifA may lead to derepression of the nif cluster. Strains with altered GlnE or AmtB activity revealed markedly increased ammonium excretion rates when compared to wild-type or derived strains without these mutations, illustrating the effect of these genotypes on ammonia assimilation and reabsorption.
[0296] [0296] Two experiments were carried out to study the interaction of PBC6.1 derivatives with corn plants and to quantify the incorporation of nitrogen fixed in plant tissues. Initially, nitrogen fixation rates of microbes were quantified in a greenhouse study using isotopic tracers. Briefly, plants are grown with 15N-labeled fertilizer, and diluted concentrations of 15N in plant tissues indicate contributions of nitrogen fixed by microbes. Corn seedlings were inoculated with selected microbial strains, and the plants were grown until stage V6 of growth. The plants were subsequently deconstructed to allow the measurement of microbial colonization and gene expression, as well as the measurement of 15N / 14N ratios in plant tissues by isotopic ratio mass spectrometry (IRMS). The aerial tissue analysis showed a small, non-significant contribution by PBC6.38 to the nitrogen levels of the plants, and a significant contribution by PBC6.94 (p = 0.011). Approximately 20% of the nitrogen found above crushed maize leaves was produced by PBC6.94, with the remainder coming from the seed, the pot mix or “bottom” fixation by other microbes in the soil. This illustrates that the microbial genetic improvement pipeline of the invention can generate strains capable of significant nitrogen contributions to plants in the presence of nitrogen fertilizer. Microbial transcription within plant tissues was measured, and the expression of the nif gene cluster was observed in derived strains, but not in the wild type strain, showing the importance of nif detoxification for BNF contribution to crops under healthy conditions. fertilized. Root colonization, measured by qPCR, demonstrated that the colonization density is different for each of the strains tested. A 50-fold difference in colonization was observed between PBC6.38 and PBC6.94. This difference may be an indication that PBC6.94 has less ability in the rhizosphere compared to PBC6.38 as a result of high levels of fixation and excretion. Methods Means
[0297] [0297] The minimum medium contains (per liter) 25 g Na2HPO4, 0.1 g CaCL2-2H2O, 3 g KH2PO4, 0.25 g MgSO4 · 7H2O, 1 g NaCl, 2.9 mg FeCl3 , 0.25 mg of Na2MoO4 · 2H2O and 20 g of sucrose. The growth medium is defined as a minimum medium supplemented with 50 ml of 200 mM glutamine per liter. Diazotrophic isolation
[0298] [0298] Corn seedlings were grown from seed (DKC 66-40, DeKalb, IL) for two weeks in a greenhouse environment with controlled temperature from 22 ºC (night) to 26 ºC (day) and exposed to 16 hour cycles of light in soil collected from San Joaquin County, CA. The roots were harvested and washed with sterile deionized water to remove soil residues. The root tissues were homogenized with 2 mm stainless steel spheres in a tissue lysis instrument (TissueLyser II, Qiagenm P / N 85300) for three minutes in configuration 30, and the samples were centrifuged for 1 minute at 13,000 rpm to separate the tissue from bacteria associated with the roots. The supernatants were divided into two fractions, and one was used to characterize the microbiome, by sequencing the amplicon 16S rRNA, and the remaining fraction was diluted and plated in a medium with Nitrogen Free Broth (NfB) supplemented with 1.5% agar. . The plates were incubated at 30ºC for 5-7 days. The colonies that emerged were tested for the presence of the nifH gene by colony PCR with the primers Ueda19f and Ueda406r. Genomic DNA from strains with nifH positive colony PCR was isolated (QIAamp DNA Mini Kit, Cat. No. 51306, QIAGEN, Germany) and sequenced (Illumina MiSeq v3, SeqMatic, Fremont, CA). After assembling and annotating the sequences, isolates that contained clusters of genes for nitrogen fixation were used in the subsequent research. Determination of the isolation seedling microbiome profile
[0299] [0299] Genomic DNA was isolated from root-associated bacteria using the ZR-96 Genomic DNA I kit (Zymo Research, P / N D3011), and 16S rRNA amplicons were generated with nextera bar code primers targeting 799f and 1114r . The amplicon libraries were purified and sequenced with the Illumina MiSeq v3 platform (SeqMatic, Fremont, CA). The readings were classified taxonomically using Kraken using the minikraken database.
[0300] [0300] A modified version of the acetylene reduction assay was used to measure nitrogenase activity in pure culture conditions. Strains were propagated from a single colony in SOB (RPI, P / N S25040-1000) at 30ºC with agitation at 200 RPM for 24 hours and then subcultured 1:25 in growth medium and grown under aerobic conditions for 24 hours ( 30ºC, 200 RPM). 1 ml of the culture in minimal medium was then added to 4 ml of minimal medium supplemented with 0 to 10 mM glutamine in airtight Hungate tubes and grown in anaerobic conditions for 4 hours (30ºC, 200 RPM). 10% of the headspace was removed and then replaced with an equal volume of acetylene per injection, and the incubation continued for 1 hour. Subsequently, 2 mL of the headspace was removed using a gas-proof syringe to quantify ethylene production using an Agilent 6850 gas chromatograph equipped with a flame ionization detector (FID). Ammonium excretion test
[0301] [0301] The excretion of nitrogen fixed in the form of ammonia was measured by batch fermentation in anaerobic bioreactors. Strains were propagated from a single colony at 1 mL / well of SOB on a 96-well DeepWell plate. The plate was incubated at 30ºC with shaking at 200 RPM for 24 hours and then diluted 1:25 in a fresh plate containing 1 mL / well of growth medium. The cells were incubated for 24 hours (30 ° C, 200 RPM) and then diluted 1:10 on a fresh plate containing minimal medium. The plate was transferred to an anaerobic chamber with a gas mixture of> 98.5% nitrogen, 1.2-1.5% hydrogen and oxygen <30 ppM and incubated at 1350 RPM, at room temperature for 66-70 hours. The initial biomass of the culture was compared to the final biomass by the optical density measured at 590 nm. The cells were then separated by centrifugation, and the supernatant from the reactor broth was analyzed for free ammonia using the Megazyme Ammonia Assay kit (P / N K-AMIAR) standardized for biomass at each time. Extraction of root-associated microbiome
[0302] [0302] The roots were gently shaken to remove loose particles, and the root systems were separated and soaked in an RNA stabilization solution (Thermo Fisher P / N AM7021) for 30 minutes. The roots were then briefly rinsed with sterile deionized water. The samples were homogenized using the bead beating technique with ½-inch stainless steel bearings on a tissue lysis instrument (TissueLyser II, Qiagen P / N 85300) in 2 mL of lysis buffer (Qiagen P / N 79216). Genomic DNA extraction was performed with the ZR-96 Quick-gDNA kit (Zymo Research, P / N D3010), and RNA extraction with the RNeasy kit (Qiagen P / N 74104). Root colonization test
[0303] [0303] Four days after planting, 1 mL of a bacterial culture overnight (approximately 109 cfu) was applied to the soil above planted seeds. The seedlings were fertilized three times a week with 25 mL of modified Hoagland's solution, supplemented with 0.5 mM ammonium nitrate. Four weeks after planting, root samples were collected and total genomic DNA (gDNA) was extracted. Root colonization was quantified by qPCR with primers designed to amplify exclusive regions of the genome of the wild type strain or derivatives. The efficiency of the QPCR reaction was measured using a standard curve generated from a known amount of gDNA from the target genome. The data were normalized to copies of the genome per g of fresh mass using tissue mass and extraction volume. For each experiment, colonization numbers were compared to untreated control seedlings.
[0304] [0304] The transcriptional profiles of root-associated microbes were measured in seedlings grown and processed as described in the Root Colonization Assay. Purified RNA was sequenced using the Illumina NextSeq platform (SeqMatic, Fremont, CA). The readings were mapped against the genome of the inoculated strain using the “--very-sensitive-local” bowtie2 parameters and minimum score for alignment of 30. Coverage through the genome was calculated using Samtools. The differential coverage was normalized for expression of housekeeping genes (constitutive) and visualized through the genome, using Circuses, and through the cluster of nif genes using DNAplotlib. In addition, the transcriptional profile in planta was quantified by means of Nanostring analysis. The purified RNA was processed on a Sprint nCounter (Core Diagnostics, Hayward, CA). Greenhouse 15N dilution study
[0305] [0305] A 15N fertilizer dilution experiment was carried out to evaluate optimized activity of strains in planta. A planting medium, containing minimum N of bottom, was prepared with a mixture of vermiculite and washed sand (5 rinses in H2O DI). The sandy mixture was autoclaved for 1 hour at 122 ° C, and approximately 600 g was measured for 40 cubic inch (656 mL) pots, which were saturated with sterile DI H2O DI and allowed to drain 24 hours before planting. Corn seeds (DKC 66-40) were sterilized with 0.625% sodium hypochlorite for 10 minutes, then rinsed five times in sterile distilled water and planted 1 cm deep. The plants were kept under fluorescent lamps for four weeks with a 16-hour-long day at ambient temperatures averaging 22 ° C (night) to 26 ° C (day).
[0306] [0306] Five days after planting, the seedlings were inoculated with 1 ml of a cell suspension, squirted directly on the emerging coleoptile. The inoculum was prepared from 5 ml of cultures overnight in SOB, which were centrifuged and resuspended twice in 5 ml of PBS to remove residual SOB SOB before final dilution to OD 1.0 ( approximately 109 CFU / mL). Control plants were treated with sterile PBS, and each treatment was applied to ten replicates of the plants.
[0307] [0307] The plants were fertilized with 25 mL of fertilizer solution, containing 2% 2 mM KNO3, enriched with 15N, at 5, 9, 14 and 19 days after planting, and the same solution without KNO3 at 7, 12, 16 and 18 days after planting. The fertilizer solution contained (per liter) CaCl2 3 mmol, KH2PO4 0.5 mmol, MgSO4 2 mmol, FeSO4 17.9 µmol, H3BO3 2.86 mg, MnCl2 • 4H2O 1.81 mg, ZnSO4 • 7H2O 0.22 mg, CuSO4 • 5H2O 51 µg, Na2MoO4 • 2H2O 0.12 mg and NiCl2 0.14 nmol. All pots were watered with sterile H2O DI necessary to maintain consistent soil moisture without runoff.
[0308] [0308] In four weeks, the plants were harvested and separated at the lowest node in samples for extraction of gDNA and RNA from the root and aerial tissue by IRMS. The aerial tissues were cleaned as necessary to remove the sand, placed whole in paper bags and dried for at least 72 hours at 60 ° C. Once completely dry, the total aerial tissue was homogenized by bead beating and samples of 5-7 mg were analyzed by δ15N isotopic ratio mass spectrometry (IRMS) by MBL Stable Isotope Laboratory (The Ecosystems Center, Woods Hole, BAD). The percentage NDFA was calculated using the following formula:% of NDFA = (δ15N of average UTC - δ15N of sample) / (δ15N of average UTC) x 100. Example 7: Methods and assays for detecting non-intergeneric modified microbes
[0309] [0309] The present invention teaches primers, probes and assays that are useful for detecting the microbes used in the various Examples mentioned above. The assays are able to detect sequences with “junctions” of unnatural nucleotides in non-intergeneric derived / mutant microbes. These unnatural nucleotide junctions can be used as a type of diagnosis that is indicative of the presence of a specific genetic change in a microbe.
[0310] [0310] The present techniques are able to detect these unnatural nucleotide junctions via the use of specialized quantitative PCR methods, including primers and probes designed exclusively. Probes can bind to sequences with unnatural nucleotide junctions. In other words, it is possible to use DNA probes for specific sequences that consist of oligonucleotides that are marked with a fluorescent reporter, which allows detection only after hybridization of the probe to its complementary sequence. Quantitative methods are able to ensure that only the junction of unnatural nucleotides will be amplified with the taught primers and that, consequently, it can be detected via a non-specific dye, or via the use of a specific hybridization probe. Another aspect of the method is the choice of primers such that the primers flank either side of a splice sequence, such that, if an amplification reaction occurs, then said splice sequence is present.
[0311] [0311] Consequently, genomic DNA can be extracted from samples and used to quantify the presence of microbes of the invention using qPCR. The primers used in the qPCR reaction can be primers designed by Primer Blast (https://www.ncbi.nlm.nih.gov/tools/iniciador-blast/) to amplify regions unique to the wild-type genome or regions unique to mutant strains modified non-intergenerational. The qPCR reaction can be performed with the SYBR GreenER qPCR SuperMix Universal kit (Thermo Fisher, P / N 11762100), using only forward and reverse primers for amplification; alternatively, the Kapa Sonda Force kit (Kapa Biosystems, P / N KK4301) can be used with amplification primers and a TaqMan probe containing a FAM dye marking on the 5 'end, an internal ZEN quencher and a narrow groove ligand and a fluorescent quencher at the 3 'end (Integrated DNA Technologies). Table 10: Microbes unmodified intergeneric strain name genotype sequence CI006 16S rDNA - contig 5 ttgaagagtt tgatcatggc tcagattgaa cgctggcggc aggcctaaca catgcaagtc gaacggtagc acagagagct tgctctcggg tgacgagtgg cggacgggtg agtaatgtct gggaaactgc ctgatggagg gggataacta ctggaaacgg tagctaatac cgcataacgt cgcaagacca aagaggggga ccttcgggcc tcttgccatc agatgtgccc agatgggatt agctagtagg tggggtaacg gctcacctag gcgacgatcc ctagctggtc tgagaggatg accagccaca ctggaactga gacacggtcc agactcctac gggaggcagc agtggggaat attgcacaat gggcgcaagc ctgatgcagc catgccgcgt gtgtgaagaa ggccttcggg ttgtaaagca ctttcagcgg ggaggaaggg agtaaggtta ataaccttat tcattgacgt tacccgcaga agaagcaccg gctaactccg tgccagcagc cgcggtaata cggagggtgc aagcgttaat cggaattact gggcgtaaag cgcacgcagg cggtctgtca agtcggatgt gaaatccccg ggctcaacct gggaactgca tccgaaactg gcaggcttga gtctcgtaga gggaggtaga attccaggtg tagcggtgaa atgcgtagag atctggagga ataccggtgg cgaaggcggc ctcctggacg aagactgacg ctcaggtgcg aaagcgtggg gagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgtcta tttggaggtt gtgcccttga ggcgtggctt ccggagctaa cgcgttaaat agaccgcctg gggagtacgg ccgcaaggtt aaaactcaaa tgaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgat gcaacgcgaa gaaccttacc tggtcttgac atccacagaa ctttccagag atggattggt gccttcggga actgtgagac aggtgctgca tggctgtcgt cagctcgtgt tgtgaaatgt tgggttaagt cccgcaacga gcgcaaccct tatcctttgt tgccagcggt ccggccggga actcaaagga gactgccagt gataaactgg aggaaggtgg ggatgacgtc aagtcatcat ggcccttacg accagggcta cacacgtgct acaatggcgc atacaaagag aagcgacctc gcgagagtaa gcggacctca taaagtgcgt cgtagtccgg attggagtct gcaactcgac tccatgaagt cggaatcgct agtaatcgtg gatcagaatg ccacggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccatg ggagtgggtt gcaaaagaag taggtagctt aaccttcggg agggcgctta ccactttgtg attcatgact ggggtgaagt cgtaacaagg taaccgtagg ggaacctgcg gttggatcac ctcctt CI006 16S rDNA - contig 8 ttgaagagtt tgatcatggc tcagattgaa cgctggcggc aggcctaaca catgcaagtc gaacggtagc acagagagct tgctctcggg tgacgagtgg cggacgggtg agtaatgtct gggaaactgc ctgatggagg gggataacta ctggaaacg g tagctaatac cgcataacgt cgcaagacca aagaggggga ccttcgggcc tcttgccatc agatgtgccc agatgggatt agctagtagg tggggtaacg gctcacctag gcgacgatcc ctagctggtc tgagaggatg accagccaca ctggaactga gacacggtcc agactcctac gggaggcagc agtggggaat attgcacaat gggcgcaagc ctgatgcagc catgccgcgt gtgtgaagaa ggccttcggg ttgtaaagca ctttcagcgg ggaggaaggn antanggtta ataacctgtg ttnattgacg ttacccgcag aagaagcacc ggctaactcc gtgccagcag ccgcggtaat acggagggtg caagcgttaa tcggaattac tgggcgtaaa gcgcacgcag gcggtctgtc aagtcggatg tgaaatcccc gggctcaacc tgggaactgc atccgaaact ggcaggcttg agtctcgtag
[0312] [0312] The 16S rRNA gene sequences used to study bacterial phylogeny and taxonomy have been the most common housekeeping genetic marker to date - and for several reasons. These reasons include (i) their presence in almost all bacteria, often existing as a family of multiple genes, or operons; (ii) the function of the 16S rRNA gene has not changed over time, suggesting that random changes in the sequence are a more accurate measure of time (evolution); and (iii) the 16S rRNA gene (1,500 bp) is large enough for computing purposes.
[0313] [0313] The nucleotide sequence of the 16S rRNA gene of Klebsiella variicola and Kosakonia sacchari was determined by PCR analysis. The primers used are universal 16S primers, with the following nucleotide sequence: 16S Front Primer 27f (AGAGTTTGATCMTGGCTCAG) Reverse Primer 165 1492r (GGTTACCTTGTTACGACTT).
[0314] [0314] The products resulting from the PCR were sequenced, and the resulting sequence was compared against the National Center for Biotechnology Information (NCBI) database for species identification during the original isolation. In any subsequent production of the microbe, the same analysis was carried out by PCR to ensure a pure microbe verified to be free from contamination.
[0315] [0315] Two parental strains have been identified by this method. The first scientific name is Klebsiella variicola. The species identity was determined by sequencing the organism's 16S rRNA, multiple times, using BLAST in the NCBI database to verify the genus and species. In addition, the entire genome of the organism was sequenced and the extracted 16S rRNA (from the genome sequence) was subjected to BLAST. This sequence was aligned to the 16 rRNA sequences previously determined to confirm that the organism was isolated for a pure culture.
[0316] [0316] The scientific name of the second organism is Kosakonia sacchari. The species identity was determined by sequencing the organism's 16S rRNA, multiple times, using BLAST in the NCBI database to verify the genus and species. In addition, the entire genome of the organism was sequenced and the extracted 16S rRNA (from the genome sequence) was subjected to BLAST. This sequence was aligned to the 16 rRNA sequences previously determined to confirm that the organism was isolated for a pure culture. Example 9: Remodeled strains with greater colonization or nitrogen fixation activity
[0317] [0317] Several mutant strains have been developed to investigate new modifications that could increase the ability to colonize, nitrogen-fixing activity or nitrogen excretion. These strains are summarized in Table 11. The promoter and gene sequences are listed in Table 12. Remodeled strains were evaluated for wild type, or parent strains, to confirm the altered expression of the modified gene ( Figures 5, 12 and 13), for nitrogen reduction activity (Figures 6, 18, 20, 22A, 22B, 24, 26A, 26B, 34 and 36), nitrogen excretion (Figures 14, 15, 16, 17, 19, 21, 23A, 23B, 25, 27A, 27B, 33 and 35), in vitro colonization
[0318] [0318] Table 12: Promoter and gene sequences. The promoter sequences are listed in lower case and the gene sequences in upper case. In the case of deletion mutations, the sequence before deletion is listed in lowercase letters and the sequence after deletion is listed in uppercase letters
[0319] [0319] Method: Ammonium excretion test
[0320] [0320] To confirm the absence of toxicity to humans or to Klebsiella variicola and Kosakonia sacchari. Mammals, a toxicity study was conducted with these organisms. The contractor was Smithers Avanza Toxicology Services, which has extensive experience in toxicology studies.
[0321] [0321] The aim of this study was to confirm the toxicity of bacterial isolates in mice after a single subcutaneous injection. CD-1 mice were used to maximize the number of bacterial isolates per kg of body weight. The subcutaneous route was selected because it is the appropriate route for the inoculation of bacterial isolates in animals, and the one that most simulates the most likely route of human exposure to microbes.
[0322] [0322] Treatments with bacterial isolates of Klebsiella variicola or Kosakonia sacchari did not affect the mortality of mice, all animals survived until the planned closure at the end of the study period. There were no abnormalities in clinical observations of any of the animals in the Klebsiella variicola and Kosakonia sacchari treatment groups throughout the study. In addition, no abnormalities were noted during observations next to the cages. In addition, there were no abnormalities with dermal observations for the Klebsiella variicola and Kosakonia sacchari treatment groups. The body temperatures of the mice were normal in all groups, with average temperatures ranging from 36.66 ° C to 37.62ºC. There was a slight loss of body weight for mice in the treatment groups with Klebsiella variicola and Kosakonia sacchari, but this was within the normal range for mice in that study and historical data for that type of study. The results of this study confirm that bacterial isolates of Klebsiella variicola and Kosakonia sacchari do not pose a risk of toxicity or pathogenicity to humans or other mammals.
[0323] [0323] The aim of this study was to determine the toxicity of bacterial isolates in mice after a single subcutaneous injection. CD-1 mice were used to maximize the number of bacterial isolates per kg of body weight. The subcutaneous route was selected because it is the appropriate route for inoculation of bacterial isolates in animals.
[0324] [0324] Forty-five female CD-1 mice approximately 8 weeks old were received from Charles River Breeding Labs on June 8, 2017. The animals were housed in groups (5 animals per cage) in polycarbonate cages containing hardwood lining. The animals were fed the Certified Global Teklad Laboratory Diet 2018 diet (pellets) at will and received drinking water through water bottles. Environmental controls in the animal room were set up to maintain a temperature of 20-26ºC, a relative humidity of 30-70%, at least 10 air changes per hour and a cycle of 12 hours in the light / 12 hours in the dark.
[0325] [0325] Forty mice were randomized into eight groups consisting of five mice each. Extra animals were taken to training / stocking colony. The animals were treated as indicated in Table 15. Table 15: Design of the study Volume of the item Females Dose of the item under test Group Treatment ID gender / species under test (isolated / animal) N Number of animals (mL / animal) 1 PB137 Klebsiella variicola> 108 0.1 5 21994-21998 8 2 PB728 Klebsiella variicola> 10 0.1 5 21999-22003 8 3 PB006 Kosakonia sacchari> 10 0.1 5 22004-22008 4 PB063 Rahrella aquatiiis> 108 0.1 5 22009- 22013 8 5 PB019 Rahnella aquatiiis> 10 0.1 5 22014-22018 6 PB747 Rahnella aquiatiiis> 108 0.1 5 22019-22023 8 7 PB910 Kluyvera intermedia> 10 0.1 5 22024-22028 8 PB1116 Enterobacter tabaci> 108 0, 1 5 22029-22033 Table 15: strains used
[0326] [0326] The items under test were provided by the Sponsor in ready-to-serve form. All animals received a single dose through subcutaneous injection in the pre-scapular region on Study Day (SD) 1.
[0327] [0327] Physical examinations were performed daily and observations next to the cage, twice a day. Dermal observations were also performed daily according to Table 16. Additional observations are listed in Tables 17-27. Table 16: Dermal observations Score Grade Edema Erythema 0 None Absence of swelling Normal color 1 Minimal Slight swelling (almost imperceptible) Light pink (almost imperceptible) 2 Slight definite swelling (distinct) Bright pink / light red 3 Moderate Defined swelling (high) Red brilliant 4 Severe pronounced swelling Dark red
[0328] [0328] Body weights were recorded before dose administration (SD 1) and before closure (SD 8). Body temperature was measured before closure (SD 8). After the final data collection in SD 8, the animals were sacrificed by CO2 inhalation followed by cervical dislocation and discarded without necropsy.
[0329] [0329] Treatment with any of the bacterial isolates did not affect mortality. All animals survived until the planned closure.
[0330] [0330] Clinical observations were noted during physical examinations. No abnormalities were observed in any of the animals throughout the study.
[0331] [0331] No abnormalities were noted during observations next to the cages.
[0332] [0332] Observations were made in the Draize skin test. One animal in Group 6 exhibited minimal edema and erythema starting at SD 7 until the end of the observation period (SD 8). One animal in Group 7 exhibited erythema starting at SD 5 until the end of the observation period (SD 8). In Group 8, minimal or mild edema was observed in two animals from one to four days. Mild erythema was observed in Animal 22029 (8f) in SD 7 through SD 8, and moderate erythema was observed in Animal 22032 (8f) in SD 5 and 6, which decreased to minimal erythema in SD 7 and 8.
[0333] [0333] Body weights and changes in body weight were noted. Groups 1, 2, 3, 5, 6 and 8 suffered minimal weight loss throughout the study, with an average percentage change ranging from - 1.91% to -6.87%. Groups 4 and 7 achieved minimum weight gain throughout the study, with an average percentage change of 0.31% and 2.73%, respectively.
[0334] [0334] Body temperatures have been tracked. Body temperatures were normal for all groups, with average temperatures ranging from 36.66ºC to 37.62ºC. Example 10: Field trial
[0335] [0335] A field trial can be conducted to assess the effectiveness of Pivot strains on maize growth and productivity under varying nitrogen schedules. Materials and methods
[0336] [0336] Experimental design: Design of Divided Plots with Rates of N as Main plots and Treatments as Subplots.
[0337] [0337] Locations: Three geographically different locations in Argentina.
[0338] [0338] Data: Provide within 1 week of planting, geographic coordinates with reference to TAG, Field Plot, Treatment Locations and Decoder board (Decoficador).
[0339] [0339] Treatments: 5 Main Installments -100%, 100% - 25 pounds, 100% - 50 pounds, 100% - 75 pounds, 0%
[0340] [0340] 14 Seed Treatment Subplots (treated seed supplied by AGT)
[0341] [0341] Nitrogen time: Pre-planting urea or other approved best management practices.
[0342] [0342] Replications: 6
[0343] [0343] Plots: 420 per location
[0344] [0344] Hybrid: Provided by AgriThority, one for northern locations and one for southern locations.
[0345] [0345] NO biological agents applied to the seed.
[0346] [0346] Minimum plot size: 4 rows 30 'long (~ 3 acres in total)
[0347] [0347] Observations: All observations (unless otherwise stated) taken from 2 rows from the center of the plot. All destructive sampling to be removed from outside queues.
[0348] [0348] Sample maintenance: Keep samples of treated seeds refrigerated until use. Remove samples from the refrigerator 1.5 to 2 hours before required for application.
[0349] [0349] Fertilizer application: Employee providing the recommended fertilizer rate and obtaining Pivot Bio's agreement before application.
[0350] [0350] Local agriculture: Practice ● Seed: Commercial corn (supplied) applied with commercial seed treatment without biological agent. Sowing rate - local practice. ● Planting date: local practice. ● Standard local production practices - weed, insect management, etc. ● Standard handling practices must be followed with the exception of fungicide applications. Fungicide applications require customer consultation. ● Record all agricultural practices followed for cultivating the crop.
[0351] [0351] Soil characterization ● Soil texture ● Soil fertility analysis ○ To determine the level of nitrogen fertilizer, take samples of pre-planting soil from each location to ensure that 0-12 ”and potentially 12” - 24 ”Of Nitrate Nitrogen is less than 50 pounds / acre. Complete a soil pattern plus Nitrate Nitrogen, Ammonium Nitrogen, Total Nitrogen, Organic Matter and CEC. Send directly to the laboratory. ○ Acceptable responsibility for nitrogen. High yield delta between 0 and 100% nitrogen fertilization. ○ pH, CEC, total K and P. Standard procedure for soil sampling, p. eg, samples of intact soil (soil colors) 0 cm to 30 cm and 30 cm - 60 cm ○ Before planting and fertilization, collect a Pivot Serum Sample - 2 mL of soil sample with 0 to 6-12 inches of
[0352] [0352] Reviews
[0353] [0353] Monitoring scheme ● AgriThority professionals visit all studies in stage V3-V4 to assess the response at the beginning of the season to treatments and during reproductive growth to monitor maturity ● Joint visit with Pivot, planned as the conditions justify and the customer wishes.
[0354] [0354] b. Climatic information Climatic data from planting to harvest, consisting of: ● Daily maximum and minimum temperatures, ● Soil temperature at sowing ● Daily rain plus irrigation (if applied) ● Unusual weather events, p. excessive rain, wind, cold, heat
[0355] [0355] c. Analysis and data reporting ● AgriThority provides the mid-season report to Pivot on August 1, functional design on November 30 and final report on December 31 ● Additional reports can be negotiated ● Employee will provide replication data ( raw data) for AgriThority upon completion of the study in Excel format (provided) on November 15, 2016 including the planting layout map. ● The report must include all relevant information, including those noted below: ● Soil texture and test report
[0356] [0356] Although preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such modalities are provided by way of example only.
Numerous variations, changes and substitutions will now occur to those skilled in the art without deviating from the invention.
It is to be understood that several alternatives to the modalities of the invention described herein can be employed in the practice of the invention.
The intention is that the following claims define the scope of the invention and that the methods and structures within the scope of those claims and their equivalents are covered by them.

权利要求:
Claims (22)
[1]
1. Bacterial composition, characterized by the fact that it comprises: at least one genetically modified bacterial strain that fixes atmospheric nitrogen in an agricultural system, in which the bacterial strain comprises a modification in one or more genes selected from the group consisting of bcsll, bcsIII, yjbE, fhaB, pehA, glgA, otsB, treZ and cysZ.
[2]
2. Bacterial composition, characterized by the fact that it comprises: a bacterial strain that promotes plant growth, in which said strain has been remodeled to increase the colonization of said bacterial strain that promotes plant growth in a plant.
[3]
3. Bacterial composition according to claim 2, characterized by the fact that said colonization of said bacterial strain that promotes plant growth occurs in a root of said plant.
[4]
4. Bacterial composition according to claim 2, characterized by the fact that said bacterial strain comprises a genetic modification in an enzyme or pathway involved in the production of exopolysaccharides.
[5]
5. Bacterial composition according to claim 0, characterized by the fact that said genetic modification is in a gene selected from the group consisting of bcsII, bcsIII and yjbE.
[6]
6. Bacterial composition according to claim 2, characterized by the fact that said bacterial strain comprises a genetic modification in an enzyme or pathway involved in the production of a filamentous hemagglutinin.
[7]
7. Bacterial composition according to claim 0, characterized by the fact that said genetic modification is in a fhaB gene.
[8]
8. Bacterial composition according to claim 2, characterized by the fact that said bacterial strain comprises a genetic modification in an enzyme or pathway involved in the production of an endo-polygalaturonase.
[9]
9. Bacterial composition according to claim 0, characterized by the fact that said genetic modification is in a pehA gene.
[10]
10. Bacterial composition according to claim 2, characterized by the fact that said bacterial strain comprises a genetic modification in an enzyme or pathway involved in the production of trehalose.
[11]
11. Bacterial composition according to claim 0, characterized by the fact that said genetic modification is in a gene selected from the group consisting of: otsB and treZ.
[12]
12. Bacterial composition according to claim 2, characterized by the fact that said bacterial composition is formulated for application to a field.
[13]
13. Bacterial composition according to any one of claims 2-12, characterized by the fact that said plant growth promoting bacteria provides a nutrient to said plant.
[14]
14. Bacterial composition according to any one of claims 2-13, characterized by the fact that said plant growth-promoting bacteria supplies fixed nitrogen to said plant.
[15]
15. Bacterial composition according to any of claims 2-14, characterized by the fact that said plant is selected from the group consisting of corn, barley, wheat, sorghum, soy, and rice.
[16]
16. Method for increasing the colonization of a bacterial strain that promotes plant growth in a plant, characterized by the fact that said method comprises: introducing in the said bacterial strain that promotes plant growth a genetic modification in a gene involved in a selected pathway a from the group consisting of: production of exopolysaccharides, production of endo-polygalaturonase and production of trehalose.
[17]
17. Method for increasing the nitrogen available to a plant, characterized by the fact that said method comprises: applying a plurality of remodeled bacteria to a plant, in which said plurality of remodeled bacteria exhibit decreased expression of glgA.
[18]
18. Method according to claim 17, characterized by the fact that said remodeled bacteria exhibit less nitrogen assimilation within said remodeled bacteria when compared to nitrogen assimilation of the same amount of non-remodeled bacteria of the same species that said remodeled bacteria.
[19]
19. Method for increasing the nitrogen available to a plant, characterized by the fact that said method comprises: applying a plurality of remodeled bacteria to a plant, wherein said plurality of remodeled bacteria contains an increased amount of at least one cofactor of nitrogenase within said remodeled bacteria.
[20]
20. Method according to claim 19, characterized by the fact that said cofactor is sulfur.
[21]
21. Method according to claim 19, characterized by the fact that said remodeled bacteria increased the expression of cysZ.
[22]
22. Method according to claim 19, characterized in that said remodeled bacteria increased the expression of a sulfur transporter.

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法律状态:
2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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