Bacillus velezensis compositions and methods thereof

Abstract

The present invention generally relates to strains of Bacillus velezensis bacteria beneficial to plants, and compositions and inoculums comprising the same. The invention is also directed to plant seeds coated with the compositions and inoculums, and methods for inducing rhizosheath and rhizonest structures on plant roots using the same.

Description

TITLE OF THE INVENTIONBACILLUS VELE / ENSIS COMPOSITIONS AND METHODS THEREOF REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States provisional application No.63 / 748,515, filed January 23, 2025, herein incorporated by reference in its entirety.INCORPORATION OF SEQUENCE LISTING

[0002] The sequence listing contained in the file named “UCTl.P_002WO.xml”, which is 6 kilobytes (measured in MS-Windows®) and created on January 20, 2026, is filed herewith by electronic submission and incorporated herein by reference.FIELD OF THE INVENTION

[0003] The present disclosure generally relates to strains of Bacillus velezensis bacteria beneficial to plants, and compositions comprising the same. The present disclosure is also directed to plant seeds coated with the compositions, and methods for inducing rhizosheath and rhizonest structures on plant roots using the same.BACKGROUND OF THE INVENTION

[0004] Current challenges to agricultural production include frequent droughts, heat waves, and soil nutrient deficiencies. Plant root systems are the primary absorption point for soil nutrients. In higher plants, the root hairs may be immersed in a secreted mucilage on the root surface that together binds soil particles, a structure known as the rhizosheath. The rhizosheath coats plant roots, promotes root-soil contact, enhances water and fertilizer uptake, protects plants against drought, and can also provide a sugar-rich, anaerobic habitat for beneficial microbes. Thus, inducing the growth and development of rhizosheaths on plant roots represents a viable strategy to overcome many of the challenges currently faced in agricultural production. However, no de novo methods are currently known to initiate and promote the development of rhizosheath structures; and initiation of the rhizosheath is primarily regulated by the plant's genome.

[0005] There is a constant need for developing new plant health-enhancing and / or plant growth stimulating compositions. Methods and compositions for inducing rhizosheath structures on root hairs de novo would be advantageous toward increasing the crop yield, reducing the use and amounts of fertilizers required, and providing other benefits for agricultural and horticultural communities.1US_ACTI VE\132140149W-1SUMMARY OF THE INVENTION

[0006] Compositions comprising microbiological strains and cultures are provided herein. Certain strains, inoculums, cultures, and compositions described herein are useful for inducing plant root structures and promoting increased nutrient use efficiency, for example, of various crop plants including corn, wheat, and other cereal plants. Plant growth promoting compositions, and methods of inducing rhizosheath and rhizonest structures on plant roots, and for increasing drought tolerance, nutrient efficiency, and yield, are also provided. Also provided are methods for the use of such compositions as plant growth promoting agents in combination with other agriculturally effective compounds.

[0007] In one aspect, the present disclosure provides a method for inducing a rhizosheath, wherein the method comprises applying a composition comprising an effective amount of Bacillus velezensis strain 4E2b (IDAC Accession No. 100125-01) to a plant, a plant seed, a plant growth medium, or an area surrounding a plant or plant seed. In some embodiments, the method further comprises inducing a rhizonest. In other embodiments, the method comprises increased nutrient use efficiency as compared to a control plant lacking said application. In further embodiments, the plant comprises increased drought resistance as compared to a control plant lacking said application. In certain embodiments, the composition is applied to the plant under drought conditions; or under nitrogen limited conditions. In specific embodiments, the plant’s root hairs or rhizosheath develops asymmetrically. In still other embodiments, applying the composition comprises coating the plant seed with the composition; or applying the composition comprises a foliar spray. In certain embodiments, the composition is applied at a concentration in excess of 101CFU / seed (colony forming units per seed) or 103CFU / plant (colony forming units per plant). In some embodiments, the composition is applied to a corn plant, a wheat plant, a barley plant, a sorghum plant a rice plant, an oat plant, or a turfgrass plant.

[0008] In another aspect, provided herein are compositions for application to plants, plant seeds, or a plant growth medium, wherein the composition comprises an effective amount of Bacillus velezensis strain 4E2b (IDAC Accession No. 100125-01); and at least one species of nitrogen fixing bacteria; or at least one plant growth regulator; in a synergistically effective amount. Tn some embodiments, the nitrogen fixing bacteria is a Klebsiella bacterium, a Stenotrophomonas bacterium, a Pantoea bacterium, a Bacillus bacterium, a Paenibacillus bacterium, an Azotobacter bacterium, a Kosakonia bacterium, an Herbaspirillum bacterium, a Gluconacetobacter bacterium, an Azospirillum bacterium, a Methylobacterium, a 2US_ACTI VE\132140149W-1Curlobaclerium bacterium, a member of the Rhizobiaceae family, or a combination of any thereof. In specific embodiments, the nitrogen fixing bacterium comprises Klebsiella variicola. In other embodiments, the plant growth regulator comprises a plant hormone, an herbicide, or a bioactive metabolite. In further embodiments, the plant growth regulator comprises indole-3-acetic acid (IAA), 4- chloroindole-3-acetic acid (4-Me-IAA), 4-methylindole-3 -acetic acid (4-Cl-IAA), a-Naphthalene acetic acid, 1 -naphthaleneacetic acid, DICAMB A, 2, 4-dichlorophenoxyacetic acid (2,4-D), 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB), 4-chloro-2-methylphenoxyacetic acid (MCPA), diflufenzopyr, halauxifen, aminopyralid, clopyralid, fluroxypyr, triclopyr, quinclorac, diflufenzopyr, picloram, cthrcl or any combination of any thereof. In some embodiments, the composition of the present disclosure are prepared as a formulation selected from the group consisting of an emulsion, a colloid, a dust, a granule, a pellet, a powder, a spray, an emulsion, and a solution.

[0009] In yet another aspect of the present disclosure, composition for application to plants, plant seeds, or a plant growth medium are provided, wherein the composition comprises a mutant of Bacillus velezensis strain 4E2b (IDAC Accession No. 100125-01), wherein the mutant is a drought-tolerant mutant, a cold-tolerant mutant, a biofilm / mucilage-secreting mutant, a fungicide tolerant mutant, an herbicide tolerant mutant, a plant host compatibility mutant, a plant microbiome compatible mutant, a soil microbiome compatible mutant, an auxin mutant, or a mutant with a combination of mutations to impart drought tolerance, cold tolerance, biofilm / mucilage-secreting ability, fungicide tolerance, herbicide tolerance, plant host compatibility, plant microbiome compatibility, soil microbiome compatibility, auxin activity and retains the ability to induce a rhizosheath or rhizonest or asymmetry on a plant root. In some embodiments, the mutant is Bacillus velezensis strain 4E2c (IDAC Accession No. 150125-01). In other embodiments, the composition further comprises at least one species of nitrogen fixing bacteria or at least one plant growth regulator; in a synergistically effective amount. Compositions of the present disclosure may also comprise an agriculturally acceptable carrier. Plant seeds coated with the compositions described herein are also provided. In certain embodiments, the coated seed is a monocotyledon seed or a dicotyledon seed.

[0010] Another further aspect of the disclosure provides a method for stimulating plant growth, wherein the method comprises applying a composition described herein to a plant, a plant seed, a plant growth medium, or an area surrounding a plant or plant seed. In some embodiments, the method comprises applying the composition to the plant growth medium prior to, concurrently3US_ACTI VE\132140149W-1with, or after planting of seeds, seedlings, cuttings, bulbs, or plants in the plant growth medium. In other embodiments, the method comprises applying the composition to plant leaves, roots, or stems. In specific embodiments, the method comprises applying the composition to plant seeds. In further embodiments, the plant exhibits an improved agronomic characteristic selected from the group consisting of: increased average grain yield and shoot biomass, wherein the average grain yield or shoot biomass is increased by at least about 1%, 3%, 5%, 10%, 15%, 20%, or 25% as compared to the height of a control plant lacking application of the composition. In still further embodiments, the plant exhibits increased root biomass by at least about 5%, 10%, 15%, 20%, or 25%; increased average primary root length by at least about 5%, 10%, 15%, 20%, or 25%; increased average root hair density by at least about 5%, 10%, 15%, 20%, or 25%; or increased average root hair length by at least about 5%, 10%, 15%, 20%, or 25%, as compared to the height of a control plant lacking application of the composition.

[0011] In yet another further aspect, the disclosure provides a method for increasing drought tolerance in a plant, wherein said method comprises applying a composition comprising an effective amount of Bacillus velezensis strain 4E2b (ID AC Accession No. 100125-01) to a plant, a plant seed, a plant growth medium, or an area surrounding a plant or plant seed. In some embodiments, applying the composition comprises coating the plant seed with the composition; or applying the composition comprises a foliar spray. In further embodiments, the plant exhibits increased yield by at least about 5%, 10%, 15%, 20%, or 25% as compared to the height of a control plant lacking application of the composition.

[0012] These and other objects and features of the invention will become more fully apparent from the following detailed description of the invention and the claims.BRIEF DESCRIPTION OF THE SEQUENCES

[0013] SEQ ID NO:1 is the partial 16s rRNA nucleic acid sequence of Bacillus velezensis strain 4E2b.

[0014] SEQ ID NO:2 is the partial 16s rRNA nucleic acid sequence of Bacillus velezensis strain 4E2c.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 : Shows an exemplary a rhizosheath on plant root surfaces comprising primarily of root hairs and mucilage.4US_ACTI VE\132140149W-1

[0016] FIG. 2: Shows images of Inbred Corn Lince W22 and Balsas leosinle roots under high nitrogen (Panels A, C, E, and G) and low nitrogen (Panels B, D, F, and H). The images show the absence of obvious rhizosheaths, rhizonests, or asymmetric rhizosheaths (adapted from Gaudin et al., 2011)

[0017] FIG. 3: Shows that Bv4E2b and its derivatives are susceptible to 17 of 18 antibiotics tested.

[0018] FIG. 4: Bv4E2b colonies secrete a sugary, biofilm-mucilage substance under water stress (30% PEG-6000) and nutrient limitation, including when plated on nitrogen-free lant growth media (Panels G). Bv4E2b colonies grown under no stress (LB media: Panel A); LGBM medium (LB plus 1% [vol / vol] glycerol and 0.1 mM MnSO4) medium, which is used to induce biofilm formation (Panel B); LB medium supplemented with 30% PEG6000 to induce water limitation after culturing for 48 h (Panel C) and 72 h (Panel D); M9 minimal medium after culturing for 48 h (Panel E) and 96 h (Panel F); Bv4E2b colonies grown on low nitrogen plant growth medium, Phytagel MS531 (same media as plants) after culturing for 72 h (Panel G).

[0019] FIG. 5: Shows that application of Bv4E2b onto corn plants stimulates rhizosheath development (Panels A-C).

[0020] FIG. 6: Shows that application of Bv4E2b onto water-limited com plants stimulates rhizosheaths only on one side of the roots to create a theoretical osmotic gradient to drive water / nutrient uptake (Panels A-K).

[0021] FIG. 7 : Shows that application of Bv4E2b onto com plants results in the development of proto-nodule-like spheres (rhizonests) constructed from intertwined root hairs and mucilage that provide a habitat for beneficial microbes (Panels A-H).

[0022] FIG. 8: Shows that application of Bv4E2b constructs rhizonests by bending and intertwining root hairs to create a mesh for mucilage (Panels A-C).

[0023] FIG. 9: Shows that Bacillus velezensis (Bv4E2b) and Klebsiella variicola co-inoculation provide synergistic effects on plant traits (Panels A-D).

[0024] FIG. 10: Shows that co-application of Bv4E2b and auxin stimulating compounds onto corn plants enlarges rhizonests (which are habitats for beneficial microbes including nitrogen fixing bacteria) (Panels A-G).5US_ACTI VE\132140149W-1

[0025] FIG. 11: Shows that Bv4E2b inoculation promotes maize seedling growth under low nitrogen conditions (Panel A, DKC3855RIB; Panel B, PHRE1).

[0026] FIG. 12: Shows that Bv4E2b survives water limitation and its application onto com seeds protects seedlings against severe water limitation (Panels A-D).

[0027] FIG. 13: Shows that application of Bv4E2b onto corn plants under dual nutrient and water limitation results in an enlarged rhizosheath and enhanced delivery of nitrogen to host plants (Panels A-G).

[0028] FIG. 14: Com Field Trial Data (2022): Grain yield impact of Bv4E2b as a foliar spray under low N-fertilizer, without irrigation.

[0029] FIG. 15: Com Field Trial Data (2023): Grain yield impact of Bv4E2b as a foliar spray with 75% N-fertilizer, without irrigation.

[0030] FIG. 16: Com Field Trial Data (2023): Grain yield impact of Bv4E2b as a seed coating agent with 75% N-fertilizer, and without irrigation.

[0031] FIG. 17: Com field trial data demonstrating that a non-Bv4E2b Bacillus velezensis strain (LG51) does not improve grain yield under low nitrogen conditions without irrigation.

[0032] FIG. 18: Winter Wheat Field Trial Data (2021-2022): Grain yield impact of Bv4E2b as a foliar spray under low N-fertilizer, without irrigation.

[0033] FIG. 19: Spring Wheat Field Trial Data (2022; Single Dose): Grain yield impact of Bv4E2b at different dosages as a foliar spray under low N-fertilizer and without irrigation.

[0034] FIG. 20: Spring Wheat Field Trial Data (2022; Double Dose); Grain yield impact of Bv4E2b at different dosages as a foliar spray under low N-fertilizer and without irrigation

[0035] FIG. 21: Shows Bv4E2c cell viability improvement at 13° C versus 30° C.

[0036] FIG. 22: Shows Bv4E2c colony growth improvement at 13° C.

[0037] FIG. 23: Shows Bv4E2c colony growth improvement at 30° C.DEFINITIONS

[0038] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of 6US_ACTI VE\132140149W-1skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

[0039] The term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. When used in conjunction with the word “comprising” or other open language in the claims, the words “a” and “an” denote “one or more,” unless specifically noted otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. The terms “comprise,” “have,” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes,” and “including,” are also open-ended. For example, any method that “comprises,” “has,” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps. Similarly, any composition or method that “comprises,” “has,” or “includes” one or more components is not limited to possessing only those components and covers other unlisted components.

[0040] A “biologically pure bacterial culture” refers to a culture of bacteria containing no other bacterial species in quantities sufficient to interfere with the replication of the culture or be detected by normal bacteriological techniques. Stated another way, it is a culture wherein virtually all of the bacterial cells present are of the selected strain.

[0041] The term “inoculant” as described herein is defined in several Federal, or State regulations as (1) “soil or plant inoculants shall include any carrier or culture of a specific microorganism or mixture of micro-organisms represented to improve the soil or the growth, quality, or yield of plants, and shall also include any seed or fertilizer represented to be inoculated with such a culture” (New York State 10-A Consolidated Law); (2) “substances other than fertilizers, manufactured, sold or represented for use in the improvement of the physical condition of the soil or to aid plant growth or crop yields” (Canada Fertilizers Act); (3) “a formulation containing pure or predetermined mixtures of living bacteria, fungi or virus particles for the treatment of seed, seedlings or other plant propagation material for the purpose of enhancing the growth7US_ACTI VE\132140149W-1UGLP:002WG capabilities or disease resistance or otherwise altering the properties of the eventual plants or crop” (Ad hoc European Working Group, 1997) or (4) “meaning any chemical or biological substance of mixture of substances or device distributed in this state to be applied to soil, plants or seeds for soil corrective puiposes; or which is intended to improve germination, growth, quality, yield, product quality, reproduction, flavor, or other desirable characteristics of plants or which is intended to produce any chemical, biochemical, biological or physical change in soil” (Section 14513 of the California Food and Agriculture Code).

[0042] As used herein, the term “isolated” as applied to a microorganism (e.g. Bacillus velezensis strain 4E2b, Bacillus velezensis strain 4E2c, or derivatives thereof) refers to a microorganism which has been removed and / or purified from an environment in which it naturally occurs. As such, an “isolated strain” of a microbe as used herein is a strain that has been removed and / or purified from its natural milieu. Thus, an “isolated microorganism” does not include one residing in an environment in which it naturally occurs. Further, the term “isolated” does not necessarily reflect the extent to which the microbe has been purified. A “substantially pure culture” of the strain of microbe refers to a culture which contains substantially no other microbes than the desired strain or strains of microbe. In other words, a substantially pure culture of a strain of microbe is substantially free of other contaminants, which can include microbial contaminants as well as undesirable chemical contaminants. Further, as used herein, a “biologically pure” strain is intended to mean the strain separated from materials with which it is normally associated in nature. Note that a strain associated with other strains, or with compounds or materials that it is not normally found with in nature, is still defined as “biologically pure.” A monoculture of a particular strain is, of course, “biologically pure.” As used herein, the term "enriched culture" of an isolated microbial strain refers to a microbial culture that contains more than 50%, 60%, 70%, 80%, 90%, or 95% of the isolated strain.

[0043] An “effective amount” refers to an amount sufficient to affect beneficial or desired results. In terms of drought protection, plant growth stimulation or promotion, or induction of a rhizosheath and rhizonest, an effective amount is that amount sufficient to protect, increase, induce, or accelerate a beneficial agronomical characteristic. For example, an “effective amount” may refer to a composition or inoculant comprising Bacillus velezensis strain 4E2b or Bacillus velezensis strain 4E2c in a quantity sufficient to result in inducing a rhizosheath and / or rhizonest structure on a plant’s roots as compared to the plant roots of a control-treated plant.8US_ACTI VE\132140149W-1Furthermore, an effective amount of a given microorganism antagonist (e.g. Bacillus v elevens is strain 4E2b, Bacillus velezensis strain 4E2c, or derivative thereof) may be defined as a concentration in excess of IO03, 101, 102, 103, 104, 105, 106, 107, 10s, or 109CFU / seed (colony forming units per seed) or 101, 102, 103, 104, 105, 106, 107, 10s, or 109CFU / plant (colony forming units per plant) induces a rhizosheath and / or rhizonest structure. An effective amount can be administered in one or more administrations. The actual rate of application of a liquid formulation will usually vary from a minimum of about 1 X 103to about 1 X IO10viable cells / mL and preferably from about 1 X 106to about 5 X 109viable cells / mL. Under most conditions, the microbial strains provided herein and described in the Examples below, would be optimally effective at application rates in the range of about 1 x 106to 1 X 109viable cells / mL, including all ranges derivable therebetween, assuming a mode of application which would achieve substantially uniform contact of at least about 10% of the plant tissues. If the microorganisms are applied as a solid formulation, the rate of application should be controlled to result in a comparable number of viable cells per unit area of plant tissue surface as obtained by the aforementioned rates of liquid treatment. Typically, the compositions of the present disclosure are biologically effective when delivered at a concentration in excess of 101CFU / seed, or in excess of 103CFU / seed, or in excess of 106CFU / seed. In regards to the applying compositions of the present disclosure to plants, the compositions of the present disclosure are biologically effective when delivered at a concentration in excess of 103CFU / plant, or in excess of 101CFU / seed, or in excess of 103CFU / seed, or in excess of 106CFU / seed.

[0044] As used herein, the term “Nutrient Use Efficiency” (NUE) refers to a measure of a plant's ability to absorb, utilize, and convert nutrients from the soil or other sources into growth, development, and yield. Approaches and techniques for assessing NUE are well known in the art, and generally include an assessment of output (e.g. biomass, yield, etc.) with respect to the input supplied to the plant (e.g. fertilizer supplied).

[0045] The terms “agriculturally acceptable carrier” and “carrier” are used interchangeably herein.

[0046] The terms “promoting plant growth” and “stimulating plant growth” are used interchangeably herein, and refer to the ability to enhance or increase at least one of the plant's height, weight, leaf size, root size, or stem size, to increase protein yield from the plant or to increase grain yield of the plant.9US_ACTI VE\132140149W-1

[0047] As used herein, the term “mutant” or “variant” in reference to a microorganism refers to a modification of the parental strain in which the desired biological activity is similar to that expressed by the parental strain. For example, in the case of Bacillus velezensis strain 4E2b the “parental strain” is defined herein as the original Bacillus velezensis strain before mutagenesis and / or directed improvement.

[0048] Percentage of percent identity: “percentage of sequence identity”, as used herein, is determined by comparing two optimally locally aligned sequences over a comparison window defined by the length of the local alignment between the two sequences. The amino acid sequence in the comparison window may comprise additions or deletions (e. g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Local alignment between two sequences only includes segments of each sequence that are deemed to be sufficiently similar according to a criterion that depends on the algorithm used to perform the alignment (e. g. BLAST). The percentage identity is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981) Add. APL. Math. 2:482, by the global homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. USA 85: 2444, 1988), by heuristic implementations of these algorithms (NCBI BLAST, WU-BLAST, BLAT, SIM, BLASTZ), or by inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to detemiine their optimal alignment. Typically, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. The term “substantial sequence identity” between polynucleotide or polypeptide sequences refers to polynucleotide or polypeptide comprising a sequence that has at least 50% sequence identity, preferably at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 96%, 97%, 98% or 99% sequence identity compared to a reference sequence using the programs.

[0049] Query nucleic acid and amino acid sequences can be searched against subject nucleic acid or amino acid sequences residing in public or proprietary databases. Such searches can be10US_ACTI VE\132140149W-1done using the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI BLAST v 2.18) program. The NCBI BLAST program is available on the internet from the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov / Blast.cgi). Typically the following parameters for NCBI BLAST can be used: Filter options set to "default”, the Comparison Matrix set to "BLOSUM62”, the Gap Costs set to “Existence: 11, Extension: 1”, the Word Size set to 3, the Expect (E threshold) set to le-3, and the minimum length of the local alignment set to 50% of the query sequence length. Sequence identity and similarity may also be determined using GenomeQuestTM software (Gene-IT, Worcester Mass. USA).

[0050] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0051] No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.DETAILED DESCRIPTION

[0052] Strategies to protect roots from water limitation and simultaneously decrease the requirement of nitrogen fertilizer (N) in agricultural production are of significant interest. Nitrogen is the predominant element in chemical fertilizers for grain crops such as com, rice and wheat which combined provide about 50% of all calories consumed by humans. N application accounts for 20% of maize production costs globally. Improving nutrient use efficiency (NUE) (defined as the amount of yield or biomass produced per input of fertilizers) is of significant interest, especially under water limitation and drought conditions.

[0053] The present disclosure relates to methods for inducing the production of rhizosheath and rhizonest structures on plant roots. As used herein, the term “rhizosheath” refers to a protective complex of mucilage and root hairs that form and encase the plant root surface, typically associated with soil particles when present. In particular, the rhizosheath is a visible coat on the root surface. In some embodiments, the rhizosheath can be further defined as comprising a thickness ranging from 1 micron to 5000 microns; a mucilage containing sugars, amino acids,11US_ACTI VE\132140149W-1lipids, and minerals; Bacillus velezensis strain 4E2b, Bacillus velezensis strain 4E2c, or mutants or variants thereof; nitrogen fixing bacteria; and / or other plant beneficial bacteria including but not limited to Stenotrophomonas pavanii, Bacillus nakamurai, and Pantoea ananatis. In other embodiments, the rhizosheath comprises a thickness ranging from 10 microns to 100 microns, 25 microns to 150 microns, 50 microns to 250 microns, 100 microns to 500 microns, 150 microns to 600 microns, 250 microns to 750 microns, 500 microns to 1000 microns, 750 microns to 2000 microns, 1000 microns to 2500 microns, 2000 microns to 3000 microns, 3000 microns to 4000 microns, 3500 microns to 4500 microns, or 4000 microns to 5000 microns, including all ranges derivable therebetween. In still other embodiments, the mucilage comprises sugars, including but not limited to, glucose, galactose, fructose, galacturonic acid, and fucose. In still further embodiments, the rhizosheath may comprise K. variicola and / or 5. maltophilia. In certain embodiments, the rhizosheath may comprise root hairs, if present.

[0054] The term “rhizonest” refers to a novel plant root surface structure consisting of an intertwined mesh of elongated root hairs and mucilage, often spherical in shape, which acts as a protective structure for beneficial microbes, analogous to a legume proto-nodule. In particular, the rhizonest is a visible structure on the root surface with a flattened or fully spherical shape. In some embodiments, the rhizonest can be further defined as comprising a diameter ranging from 1 micron to 5000 microns, 10 microns to 100 microns, 25 microns to 150 microns, 50 microns to 250 microns, 100 microns to 500 microns, 150 microns to 600 microns, 250 microns to 750 microns, 500 microns to 1000 microns, 750 microns to 2000 microns, 1000 microns to 2500 microns, 2000 microns to 3000 microns, 3000 microns to 4000 microns, 3500 microns to 4500 microns, or 4000 microns to 5000 microns, including all ranges derivable therebetween. In other embodiments, the rhizonest root hairs may intertwine with one another to form an interwoven mesh. In further embodiments, the rhizonest root hairs may internally bend at angles ranging from 75° to 105° relative to their base, or form spirals. For example, the root hairs of the rhizonest may bend at a 75°, 76°, 77°, 78°, 79°, 80°, 81°, 82°, 83°, 84°, 85°, 86°, 87°, 88°, 89°, 90°, 91°, 92°, 93, 94°, 95°, 96°, 97°, 98°, 99°, 100°, 101°, 102°, 103°, 104°, 105° angle relative to their base. The rhizonest may further comprise Bacillus velezensis strain 4E2b, Bacillus velezensis strain 4E2c, or mutants or variants thereof; nitrogen fixing bacteria, including but not limited to K. variicola and .S', maltophilia; other plant beneficial bacteria including but not limited to Stenotrophomonas pavanii, Bacillus nakamurai, and Pantoea ananatis; and / or root hairs complexed with mucilage containing sugars, amino acids, lipids and / or minerals.12US_ACTI VE\132140149W-1

[0055] The present disclosure further describes the discovery and development of methods and compositions to stimulate rhizosheath-root hair formation asymmetrically, preferentially or primarily on one side of roots. Asymmetric root hair formation is not known to exist in nature. Such structures can create an extreme osmotic gradient driving improved water / nutrient uptake from soil. Specifically, asymmetry creates an osmotic gradient to drive water uptake under water limitation (from low to high), which would also facilitate uptake of dissolved mineral nutrients (fertilizers) by maintaining a continuous zone along the root that is low in water (i.e. with no water-rich rhizosheath). The asymmetric growth of individual root hairs can result in elaborate nest-like cross-stitch patterns including rhizonest structures. These rhizoncsts arc characterized as proto-nodules creating a protective mucilage rich habitat for beneficial microbes on root surfaces (including as shown later, nitrogen-fixing bacteria), to protect them against competition from soil predators, with the walls being intertwined root hairs that elaborate cross-stich patterns. Rhizonest structures induced by Bv4E2b are not believed to have been previously observed in any plant species (Van Norman, 2016; Aslam et al., 2022).

[0056] The microbiological compositions provided herein that comprise isolated microbial strains or cultures thereof, such as Bacillus velezensis strain 4E2b, Bacillus velezensis strain 4E2c, or mutants or variants thereof, can be in a variety of forms, including, but not limited to, still cultures, whole cultures, stored stocks of cells (particularly glycerol stocks), agar strips, stored agar plugs in glycerol / water, freeze dried stocks, and dried stocks such as lyophilisate dried onto filter paper or grain seeds.

[0057] The present disclosure likewise relates to the use of the composition and inoculums provided herein for treating plants and seed for the purpose of inducing rhizosheath and / or rhizonest structures, increasing nutrient use efficiency, or increasing drought resistance.

[0058] Additional methods and compositions are described to induce rhizosheath and / or rhizonest structures, increase nutrient use efficiency, increase drought resistance, and improve the plant growth promotion efficacy of Bacillus velezensis by co-application with nitrogenfixing bacteria and / or plant growth regulator. For example, compositions and inoculums of the present disclosure may comprise Bacillus velezensis strain 4E2b, Bacillus velezensis strain 4E2c, or mutants derived therefrom, and at least one species of nitrogen fixing bacteria. Examples of such nitrogen fixing bacteria include, but are not limited to, a Klebsiella bacterium, a Stenotrophomonas bacterium, a Pantoea bacterium, a Bacillus bacterium, a Paenibacillus bacterium, an Azotobacter bacterium, a Kosakonia bacterium, an Herbaspirilluni bacterium, a13US_ACTI VE\132140149W-1Gluconacetobacler bacterium, an Azospirillum bacterium, a Melhylobaclerium, a Curtobacterium bacterium, a member of the Rhizobiaceae family, or a combination of any thereof. Alternatively, or in addition, the composition or inoculum may comprise at least one plant growth regulator such as an auxin-altering compounds. Examples of such plant growth regulators include, but are not limited to, indole-3 -acetic acid (IAA), 4- chloroindole-3 -acetic acid (4-Me-IAA), 4-methylindole-3-acetic acid (4-C1-IAA), a-Naphthalene acetic acid, 1-naphthaleneacetic acid, DICAMBA, 2, 4-dichlorophenoxyacetic acid (2,4-D), 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB), 4-chloro-2-methylphenoxyacetic acid (MCPA), diflufcnzopyr, halauxifcn, aminopyralid, clopyralid, fluroxypyr, triclopyr, quinclorac, diflufenzopyr, picloram, ethrel or any combination of any thereof.

[0059] In preferred embodiments, the combination of microorganism (e.g. Bacillus velezensis strain 4E2b, Bacillus velezensis strain 4E2c, or a mutant or variant derived therefrom) and nitrogen-fixing bacteria and / or plant growth regulator is provided in a synergistically effective amount. A “synergistically effective amount” according to the present disclosure represents a quantity of a combination of the microorganism and at least one particular nitrogen-fixing bacteria or plant growth regulator as described herein that more effectively induces a rhizosheath or rhizonest structure than the microorganism or such nitrogen-fixing bacteria or plant growth regulator alone. A “synergistically effective amount” according to the present disclosure also represents quantity of a combination of the microorganism and at least one particular nitrogenfixing bacteria or plant growth regulator as described herein that more effectively increases drought tolerance than the microorganism or such nitrogen-fixing bacteria or plant growth regulator alone. A “synergistically effective amount” according to the present disclosure represents a quantity of a combination of the microorganism and at least one particular nitrogenfixing bacteria as described herein that more effectively increases nitrogen use efficiency in a plant than such nitrogen-fixing bacteria.

[0060] A synergistic effect of active ingredients is present when the activity of the active ingredient combinations exceeds the total of the activities of the active ingredients when applied individually. The expected activity for a given combination of two active ingredients can be calculated as follows (cf. Colby, S.R., “Calculating Synergistic and Antagonistic Responses of Herbicide Combinations”, Weeds 1967, 75, 20-22). If X is the efficacy when active ingredient A is applied at an application rate of m ppm (or g / ha). Y is the efficacy when active ingredient B is applied at an application rate of n ppm (or g / ha), E is the efficacy when the active ingredients14US_ACTI VE\132140149W-1A and B are applied at application rates of m and n ppm (or g / ha), respectively, and then E = X + Y - X*Y / 100. If the actual activity exceeds the calculated value, then the activity of the combination is super additive, i.e.. a synergistic effect exists. In this case, the efficacy which was actually observed must be greater than the value for the expected efficacy (E) calculated from the above-mentioned formula.

[0061] For instance, the formula and analysis can be applied to an evaluation of rhizosheath or rhizonest growth. Such an assay is evaluated several days or weeks after the applications to plants. 100% means rhizosheath or rhizonest diameter which corresponds to that of the untreated control plant. Efficacy means in this case the additional % of rhizosheath or rhizonest diameter in comparison to that of the untreated control. For example, a treatment that resulted in rhizosheath or rhizonest diameter that were 120% compared to the untreated control plant would have an efficacy of 20%. If the plant rhizosheath or rhizonest induction effect for the combination (i.e., the observed efficacy for % rhizosheath or rhizonest diameter of plants treated with the combination) exceeds the calculated value, then the activity of the combination is super additive, i.e., a synergistic effect exists.

[0062] A further way of demonstrating a synergistic effect is the method of Tammes (cf. “Isoboles, a graphic representation of synergism in pesticides” in Neth. I. Plant Path., 1964, 70, 73-80). Those skilled in the art may also apply additional models or formulas to evaluate synergism, including but not limited to Bliss Independence, Loewe Additivity, or the Chou-Talalay Combination Index.

[0063] The amount of the microorganism which is used or employed in combination with at least one particular nitrogen-fixing bacteria or plant growth regulator disclosed herein, depends on the final formulation as well as size or type of the plant, plant parts, seeds, harvested fruits and vegetables to be treated. Usually, the microorganism to be employed or used according to the present disclosure is present in about 1% to about 80% (w / w), preferably in about 1% to about 60% (w / w), more preferably about 10% to about 50% (w / w) of its solo-formulation or combined-formulation with the at least one particular nitrogen-fixing bacteria or plant growth regulator disclosed herein.

[0064] Also the amount of the at least one particular nitrogen-fixing bacteria or plant growth regulator disclosed herein which is used or employed in combination with the microorganism (e.g. Bacillus velezensis strain 4E2b, Bacillus velezensis strain 4E2c, or a mutant or variant derived therefrom) depends on the final formulation as well as size or type of the plant, plant15US_ACTI VE\132140149W-1parts, seeds, harvested fruit or vegetable to be treated. Usually, the particular nitrogen-fixing bacteria or plant growth regulator to be employed or used according to the present disclosure is present in about 0.1% to about 80% (w / w), preferably 1% to about 60% (w / w), more preferably about 10% to about 50% (w / w) of its solo-formulation or combined-formulation with the microorganism.

[0065] Application of the microorganism may be effected as a foliar spray, as a soil treatment, and / or as a seed treatment / dressing. When used as a foliar treatment, in one embodiment, about 1 / 16 to about 5 gallons of whole broth are applied per acre. When used as a soil treatment, in one embodiment, about 1 to about 5 gallons of whole broth arc applied per acre. When used for seed treatment about 1 / 32 to about 1 / 4 gallons of whole broth are applied per acre. For seed treatment, the end -use formulation contains at least at least 1 x 104, at least 1 x 105, at least 1 x 106, 1 x 107, at least 1 x 10s, at least 1 x 109, at least 1 x 1010colony forming units per gram.

[0066] The microorganism and at least one particular nitrogen-fixing bacteria or plant growth regulator disclosed herein are used or employed in a synergistic weight ratio. The skilled person is able to determine the synergistic weight ratios for the present invention by routine methods. The skilled person understands that these ratios refer to the ratio within a combined-formulation as well as to the calculative ratio of the microorganism and at least one particular nitrogen-fixing bacteria or plant growth regulator disclosed herein when both components are applied as monoformulations to a plant to be treated. The skilled person can calculate this ratio by simple mathematics since the volume and the amount of the microorganism and at least one particular nitrogen-fixing bacteria or plant growth regulator, respectively, in a mono-formulation is known to the skilled person.

[0067] The ratio can be calculated based on the amount of the at least one particular nitrogenfixing bacteria or plant growth regulator disclosed herein, at the time point of applying said component of a combination according to the present disclosure to a plant or plant part and the amount of microorganism cells shortly prior (e.g., 48 h, 24 h, 12 h, 6 h, 2 h, 1 h) or at the time point of applying said component of a combination according to the present disclosure to a plant or plant part.

[0068] The application of the microorganism and the at least one particular nitrogen-fixing bacteria or plant growth regulator disclosed herein to a plant or a plant part can take place simultaneously or at different times as long as both components are present on or in the plant after the application(s). In cases where the microorganism and at least one particular nitrogen-16US_ACTI VE\132140149W-1fixing bacteria or plant growth regulator disclosed herein are applied at different times and the particular nitrogen-fixing bacteria or plant growth regulator disclosed herein is applied prior to the microorganism, the skilled person can determine the concentration of nitrogen-fixing bacteria or plant growth regulator on / in a plant by chemical analysis known in the art, at the time point or shortly before the time point of applying the microorganism or inoculum thereof. Vice versa, when the microorganism is applied to a plant first, the concentration of the microorganism can be determined using tests which are also known in the art, at the time point or shortly before the time point of applying the nitrogen-fixing bacteria or plant growth regulator.

[0069] In particular, in one embodiment the synergistic weight ratio of the microorganism cells and the at least one particular nitrogen- fixing bacteria or plant growth regulator disclosed herein lies in the range of 1:1000 to 1000:1, preferably in the range of 1:500 to 500:1, more preferably in the range of 1:300 to 500:1. In another embodiment, ratios are between 20:1 and 1:20, such as 10:1, 5:1 or 2:1. It has to be noted that these ratio ranges refer to the microorganism (to be combined with at least one particular nitrogen-fixing bacteria or plant growth regulator disclosed herein or a preparation of at least one particular nitrogen- fixing bacteria or plant growth regulator disclosed herein). For example, a ratio of 100:1 means 100 weight parts of a microorganism such as a Bacillus velezensis strain 4E2b, Bacillus velezensis strain 4E2c, or a mutant or variant derived therefrom, and 1 weight part of the particular nitrogen-fixing bacteria or plant growth regulator disclosed herein are combined (either as a solo formulation, a combined formulation or by separate applications to plants so that the combination is formed on the plant).

[0070] In some embodiments, the synergistic weight ratio of the microorganism and at least one particular nitrogen-fixing bacteria or plant growth regulator disclosed herein is in the range of 1:100 to 20,000:1, including all ranges derivable therebetween. In particular embodiments, the synergistic weight ratio is in the range of 1 :50 to 10,000: 1 or even in the range of 1 :50 to 1000: 1.

[0071] In another embodiment, the concentration of the microorganism after dispersal is at least 50 g / ha, such as 50 - 7500 g / ha, 50 - 2500 g / ha, 50 - 1500 g / ha; at least 250 g / ha (hectare), at least 500 g / ha or at least 800 g / ha, including all ranges derivable therebetween

[0072] The application rate of composition to be employed or used according to the present disclosure may vary. The skilled person is able to find the appropriate application rate by way of routine experiments.

[0073] In another aspect, a seed treated with the composition as described above is provided.17US_ACTI VE\132140149W-1

[0074] For the purposes of the present disclosure, the composition is applied alone or in a suitable formulation to the seed. The seed is preferably treated in a condition in which its stability is such that no damage occurs in the course of the treatment. Generally speaking, the seed may be treated at any point in time between harvesting and sowing. Typically, seed is used which has been separated from the plant and has had cobs, hulls, stems, husks, hair, or pulp removed. Thus, for example, seed may be used that has been harvested, cleaned, and dried to a moisture content of less than 15% by weight. Alternatively, seed can also be used that after drying has been treated with water, for example, and then dried again.

[0075] When treating seed it is necessary, generally speaking, to ensure that the amount of the composition, and / or of other additives, that is applied to the seed is selected such that the germination of the seed is not adversely affected, and / or that the plant which emerges from the seed is not damaged. This is the case in particular with active ingredients which may exhibit phytotoxic effects at certain application rates.

[0076] The compositions of the present disclosure can be applied directly, in other words without comprising further components and without having been diluted. As a general rule, it is preferable to apply the compositions in the form of a suitable formulation to the seed. Suitable formulations and methods for seed treatment are known to the skilled person and are described in, for example, the following documents: U.S. Patent Nos. 4,272,417 A; 4,245,432 A; 4,808,430 A; 5,876,739 A; U.S. Patent Publication No. 2003 / 0176428 Al; WO 2002 / 080675 Al; WO 2002 / 028186 A2.

[0077] The combinations which can be used in accordance with the present disclosure may be converted into the customary seed-dressing formulations, such as solutions, emulsions, suspensions, powders, foams, slurries, or other coating compositions for seed, and also ULV formulations.

[0078] These formulations are prepared in a known manner, by mixing composition with customary adjuvants, such as, for example, customary extenders and also solvents or diluents, colorants, wetters, dispersants, emulsifiers, antifoams, preservatives, secondary thickeners, stickers, gibberellins, and also water.

[0079] The methods described herein can promote plant growth by multiple mechanisms, including but not limited to, improving nutrient use efficiency, providing sugar-rich habitat for biological nitrogen fixing bacteria, and protecting against water limitation.18US_ACTI VE\132140149W-1

[0080] The strains disclosed herein were identified by 16S rRNA sequencing and biochemical assays. Thus, Bacillus velezensis strain 4E2b has a 16S ribosomal RNA sequence having at least about 98%, at least about 99%, or 100% sequence identity with the sequence of SEQ ID NO: 1; and Bacillus velezensis strain 4E2c has a 16S ribosomal RNA sequence having at least about 98%, at least about 99%, or 100% sequence identity with the sequence of SEQ ID NO: 2. These sequences are shown in Table 1 below.

[0081] Thus, the compositions and methods provided herein further relate to Bacillus velezensis strains comprising a 16S ribosomal RNA sequence having at least about 98%, at least about 99%, or 100% sequence identity with the sequence of SEQ ID NO: 1, wherein the strain is capable of inducing a rhizosheath or rhizonest structure.TABLE 119US_ACTI VE\132140149W-1

[0082] Methods for determining sequence identity are well known by one of ordinary skill in the art. By way of example and not of limitation, the BLASTn algorithm available through National Center for Biotechnology Information (NCBI) can be used to align sequences and determine their identity. The foregoing bacterial strains were identified at least to their genus designation by means of conventional biochemistry and morphological indicators.

[0083] The present disclosure also relates to a biologically pure bacterial culture wherein bacteria, i.e. the bacterial strain in the bacterial culture, are mutants of any of the foregoing bacterial strains, which comprises one or more mutations that retain the ability to induce rhizosheath and / or rhizonest structures; increase nutrient use efficiency; or increase drought resistance. Thus, the mutant of any of the foregoing strains will be capable of inducing rhizosheath and / or rhizonest structures, increasing nutrient use efficiency, or increasing drought resistance when compared to plants to which the mutant was not applied. For example, theUS_ACTI VE\132140149W-1mutant can comprise a cold-tolerant mutant (e.g., cold-tolerant mutant Bacillus velezensis strain 4E2c).

[0084] A composition or inoculum which includes an effective amount of a mixture of bacteriologically pure bacterial cultures can include Bacillus velezensis strain 4E2b, Bacillus velezensis strain 4E2c, a Klebsiella bacterium, a Stenotrophomonas bacterium, a Pantoea bacterium, a Bacillus bacterium, a Paenibacillus bacterium, an Azotobacter bacterium, a Kosakonia bacterium, an Herbaspirillum bacterium, a Gluconacetobacter bacterium, an Azospirillum bacterium, a Methylobacterium, a Curtobacterium bacterium, or a member of the Rhizobiaccac family. Such mixtures of bacteriologically pure bacterial cultures arc favorable for use in increasing drought resistance or nutrient use efficiency in a plant.

[0085] In addition to one or more biologically pure bacterial cultures as described in the foregoing sections, an inoculum of the present disclosure also comprises an agriculturally acceptable carrier. The carrier can include a dispersant, a surfactant, an additive, water, a thickener, an anti-caking agent, residue breakdown, a composting formulation, a granular application, diatomaceous earth, an oil, a coloring agent, a stabilizer, a preservative, a polymer, a coating, or a combination thereof. One of ordinary skill in the art can readily determine the appropriate carrier to be used taking into consideration factors such as a particular bacterial strain, plant to which the inoculum is to be applied, type of soil, climate conditions, whether the inoculum is in liquid, solid or powder form, and the like.

[0086] The additive can comprise an oil, a gum, a resin, a clay, a polyoxyethylene glycol, a terpene, a viscid organic, a fatty acid ester, a sulfated alcohol, an alkyl sulfonate, a petroleum sulfonate, an alcohol sulfate, a sodium alkyl butane diamate, a polyester of sodium thiobutant dioate, a benzene acetonitrile derivative, a proteinaceous material, or a combination thereof.

[0087] The proteinaceous material can include a milk product, wheat flour, soybean meal, alfalfa meal, yeast extract, blood, albumin, gelatin, or a combination thereof.

[0088] The thickener can comprise a long chain alkylsulfonate of polyethylene glycol, polyoxyethylene oleate, or a combination thereof.

[0089] The surfactant can contain a heavy petroleum oil, a heavy petroleum distillate, a polyol fatty acid ester, a polyethoxylated fatty acid ester, an aryl alkyl polyoxyethylene glycol, an alkyl amine acetate, an alkyl aryl sulfonate, a polyhydric alcohol, an alkyl phosphate, or a combination thereof.21US_ACTI VE\132140149W-1

[0090] The anti-caking agent can include a sodium salt such as a sodium sulfite, a sodium sulfate, a sodium salt of monomethyl naphthalene sulfonate, a sodium salt of dimethyl naphthalene sulfonate, or a combination thereof; or a calcium salt such as calcium carbonate, diatomaceous earth, or a combination thereof.

[0091] Any agriculturally acceptable carrier can be used. Such carriers include, but are not limited to, vermiculite, charcoal, sugar factory carbonation press mud, rice husk, carboxymethyl cellulose, peat, perlite, fine sand, calcium carbonate, flour, alum, a starch, talc, polyvinyl pyrrolidone, or a combination thereof.

[0092] Compositions and inoculants can be prepared as solid, liquid, or powdered formulations as is known in the art. The composition or inoculum of the present disclosure can be formulated as a seed coating formulation, a liquid formulation for application to plants or to a plant growth medium, or a solid formulation for application to plants or to a plant growth medium.

[0093] When the composition or inoculum is prepared as a liquid formulation for application to plants or to a plant growth medium, it can be prepared in a concentrated formulation or a ready-to-use formulation. In some instances, the seed coating formulation of the present disclosure is an aqueous or oil-based solution for application to seeds.

[0094] When the composition or inoculum of the present disclosure is prepared as a solid formulation for application to plants or to a plant growth medium, it can be prepared as a granular formulation or a powder agent. The seed coating formulation can be a powder or granular formulation for application to seeds.

[0095] The composition or inoculum can further include an agrochemical such as a fertilizer, a micronutrient fertilizer material, an insecticide, an herbicide, a plant growth amendment, a fungicide, a molluscicide, an algicide, a bacterial inoculant, a fungal inoculant, or a combination thereof. In some instances, the fertilizer is a liquid fertilizer. The agrochemical can either be applied to a plant growth medium or to plants and / or seeds. Liquid fertilizer can include, without limitation, ammonium sulfate, ammonium nitrate, ammonium sulfate nitrate, ammonium chloride, ammonium bisulfate, ammonium polysulfide, ammonium thiosulfate, aqueous ammonia, anhydrous ammonia, ammonium polyphosphate, aluminum sulfate, calcium nitrate, calcium ammonium nitrate, calcium sulfate, calcined magnesite, calcitic limestone, calcium oxide, calcium nitrate, dolomitic limestone, hydrated lime, calcium carbonate, diammonium phosphate, monoammonium phosphate, magnesium nitrate, magnesium sulfate, potassium nitrate, potassium chloride, potassium magnesium sulfate, potassium sulfate, sodium nitrates,22US_ACTI VE\132140149W-1magnesian limestone, magnesia, urea, urea-formaldehydes, urea ammonium nitrate, sulfur-coated urea, polymer-coated urea, isobutylidene diurea, K2SO4-2MgSO4, kainite, sylvinite, kieserite, Epsom salts, elemental sulfur, marl, ground oyster shells, fish meal, oil cakes, fish manure, blood meal, rock phosphate, super phosphates, slag, bone meal, wood ash, manure, bat guano, peat moss, compost, green sand, cottonseed meal, feather meal, crab meal, fish emulsion, or a combination thereof.

[0096] The micronutrient fertilizer material can comprise boric acid, a borate, a boron frit, copper sulfate, a copper frit, a copper chelate, a sodium tetraborate decahydrate, an iron sulfate, an iron oxide, iron ammonium sulfate, an iron frit, an iron chelate, a manganese sulfate, a manganese oxide, a manganese chelate, a manganese chloride, a manganese frit, a sodium molybdate, molybdic acid, a zinc sulfate, a zinc oxide, a zinc carbonate, a zinc frit, zinc phosphate, a zinc chelate, or a combination thereof.

[0097] The insecticide can include an organophosphate, a carbamate, a pyrethroid, an acaricide, an alkyl phthalate, boric acid, a borate, a fluoride, sulfur, a haloaromatic substituted urea, a hydrocarbon ester, a biologically-based insecticide, or a combination thereof.

[0098] The herbicide can comprise a chlorophenoxy compound, a nitrophenolic compound, a nitrocresolic compound, a dipyridyl compound, an acetamide, an aliphatic acid, an anilide, a benzamide, a benzoic acid, a benzoic acid derivitive, anisic acid, an anisic acid derivitive, a benzonitrile, benzothiadiazinone dioxide, a thiocarbamate, a carbamate, a carbanilate, chloropyridinyl, a cyclohexenone derivative, a dinitroaminobenzene derivative, a fluorodinitrotoluidine compound, isoxazolidinone, nicotinic acid, isopropylamine, an isopropylamine derivative, oxadi azolinone, a phosphate, a phthalate, a picolinic acid compound, a triazine, a triazole, a uracil, a urea derivative, endothall, sodium chlorate, or a combination thereof.

[0099] The fungicide can comprise a substituted benzene, a thiocarbamate, an ethylene bis dithiocarbamate, a thiophthalidamide, a copper compound, an organomercury compound, an organotin compound, a cadmium compound, anilazine, benomyl, cyclohexamide, dodine, etridiazole, iprodione, metlaxyl, thiamimefon, triforine, or a combination thereof.

[0100] All of the biologically pure bacterial cultures, compositions, and inoculums provided herein can be used in methods for inducing rhizosheath and / or rhizonest structures, increasing nutrient use efficiency, or increasing drought resistance. Such methods include applying the foregoing cultures, compositions, and inoculums to a plant, plant seed, or plant growth medium23US_ACTI VE\132140149W-1in order to induce rhizosheath and / or rhizonest structures, increase nutrient use efficiency, or increase drought resistance of the plant. Techniques for applying such compositions and inoculants to plants are known in the art, including appropriate modes of administration, frequency of administration, dosages, and the like. The composition or inoculant can be applied to the soil prior to, contemporaneously with, or after sowing seeds, after planting, or after plants have emerged from the ground. The composition and inoculant can also be applied to seeds themselves prior to or at the time of planting (e.g., packaged seed may be sold with the inoculant already applied). The composition or inoculant can also be applied to the plant after it has emerged from the ground, or to the leaves, stems, roots, or other parts of the plant. The composition or inoculant can also be applied to the plant under stress conditions, e.g. under drought conditions or nitrogen limiting conditions before or after planting.

[0101] The methods provided herein can include applying a substance such as glycerol, pyruvate, yeast extract, a polyol e.g., mannitol, sorbitol, galactitol, fucitol, iditol, inositol, arabitol, xylitol, ribitol), polyethylene glycol or combination thereof to the plant growth medium. For the preparation of yeast extract, Saccharomyces cerevisiae is a preferred yeast starting material. Additional yeast strains that can be used instead of or in addition to Saccharomyces cerevisiae include Kluyveromyces marxianus, Kluyveromyces lactis, Candida utilis (Torula yeast), Zygosaccharomyces, Pichia pastoris, and Hansanula polymorpha, and others known to those skilled in the art.

[0102] In instances in which the substance is applied to a plant growth medium, at least one bacterial culture, at least one composition, or at least one inoculum provided herein can be applied to a plant or plant seed in the plant growth medium, or to the plant growth medium. Preferably, the composition or inoculum is applied to the plant growth medium as a solid or liquid formulation. The bacterial culture, composition, or inoculum and the chemical can be applied contemporaneously or at separate times. The exact order is not of great relevance, and the optimal combination can be determined empirically by one of ordinary skill in the art without due experimentation. For example, a skilled artisan can set up experimental conditions wherein: (1) the composition, inoculum or bacterial culture and the substance are administered concurrently, (2) the composition, inoculum or bacterial culture is administered on a separate occasion after the substance is added to a plant growth medium, (3) the composition, inoculum or bacterial culture is administered on a separate occasion prior to the substance being added to a plant growth medium, and the like. The results of such and similar experimental designs can24US_ACTI VE\132140149W-1easily demonstrate the most suitable methods for application of the composition, inoculum or bacterial culture, and the substance. Thus, the composition, inoculum or bacterial culture provided herein can be applied to a plant growth medium prior to, concurrently with, or after planting of seeds, seedlings, cuttings, bulbs, or plants in the plant growth medium.

[0103] The plant growth medium includes soil, water, an aqueous solution, sand, gravel, a polysaccharide, mulch, compost, peat moss, straw, logs, clay, or a combination thereof. Preferably, the plant growth medium is soil or compost. As is known in the art, the plant growth medium can be stored for future planting.

[0104] Furthermore, depending on the plant species or plant cultivars, their location and growth conditions (soils, climate, vegetation period, diet), using or employing the compositions described herein may also result in super-additive (“synergistic”) effects. Thus, for example, by using or employing inventive composition in the treatment according to the present disclosure, reduced water or nitrogen application rates and / or an increase in plant growth, increased tolerance to high or low temperatures, increased tolerance to drought or to water or soil salt content, increased flowering performance, easier harvesting, accelerated maturation, higher harvest yields, bigger seeds, larger plant height, greener leaf color, earlier flowering, higher quality and / or a higher nutritional value of the harvested products, higher sugar concentration within the seeds, better storage stability and / or processability of the harvested products are possible, which exceed the effects which were actually to be expected.

[0105] Plants and plant cultivars which may also be treated according to the present disclosure, are those plants characterized by enhanced yield characteristics, i.e., that already exhibit an increased plant health with respect to this feature. Increased yield in said plants can be the result of, for example, improved plant physiology, growth and development, such as water use efficiency, water retention efficiency, improved nitrogen use, enhanced carbon assimilation, improved photosynthesis, increased germination efficiency and accelerated maturation.

[0106] Yield can furthermore be affected by improved plant architecture (under stress and nonstress conditions), including but not limited to, root growth, rhizosheath growth, and rhizonest growth. Further yield traits include seed composition, such as carbohydrate content, protein content, oil content and composition, nutritional value, reduction in anti-nutritional compounds, improved processability and better storage stability. Preferably, the treatment of these plants and cultivars with the composition or inoculum described herein additionally increases the overall plant health.25US_ACTI VE\132140149W-1

[0107] For purposes of the compositions and methods described herein, the plant can be a dicotyledon, a monocotyledon or a gymnosperm.

[0108] The dicotyledon can be selected from the group consisting of bean, pea, tomato, pepper, squash, alfalfa, almond, aniseseed, apple, apricot, arracha, artichoke, avocado, bambara groundnut, beet, bergamot, black pepper, black wattle, blackberry, blueberry, bitter orange, bok-choi, Brazil nut, breadfruit, broccoli, broad bean, Brussels sprouts, buckwheat, cabbage, camelina, Chinese cabbage, cacao, cantaloupe, caraway seeds, cardoon, carob, carrot, cashew nuts, cassava, castor bean, cauliflower, celeriac, celery, cherry, chestnut, chickpea, chicory, chili pepper, chrysanthemum, cinnamon, citron, clementine, clove, clover, coffee, cola nut, colza, com, cotton, cottonseed, cowpea, crambe, cranberry, cress, cucumber, currant, custard apple, drumstick tree, earth pea, eggplant, endive, fennel, fenugreek, fig, filbert, flax, geranium, gooseberry, gourd, grape, grapefruit, guava, hemp, hempseed, henna, hop, horse bean, horseradish, indigo, jasmine, Jerusalem artichoke, jute, kale, kapok, kenaf, kohlrabi, kumquat, lavender, lemon, lentil, lespedeza, lettuce, lime, liquorice, litchi, loquat, lupine, macadamia nut, mace, mandarin, mangel, mango, medlar, melon, mint, mulberry, mustard, nectarine, niger seed, nutmeg, okra, olive, opium, orange, papaya, parsnip, pea, peach, peanut, pear, pecan nut, persimmon, pigeon pea, pistachio nut, plantain, plum, pomegranate, pomelo, poppy seed, potato, sweet potato, prune, pumpkin, quebracho, quince, trees of the genus Cinchona, quinoa, radish, ramie, rapeseed, raspberry, rhea, rhubarb, rose, rubber, rutabaga, safflower, sainfoin, salsify, sapodilla, Satsuma, scorzonera, sesame, shea tree, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, swede, sweet pepper, tangerine, tea, teff, tobacco, tomato, trefoil, tung tree, turnip, urena, vetch, walnut, watermelon, yerba mate, wintercress, shepherd’s purse, garden cress, peppercress, watercress, pennycress, star anise, laurel, bay laurel, cassia, jamun, dill, tamarind, peppermint, oregano, rosemary, sage, soursop, pennywort, calophyllum, balsam pear, kukui nut, Tahitian chestnut, basil, huckleberry, hibiscus, passionfruit, star apple, sassafras, cactus, St. John’s wort, loosestrife, hawthorn, cilantro, curry plant, kiwi, thyme, zucchini, ulluco, jicama, waterleaf, spiny monkey orange, yellow mombin, starfruit, amaranth, wasabi, Japanese pepper, yellow plum, mashua, Chinese toon, New Zealand spinach, bower spinach, ugu, tansy, chickweed, jocote, Malay apple, paracress, sowthistle, Chinese potato, horse parsley, hedge mustard, campion, agate, cassod tree, thistle, burnet, star gooseberry, saltwort, glasswort, sorrel, silver lace fern, collard greens, primrose, cowslip, purslane, knotgrass, terebinth, tree lettuce, wild betel, West African pepper, yerba santa, tarragon, parsley, chervil, land cress, burnet26US_ACTI VE\132140149W-1saxifrage, honeyherb, butterbur, shiso, water pepper, perilla, bitter bean, oca, kampong, Chinese celery, lemon basil, Thai basil, water mimosa, cicely, cabbage-tree, moringa, mauka, ostrich fern, rice paddy herb, yellow sawah lettuce, lovage, pepper grass, maca, bottle gourd, hyacinth bean, water spinach, catsear, fishwort, Okinawan spinach, lotus sweetjuice, gallant soldier, culantro, arugula, cardoon, caigua, mitsuba, chipilin, samphire, mampat. ebolo, ivy gourd, cabbage thistle, sea kale, chaya, huauzontle, Ethiopian mustard, magenta spreen, good king henry, epazole, lamb’s quarters, centella plumed cockscomb, caper, rapini, napa cabbage, mizuna, Chinese savoy, kai-lan, mustard greens, Malabar spinach, chard, marshmallow, climbing wattle, China jute, paprika, annatto seed, spearmint, savory, marjoram, cumin, chamomile, lemon balm, allspice, bilberry, cherimoya, cloudberry, damson, pitaya, durian, elderberry, feijoa, jackfruit, jambul, jujube, physalis, purple mangosteen, rambutan, redcurrant, blackcurrant, salal berry, satsuma, ugli fruit, azuki bean, black bean, black-eyed pea, borlotti bean, common bean, green bean, kidney bean, lima bean, mung bean, navy bean, pinto bean, runner bean, mangetout, snap pea, broccoflower, calabrese, nettle, bell pepper, raddichio, daikon, white radish, skirret, tat soi, broccolini, black radish, burdock root, fava bean, broccoli raab, lablab, lupin, sterculia, velvet beans, winged beans, yam beans, mulga, ironweed, umbrella bush, tjuntjula, wakalpulka, witchetty bush, wiry wattle, chia, beech nut, candlenut, colocynth, mamoncillo, Maya nut, mongongo, ogbono nut, paradise nut, and cempedak.

[0109] The dicotyledon can be from a family selected from the group consisting of Acanthaceae (acanthus), Aceraceae (maple), Achariaceae, Achatocarpaceae (achatocarpus), Actinidiaceae (Chinese gooseberry), Adoxaceae (moschatel), Aextoxicaceae, Aizoaceae (fig marigold), Akaniaceae, Alangiaceae, Alseuosmiaceae, Alzateaceae, Amaranthaceae (amaranth), Amborellaceae, Anacardiaceae (sumac), Ancistrocladaceae, Anisophylleaceae, Annonaceae (custard apple), Apiaceae (carrot), Apocynaceae (dogbane), Aquifoliaceae (holly), Araliaceae (ginseng), Aristolochiaceae (birthwort), Asclepiadaceae (milkweed), Asteraceae (aster), Austrobaileyaceae, Balanopaceae, Balanophoraceae (balanophora), Balsaminaceae (touch-me-not), Barbeyaceae, Barclayaceae, Basellaceae (basella), Bataceae (saltwort), Begoniaceae (begonia), Berberidaceae (barberry), Betulaceae (birch), Bignoniaceae (trumpet creeper), Bixaceae (lipstick tree), Bombacaceae (kapok tree), Boraginaceae (borage), Brassicaceae (mustard, also Cruciferae), Bretschneideraceae, Brunelliaceae (brunellia), Bruniaceae, Brunoniaceae, Buddlejaceae (butterfly bush), Burseraceae (frankincense), Buxaceae (boxwood), Byblidaceae, Cabombaceae (water shield), Cactaceae (cactus), Caesalpiniaceae, Callitrichaceae27US_ACTI VE\132140149W-1(water starwort), Calycanthaceae (strawberry shrub), Calyceraceae (calycera), Campanulaceae (bellflower), Canellaceae (canella), Cannabaceae (hemp), Capparaceae (caper), Caprifohaceae (honeysuckle), Cardiopteridaceae, Caricaceae (papaya), Caryocaraceae (souari), Caryophyllaceae (pink), Casuarinaceae (she-oak), Cecropiaceae (cecropia), Celastraceae (bittersweet), Cephalotaceae, Ceratophyllaceae (hornwort), Cercidiphyllaceae (katsura tree), Chenopodiaceae (goosefoot), Chloranthaceae (chloranthus), Chrysobalanaceae (cocoa plum), Circaeasteraceae, Cistaceae (rockrose), Clethraceae (clethra), Clusiaceae (mangosteen, also Guttiferae), Cneoraceae, Columelliaceae, Combretaceae (Indian almond), Compositae (aster), Connaraccac (cannarus), Convolvulaccac (morning glory), Coriariaccac, Comaccac (dogwood), Corynocarpaceae (karaka), Crassulaceae (stonecrop), Crossosomataceae (crossosoma), Crypteroniaceae, Cucurbitaceae (cucumber), Cunoniaceae (cunonia), Cuscutaceae (dodder), Cyrillaceae (cyrilla), Daphniphyllaceae, Datiscaceae (datisca), Davidsoniaceae, Degeneriaceae, Dialypetalanthaceae, Diapensiaceae (diapensia), Dichapetalaceae, Didiereaceae, Didymelaceae, Dilleniaceae (dillenia), Dioncophyllaceae, Dipentodontaceae, Dipsacaceae (teasel), Dipterocarpaceae (meranti), Donatiaceae, Droseraceae (sundew), Duckeodendraceae, Ebenaceae (ebony), Elaeagnaceae (oleaster), Elaeocarpaceae (elaeocarpus), Elatinaceae (waterwort), Empetraceae (crowberry), Epacridaceae (epacris), Eremolepidaceae (catkin-mistletoe), Ericaceae (heath), Erythroxylaceae (coca), Eucommiaceae, Eucryphiaceae, Euphorbiaceae (spurge), Eupomatiaceae, Eupteleaceae, Fabaceae (pea or legume), Fagaceae (beech), Flacourtiaceae (flacourtia), Fouquieriaceae (ocotillo), Frankeniaceae (frankenia), Fumariaceae (fumitory), Garryaceae (silk tassel), Geissolomataceae, Gentianaceae (gentian), Geraniaceae (geranium), Gesneriaceae (gesneriad), Globulariaceae, Gomortegaceae, Goodeniaceae (goodenia), Greyiaceae, Grossulariaceae (currant), Grubbiaceae, Gunneraceae (gunnera), Gyrostemonaceae, Haloragaceae (water milfoil), Hamamelidaceae (witch hazel), Hernandiaceae (hemandia), Himantandraceae, Hippocastanaceae (horse chestnut), Hippocrateaceae (hippocratea), Hippuridaceae (mare’s tail), Hoplestigmataceae, Huaceae, Hugoniaceae, Humiriaceae, Hydnoraceae, Hydrangeaceae (hydrangea), Hydrophyllaceae (waterleaf), Hydrostachyaceae, Icacinaceae (icacina), Idiospermaceae, Illiciaceae (star anise), Ixonanthaceae, Juglandaceae (walnut), Julianiaceae, Krameriaceae (krameria), Lacistemataceae, Lamiaceae (mint, also Labiatae), Lardizabalaceae (lardizabala), Lauraceae (laurel), Lecythidaceae (brazil nut), Leeaceae, Leitneriaceae (corkwood), Lennoaceae (lennoa), Lentibulariaceae (bladderwort), Limnanthaceae (meadow foam), Linaceae (flax), Lissocarpaceae, Loasaceae (loasa),28US_ACTI VE\132140149W-1Loganiaceae (logania), Loranthaceae (showy mistletoe), Lythraceae (loosestrife), Magnoliaceae (magnolia), Malesherbiaceae, Malpighiaceae (Barbados cherry), Malvaceae (mallow), Marcgraviaceae (shingle plant), Medusagynaceae, Medusandraceae, Melastomataceae (melastome), Meliaceae (mahogany), Melianthaceae, Mendonciaceae, Menispermaceae (moonseed), Menyanthaceae (buckbean), Mimosaceae, Misodendraceae, Mitrastemonaceae, Molluginaceae (carpetweed), Monimiaceae (monimia), Monotropaceae (Indian pipe), Moraceae (mulberry), Moringaceae (horseradish tree), Myoporaceae (myoporum), Myricaceae (bayberry), Myristicaceae (nutmeg), Myrothamnaceae, Myrsinaceae (myrsine), Myrtaceae (myrtle), Nclumbonaccac (lotus lily), Ncpcnthaccac (East Indian pitcherplant), Ncuradaccac, Nolanaccac, Nothofagaceae, Nyctaginaceae (four-o’clock), Nymphaeaceae (water lily), Nyssaceae (sour gum), Ochnaceae (ochna), Olacaceae (olax), Oleaceae (olive), Oliniaceae, Onagraceae (evening primrose), Oncothecaceae, Opiliaceae, Orobanchaceae (broom rape), Oxalidaceae (wood sorrel), Paeoniaceae (peony), Pandaceae, Papaveraceae (poppy), Papilionaceae, Paracryphiaceae, Passifloraceae (passionflower), Pedaliaceae (sesame), Pellicieraceae, Penaeaceae, Pentaphragmataceae, Pentaphylacaceae, Peridiscaceae, Physenaceae, Phytolaccaceae (pokeweed), Piperaceae (pepper), Pittosporaceae (pittosporum), Plantaginaceae (plantain), Platanaceae (plane tree), Plumbaginaceae (leadwort), Podostemaceae (river weed), Polemoniaceae (phlox), Polygalaceae (milkwort), Polygonaceae (buckwheat), Portulacaceae (purslane), Primulaceae (primrose), Proteaceae (protea), Punicaceae (pomegranate), Pyrolaceae (shinleaf), Quiinaceae, Rafflesiaceae (rafflesia), Ranunculaceae (buttercup orranunculus), Resedaceae (mignonette), Retziaceae, Rhabdodendraceae, Rhamnaceae (buckthorn), Rhizophoraceae (red mangrove), Rhoipteleaceae, Rhynchocalycaceae, Rosaceae (rose), Rubiaceae (madder), Rutaceae (rue), Sabiaceae (sabia), Saccifoliaceae, Salicaceae (willow), Salvadoraceae, Santalaceae (sandalwood), Sapindaceae (soapberry), Sapotaceae (sapodilla), Sarcolaenaceae, Sargentodoxaceae, Sarraceniaceae (pitcher plant), Saururaceae (lizard’s tail), Saxifragaceae (saxifrage), Schisandraceae (schisandra), Scrophulariaceae (figwort), Scyphostegiaceae, Scytopetalaceae, Simaroubaceae (quassia), Simmondsiaceae (jojoba), Solanaceae (potato), Sonneratiaceae (sonneratia), Sphaerosepalaceae, Sphenocleaceae (spenoclea), Stackhousiaceae (stackhousia), Stachyuraceae, Staphyleaceae (bladdemut), Sterculiaceae (cacao), Stylidiaceae, Styracaceae (storax), Surianaceae (suriana), Symplocaceae (sweetleaf), Tamaricaceae (tamarix), Tepuianthaceae, Tetracentraceae, Tetrameristaceae, Theaceae (tea), Theligonaceae, Theophrastaceae (theophrasta), Thymelaeaceae (mezereum),29US_ACTI VE\132140149W-1Ticodendraceae, Tiliaceae (linden), Tovariaceae, Trapaceae (water chestnut), Tremandraceae, Trigoniaceae, Trimeniaceae, Trochodendraceae, Tropaeolaceae (nasturtium), Tumeraceae (turnera), Ulmaceae (elm), Urticaceae (nettle), Valerianaceae (valerian), Verbenaceae (verbena), Violaceae (violet), Viscaceae (Christmas mistletoe), Vitaceae (grape), Vochysiaceae, Winteraceae (wintera), Xanthophyllaceae, and Zygophyllaceae (creosote bush).

[0110] The monocotyledon can be selected from the group consisting of corn, wheat, oat, rice, barley, millet, banana, onion, garlic, asparagus, ryegrass, millet, fonio, raishan, nipa grass, turmeric, saffron, galangal, chive, cardamom, date palm, pineapple, shallot, leek, scallion, water chestnut, ramp, Job’s tears, bamboo, ragi, spotless watermeal, arrowlcaf elephant car, Tahitian spinach, abaca, areca, bajra, betel nut, broom millet, broom sorghum, citronella, coconut, cocoyam, maize, dasheen, durra, durum wheat, edo, fique, formio, ginger, orchard grass, esparto grass, Sudan grass, guinea corn, Manila hemp, henequen, hybrid maize, jowar, lemon grass, maguey, bulrush millet, finger millet, foxtail millet, Japanese millet, proso millet, New Zealand flax, oats, oil palm, palm palmyra, sago palm, redtop, sisal, sorghum, spelt wheat, sweet com, sweet sorghum, taro, teff, timothy grass, triticale, vanilla, wheat, and yam.

[0111] Alternatively, the monocotyledon can be selected from a family selected from the group consisting of Acoraceae (calamus), Agavaceae (century plant), Alismataceae (water plantain), Aloeaceae (aloe), Aponogetonaceae (cape pondweed), Araceae (arum), Arecaceae (palm), Bromeliaceae (bromeliad), Burmanniaceae (burmannia), Butomaceae (flowering rush), Cannaceae (canna), Centrolepidaceae, Commelinaceae (spiderwort), Corsiaceae, Costaceae (costus), Cyanastraceae, Cyclanthaceae (Panama hat), Cymodoceaceae (manatee grass), Cyperaceae (sedge), Dioscoreaceae (yam), Eriocaulaceae (pipewort), Flagellariaceae, Geosiridaceae, Haemodoraceae (bloodwort), Hanguanaceae (hanguana), Heliconiaceae (heliconia), Hydatellaceae, Hydrocharitaceae (tape grass), Iridaceae (iris), Joinvilleaceae (joinvillea), Juncaceae (rash), Juncaginaceae (arrow grass), Lemnaceae (duckweed), Liliaceae (lily), Limnocharitaceae (water poppy), Lowiaceae, Marantaceae (prayer plant), Mayacaceae (mayaca), Musaceae (banana), Najadaceae (water nymph), Orchidaceae (orchid), Pandanaceae (screw pine), Petrosaviaceae, Philydraceae (philydraceae), Poaceae (grass), Pontederiaceae (water hyacinth), Posidoniaceae (posidonia), Potamogetonaceae (pondweed), Rapateaceae, Restionaceae, Ruppiaceae (ditch grass), Scheuchzeriaceae (scheuchzeria), Smilacaceae (catbrier), Sparganiaceae (bur reed), Stemonaceae (stemona), Strelitziaceae, Taccaceae (tacca), Thurniaceae, Triuridaceae, Typhaceae (cattail), Velloziaceae, Xanthorrhoeaceae,, Xyridaceae30US_ACTI VE\132140149W-1(yellow-eyed grass), Zannichelliaceae (horned pondweed), Zingiberaceae (ginger), and Zosteraceae (eelgrass).

[0112] The gymnosperm can be selected from a family selected from the group consisting of Araucariaceae, Boweniaceae, Cephalotaxaceae, Cupressaceae, Cycadaceae, Ephedraceae, Ginkgoaceae, Gnetaceae, Pinaceae, Podocarpaceae, Taxaceae, Taxodiaceae, Welwitschiaceae, and Zamiaceae.

[0113] The present disclosure is also directed to plant seeds, which are coated with any of the compositions, inoculums, or bacteriologically pure bacterial cultures as described herein. The seed can be from any of the plants discussed in the foregoing sections belonging to monocotyledons, dicotyledons or gymnosperms. The bacterial inoculant or culture can be applied to the seeds through the use of a suitable coating mechanism prior to the seeds being sold into commerce for planting. The process of coating seeds with such an inoculum is generally well known to those skilled in the art. For example, the bacteria can be mixed with a porous, chemically inert granular carrier as described by U.S. Pat. No. 4,875,921, which is incorporated herein by reference with respect to such carriers. Alternatively, the bacterial inoculant can be prepared with or without a carrier and sold as a separate inoculant to be inserted directly into the furrows into which the seed is planted. The process for inserting such inoculants directly into the furrows during seed planting is also generally well known in the art. The density of inoculation of these bacterial cultures onto seeds or into the furrows should be sufficient to populate the sub-soil region adjacent to the roots of the plant with viable bacterial growth.

[0114] The present disclosure also relates to kits for inducing rhizosheath and / or rhizonest structures, increasing nutrient use efficiency, or increasing drought resistance in a plant, which include a composition or inoculum as described herein, and instructions for applying the same to plants, plant seeds, or a plant growth medium. Kits containing inoculants provided herein will typically include one or more containers of the inoculant, and printed instructions for using the inoculant. The kit can also include tools or instruments for reconstituting, measuring, mixing, or applying the inoculant, and will vary in accordance with the particular formulation and intended use of the inoculant.

[0115] As shown in Example 20, microorganisms provide a good system in which to select mutations for desired characteristics. It is possible to force such mutations through proper selection of desirable traits, while retaining the desired beneficial capabilities described herein. Accordingly, traits that may be desirable to induce in bacterial strains disclosed herein by31US_ACTI VE\132140149W-1forcing mutations without affecting promotion of rhizosheath and / or rhizonest structures, increased nutrient use efficiency, or increased drought resistance include, but are not limited to, drought tolerance, cold tolerance, biofilm / mucilage secretion, fungicide tolerance, herbicide tolerance, plant host compatibility, microbiome compatibility, soil microbiome compatibility, auxin activity. These attributes can be garnered by use of selective pressure or through manmade manipulation of a microorganism’s genetics.

[0116] Further details concerning the preparation of bacterial inoculants and methods for inoculating plants with bacterial inoculants are found in e.g. U.S. Pat. Nos. 5,586,411; 5,697,186; 5,484,464; 5,906,929; 5,288,296; 4,875,921; 4,828,600; 5,951,978; 5,183,759; 5,041,383; 6,077,505; 5,916,029; 5,360,606; 5,292,507; 5,229,114; 4,421,544; and 4,367,609, each of which is incorporated herein by reference with respect to such methods.

[0117] Having described the invention in detail, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims.DEPOSIT OF BIOLOGICAL MATERIAL

[0118] Purified cultures of microbial strains disclosed herein were deposited in the International Depositary Authority of Canada (IDAC), a National Microbiology Laboratory, Public Health Agency of Canda, located at 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2, in accordance with the Budapest Treaty for the purpose of patent procedure and the regulations thereunder (Budapest Treaty). The deposits have been accepted under the Budapest Treaty. The Accession No. for Bacillus velezensis strain 4E2b is 100125-01; and the Accession No. for Bacillus velezensis strain 4E2c is 150125-01. The date of deposit for these strains is January 10, 2025, and January 15, 2025, respectively. The microbial strains have been deposited under conditions that ensure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. The deposits represent substantially pure cultures of the deposited strains. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action. Preferred microorganisms of the present disclosure have all of the identifying characteristics of32US_ACTI VE\132140149W-1the deposited strains and, in particular, the identifying characteristics of being able to induce rhizosheath and / or rhizonest structures; increase nutrient use efficiency; or increase drought resistance as described herein. In particular, the preferred microorganisms provided herein refer to the deposited microorganisms as described above, and mutants thereof.EXAMPLES

[0119] The following non-limiting examples are provided to further illustrate the present invention.Example 1: Materials and Methods

[0120] Maize Root Growth Testing in Glass Jars: Com seeds were surface-sterilized (70% ethanol for 3 min with shaking; and washed 5 times with sterile ddfhO) and placed at 4°C in the dark overnight in water to promote germination. Seeds were dried under sterile conditions on filter paper and then inoculated with Bacillus velezensis strain 4E2b or the LB buffer control. The inoculant was prepared by adding 500 pl of the overnight bacterial culture in LB (OD595 = 0.53) into 10 ml of 10% PVP solution, which was then pipetted into a Petri dish containing the seeds. The seed-inoculant mixture was incubated for 2 h at room temperature on a shaker (180 rpm).

[0121] For growth in baby jars, 50 ml of media was used, and two seeds were placed into each sterile baby jar which was covered with a breathable lid. For growth in 4 L jars (height, 25 cm; diameter, 15 cm; 1 gallon, Catalog #S-19317B-M, Uline, Canada), 200 ml of media was used, and three seeds were placed into each sterile jar, which was closed with a cap. The jars were kept in the dark for one week and then transferred to light shelves (25 °C, full spectrum LED lights with an intensity of -90 mmol / m2 / sec, set at a 16 h light:8 h dark photoperiod). Inoculated and control tubes were randomized; tubes were continuously rotated every 3-4 days. Each treatment was conducted in triplicate (i.e., 3 jars). Roots were then photographed and examined for root growth promotion qualitatively.

[0122] Bacterial GFP Tagging, Plant Inoculation of Transgenic Strains, Light and Confocal Microscopy: Preparation of Bacillus competent cells: a single colony of Bacillus was inoculated into 5 mL of Neutral Complex Medium (NCM) broth (per L, 17.4 g K2HPO4, 11.6 g NaCl, 5 g glucose, 5 g tryptone, 1 g yeast extract, 0.3 g trisodium citrate, 0.05 g MgSO4-7H2O, 91.1 g sorbitol, pH 7.2) at 37°C for 48 h at 240 rpm. The overnight culture was then diluted 10-fold into fresh medium (50 mL final volume) and incubated at 37°C / 300 rpm for 7 h until the OD59533US_ACTI VE\132140149W-1reached 0.95-1.0. The tube was then kept on ice for 30 min, and the cell culture was collected by centrifugation at 4000 x g for 15 min at 4°C (Legend X1R Refrigerated Centrifuge, Thermo Scientific, USA). The supernatant was then carefully poured off and discarded. The pellet was gently resuspended in 12.5 mb cold ddlLO by gently up and down pipetting with a 1 ml pipette. The centrifugation and resuspension process were repeated three times to wash the pellet. The pellet was then resuspended in 5 mL of ice-cold 10% glycerol, then centrifuged again (as above); the supernatant was discarded, and the pellet resuspended in 100 pL of 10% ice-cold glycerol. The final cell suspension was dispensed into 50 pL aliquots, frozen in liquid nitrogen, and then stored at -80 °C for six months.

[0123] DNA transformation and selection'. Plasmid DNA (vector pDSK-GFPuv) was isolated using a plasmid miniprep kit (cat# 732-6100, Bio-Rad, USA). Into a chilled 1.5 mL tube, 40 pl of competent cells and 2 pl of plasmid DNA (180 ng / pl) were added, which were gently mixed then placed on ice for 1 min. The mixture was gently pipetted into a cuvette tube (2 mm, Catalog No# FB102, Bio-Rad, USA) that was pre-chilled in the freezer overnight. The cuvette was gently finger tapped, electroporated (Minipulser, Bio-Rad, USA according to manufacturer’s instructions) then immediately transferred to 1 mL SOC media and incubated on a shaker at 30°C or 37°C / 225 rpm for 3 h to allow the expression of the Kanamycin resistance gene. The transformed cells were then spread onto agar plates containing Kanamycin (for Bacillus, 100 pL on SOC plates; for Klebsiella, 50 pL on_LB plates) and incubated for 1-2 days at 30°C. The plates were examined for the presence of fluorescent GFP colonies, from which individual colonies were used to make glycerol stocks and stored at -80°C.

[0124] Plant inoculation of transgenic strains: To visualize GFP -tagged endophyte cells inside com roots, seeds were sterilized and coated with fluorescent bacterial strains. For the fungicide pre-treated seeds (Dekalb hybrid corn DKC38-55RIB, Bayer, Canada) were surface-sterilized (70% ethanol for 3 min with shaking, then washed 5 times with sterile ddFLO). For the inbred PHRE1, seeds were placed into a sterile 50 mL conical flask and surface-sterilized in 70% ethanol (v / v) for 3 min, 2.5% (v / v) sodium hypochlorite for 1.5 min, followed by washing 5-6 times in autoclaved ddlLO for 3 min. For both genotypes, seeds placed at 4°C in the dark overnight in water to promote germination. Seeds were dried under sterile conditions on filter paper and then inoculated with the fluorescent bacteria as described above. However, in these experiments, seeds were inoculated with one or two strains as indicated [each, 500 pl, OD595=0.5 added to 10 mL filtered 10% (w / v) PVP], placed on a shaker (180 rpm) for 2 h at34US_ACTI VE\132140149W-1room temperature, and then germinated in sterile glass jars (4 L, height, 25 cm; diameter, 15 cm; 1 gallon, Catalog #S-19317B-M, Uline, Canada), with tightened lids. Three seeds were added to each jar. Each jar contained 200 mL of Control Medium or No Nitrogen Medium. All jars were kept in the dark for one week before moved to the light shelves (conditions described above). The negative control seeds were treated with the LB buffer only. Each treatment was performed in triplicate (3 jars) and randomized. Jars were continuously rotated every 3-4 days. Plants were harvested after one month for light and confocal microscopy.

[0125] Confocal fluorescent microscopy. Three to four biological replicates per treatment were used to localize the fluorescent bacteria inside corn roots. Primary roots were cut at a distance of 0.5-1 cm (before the root hair initiation zone) from the seed by carbon steel surgical blades (size 11, #12-460-441, Fisher Scientific, USA). Each detached root system was placed onto Kimwipes to remove the attached gel media carefully and then put in Petri dishes. Root systems were left intact or cut as needed into longitudinal sections or cross-sections. Roots were then stained with propidium iodide solution (1.0 mg / ml in water) (#P4864-10ML, Sigma- Aldrich, USA) for 10 min, then gently washed 3-5 times using sterile ddH2O. Roots were examined using a Leica TCS SP5 confocal laser scanning microscope (DM 6000B upright microscope, Leica, Germany) at the Molecular and Cellular Imaging Facility at the University of Guelph, Canada. Root samples were placed onto the bottoms of 27 mm Nunc glass dishes (#150682, Thermo Scientific, USA). The excitation and emission wavelengths were 535 nm and 510-550 nm, respectively. The laser intensity was set at a maximum of 30%. and 543 nm with a HeNe laser set at 50%. The argon laser and the HeNe laser were used in sequence, by frame, and the intensity and the background of the image were adjusted with the smart gain and smart offset dial for each laser. The pinhole (Airy Unit, AU) was set to Airyl, and the objective lens to 10X dry. The default acquisition mode was xyz, the default format (pixel size) was 1024x1024 pixels at a speed between 10-100 Hz (lines / second) with different magnifications using an 10X objective lens. To optimize the image quality and adjust the intensity, the smart gain was between 560-890, the smart offset was 0.0-0.1% the pinhole was 180-190 pm, and the line average was 2.

[0126] Light microscopy. Following fluorescent microscopy, the same root samples were examined using a light microscope (MZ8 Stereomicroscope, Leica, Germany). Pictures were captured using a microscope digital camera 3.1 MP colour CCD camera (MU313-BI, COMOS, SONY, Japan) with AmScope software (version 3.7) (AmScope, USA) with a resolution 3584 x35US_ACTI VE\132140149W-12748 (approx. 10,000,000 pixels) attached to a light microscope. There were three biological replicates per treatment.

[0127] Plant Growth Media Recipes'. Control medium (no stress): A modified MS medium with nitrogen and phosphate was used, prepared as follows (per 1 L): Pi strength Murashige & Skoog basal salt mixture (Catalog #M571, Phytotechnology Laboratories, USA), 0.166 g of CaCh, 0.098 g of MgSCL (to solidify the medium), and 2 g Phytagel (P8169, Sigma, USA), supplemented with the following: 250 pl of 1 mg / ml nicotinic acid, 500 pl of 2 mg / ml glycine, 500 pl of 0.5 mg / ml pyridoxine HC1, and 5 ml of 100 mg / L thiamine HC1. The pH was adjusted to 5.8 with KOH before autoclaving. Water stress medium: this was the same as the control medium but supplemented with PEG 6000 (CAS Number: 25322-68-3, Sigma-Aldrich, USA) ranging from 10% to 30% (w / v) as indicated. PEG was added prior to autoclaving. Nitrogen stress medium: the MS medium without nitrogen consisted of (per 1 L): Pi strength Murashige & Skoog modified salts lacking nitrogen (Catalog #M531, Phytotechnology Laboratories, USA), 1.9 g / L potassium carbonate (K2CO3, to balance potassium levels), 0.166 g of CaCF, 0.098 g of MgSCL (to solidify the medium), and 2 g Phytagel (P8169, Sigma, USA), supplemented with the following: 250 pl of 1 mg / ml nicotinic acid, 500 pl of 2 mg / ml glycine, 500 pl of 0.5 mg / ml pyridoxine HO, and 5 ml of 100 mg / L thiamine HC1. The pH was adjusted to 5.8 with KOH before autoclaving.Example 2: 16S rRNA and whole genome sequencing of Bacillus velezensis 4E2b (Bv4E2b)DNA.

[0128] Bv4E2b was isolated in a greenhouse at the University of Guelph from a container growing an extant ancestor of com (maize) that grows in the wild in Mexico (Parviglumis teosinte). Although it is assumed that Bv4E2b originated from Parviglumis seeds, it is also possible that the roots captured it from the Canadian greenhouse environment, including sand, soil, water, air, contaminated pots or nearby plants that were simultaneously grown in the same greenhouse.

[0129] 16 rRNA sequencing of Bv4E2'. Initially, 16 rRNA sequencing was used to determine the bacterial taxonomy of the isolated strain. From a glycerol stock, a single colony from a restreak plate was cultivated in 5 mL of LB media (10 g tryptone, 3 g yeast extract, 5 g NaCl, and 12 g Bacto™ Agar) at 30°C with constant shaking at 200 ipm for two days. Following centrifugation, genomic DNA was extracted using the QIAamp DNA Mini kit (cat# 51306, Qiagen, Gemrany) according to the manufacturer's instructions. The PCR primer set was 27F36US_ACTI VE\132140149W-1[5'-AGAGTTTGATCMTGGCTCAG-3'] and 1492R [5' GGTTACCTTGTTACGACTT-3 ']. The 40 (il reaction mixture contained 20 pl Master Mix (M0486G, Biolabs), 1 il Forward Primer (10 (iM), 1 pl Reverse Primer (10 (iM), 2 pl genomic DNA (10 ng / pl), and 16 pl molecular grade H2O. PCR was conducted in an PTC200 Gradient Cycler (MJ Research, USA) using the following amplification conditions: initial denaturation at 96°C for 3 min, followed by 35 cycles of denaturation for 30 sec at 94°C, annealing for 30 s at 48°C, and extension for 90 sec at 72°C; then a final extension for 7 min at 72°C. PCR fragments (-1465 bp) were then confirmed and visualized via gel electrophoresis (1.5% agarose) using a Gel Doc XR+ System (Bio-Rad, USA). Amplicon DNA was purified from the PCR reaction using the Illustra GFX DNA and Gel Band Purification Kit (GE Healthcare, USA) with 30 pE of elution buffer, then submitted for sequencing to the Advanced Analysis Centre (AAC), Genomics Facility, University of Guelph. The 16S rRNA sequences were cleaned using BioEdit software (Informer Technologies, USA) and analyzed using nucleotide BLASTn against the 16S ribosomal RNA sequence database at the National Center for Biotechnology Information (NCBI) database. The results showed that this strain is a unique Bacillus strain.

[0130] Whole- genome sequencing of Bv4E2b: For better strain-level taxonomic resolution, whole genome sequencing was conducted. Bacterial genomic DNA was isolated from single colonies as described above and submitted to the Microbial Genome Sequencing Center (MiGS, Pittsburgh, USA) using Illumina (NextSeq 2000 platform). At MiGS, quality control, and adapter trimming were undertaken with bcl2fastq (version 2.20.0.445, default parameters). Species-level taxonomy was performed using MetaPhlAn3 (Tool: 3.0.7, December 9, 2020, database version; default parameters +£add_viruses). The short-read mode of Unicycler (version 0.4.8, default parameters) was employed as a SPAdes optimizer to generate short-read assemblies. Gene annotation employed PGAP (Tool: Build5132, database version January 11, 2021, using default parameters) and prokka (tool and database version: 1.14.5; using default parameters + ‘ — rnammer’ + ‘ — rfam’, added ‘ — metagenome’ when processing metagenomic / unclassified samples, added ‘ — kingdom Viruses’ when processing viral & bacteriophage samples). The whole genome sequencing confirmed that Bv4E2b is an authentic and previously unidentifiable strain of Bacillus velezensis.Example 3: Evidence that Bv4E2b and its derivatives are medically safe and clinically treatable strains for humans and livestock.37US_ACTI VE\132140149W-1

[0131] Taxonomically, B. velezensis belongs to biosafety risk group 1, and this example provides evidence that the Bv4E2b strain will be safe human exposure, including in agricultural fields: Bv4E2b was tested for antibiotic resistance. Out of 18 clinical antibiotics tested (FIG. 3), it was resistant to only one (FIG. 3). Bv4E2b was grown in overnight liquid cultures of LB broth to an ODeoo of >0.5. Based on the Kirby-Bauer protocol for antibiotic susceptibility (FIG. 3): Briefly, 500 pL of liquid culture was dispensed onto a 150 mm x 15 mm Petri dish containing 50 mL of Mueller-Hinton agar, spread using a sterilized bacterial cell spreader and a plate spinner, and left to dry for 5 min. Antibiotic discs (OxoidTM, UK) were placed onto the plate using sterile forceps. Plates were prepared in triplicate and incubated at 30 °C overnight, and zones of inhibition were measured with a transparent ruler. A ring of inhibition indicated that the bacterial isolate was susceptible to the antibiotic. These results indicate that application of Bv4E2b would be safe for farm workers or livestock, including those that are immunocompromised, because it should not cause infection, and if it did result in an unlikely infection, it would be easily treatable with a wide range of antibiotics.Example 4: Bv4E2b secretes a sugary, biofilm-mucilage.

[0132] Bv4E2b colonies secrete a sugary, biofilm-mucilage substance under water stress (30% PEG-6000) and nutrient limitation (FIG. 4), including when plated on nitrogen-free plant growth media (FIG. 4G). Bv4E2b colonies were grown under the following conditions on agar plates: (FIG. 4A): LB medium (no stress); (FIG. 4B): LGBM medium (LB plus 1% [vol / vol] glycerol and 0.1 mM MnSO4) medium, which used to induce biofilm formation; (FIG. 4C and D): LB medium supplemented with 30% PEG6000 to induce water limitation after culturing for (FIG.4C) 48 h and (FIG. 4D) 72 h; (FIG. 4E and F): M9 minimal medium after culturing for (FIG.4E) 48 h and (FIG. 4F) 96 h; (FIG. 4G and H): Bv4E2b colonies grown on low nitrogen plant growth medium, Phytagel MS531 (same media as plants) after culturing for (FIG. 4G) 72 h. For all experiments, representative colonies are shown, based on 4 agar plate replicates per treatment. These results demonstrate that Bv4E2b secretes a sugary, biofilm-mucilage under water stress (30% PEG-6000) and nutrient limitation.Example 5: Application of Bv4E2b on corn plants stimulates rhizosheath development.

[0133] In corn, light microscopy showed no interesting features on the maize primary root with or without abiotic stresses, nor when Bv4E2b was coated onto seeds and grown under non-stress conditions in Phytagel. However, when seed-coated Bv4E2b was grown under abiotic stress in Phytagel (low nitrogen or severe water limitation using 30% PEG-6000), formation of a38US_ACTI VE\132140149W-1mucilage-rich rhizosheath on the surface of the primary root as a continuous rhizoplane structure (FIG. 5A) was observed, between roots (FIG. 5B) or as discrete beads. After tagging Bv4E2b with green fluorescent protein (GFP) and imaging using confocal laser fluorescence microscopy, seed-coated Bv4E2-GFP was observed to colonize the surfaces of primary roots, root hairs and mucilage (FIG. 5C). In the confocal images, roots were stained with propidium iodide. The root hairs became elongated and appeared to serve as a structural scaffold for the mucilage (FIG.5C). In the early stages, there was an apparent air gap between the root surface and mucilage, bridged by the root hairs (FIG. 5C). The rhizosheath was also observed on primary root tips after Bv4E2-GFP-coatcd seeds were germinated in field soil, but not in non-inoculatcd seeds. In these earlier images, the root was washed prior to imaging. However, without washing, Bv4E2-GFP was observed to form biofilms on the root surface with soil particles. When plants were grown on a Turface clay substrate which allowed for less damaging excavation prior to imaging, Bv4E2-GFP was similarly shown to complex with clay particles on root surfaces, but sometimes a visible mesh of root hairs was observed inside the mucilage.

[0134] These results demonstrate that the application of Bv4E2b onto com plants surprisingly causes the bacteria to act as a master switch to stimulate rhizosheath development on root surfaces. This was unexpected given the fact that it was believed that rhizosheath formation was primarily under plant genome control. However, as described herein, effective amounts of compositions comprising Bv4E2b represent a novel inducer of the rhizosheath fomration.Example 6: Application of Bv4E2b onto water-limited corn plants stimulates rhizosheaths only on one side of the roots to create an osmotic gradient to drive water / nutrient uptake.

[0135] A fundamental property of eukaryotes is biological symmetry. In plants, the primary root is bilaterally symmetrical while root hairs display radial symmetry. In maize, alternating longitudinal files of epidermal cells can initiate root hairs, and as a result, root hairs are observed radially, i.e. 360°, around the axis of the primary root. Under mild nitrogen or water stress (10% PEG), Bv4E2b stimulated root hair elongation but not mucilage, making it easier to observe root hair developmental patterning (FIG. 6A-C). Under these conditions, seeds coated with Bv4E2b maintained root hair radial symmetry in the absence of stress (FIG. 6A) and under low nitrogen stress (FIG. 6B). However, remarkably, under mild water stress (10% PEG-6000), Bv4E2b suppressed root hair initiation on half (i.e., -180°) of the primary root cylinder, resulting in an asymmetric pattern of root hairs (FIG. 6C). The root hair asymmetry on primary roots was maintained into late-stage development (FIG. 6D-G). Under more severe stress conditions when39US_ACTI VE\132140149W-1mucilage development was stimulated, Bv4E2b further restricted its root hair initiation and mucilage formation relative to the primary root axis: under low nitrogen stress, bilaterally symmetric thin rhizosheaths (two) formed on opposite sides of the root cylinder (FIG. 6H); however under severe water limitation (30% PEG-6000), only a single thin asymmetric sheath formed, perpendicular to the root axis (FIG. 61). Bv4E2-GFP showed sporadic or symmetric colonization on the epidermal surface in the absence of stress, low phosphorus or low nitrogen at different stages. However, under water limitation, Bv4E2-GFP strictly colonized asymmetrically at all developmental stages, initiating endophytically inside the primary root endodermis and later on the primary root surface. Bv4E2-GFP colonization was always on the same side as the root hairs and mucilage (FIG. 6J). This result demonstrates that the Bv4E2b colonization is not coincident with the side of the primary root in which RH initiation is inhibited, but rather where it occurs. The asymmetry was also observed on post-embryonic crown roots under severe water limitation.

[0136] Furthermore, Bv4E2-GFP inoculated seeds were germinated in sand:Phytagel media (50:50) or in pure sand (FIG. 6K): the asymmetry was observed in both substrates under water limitation. The Bv4E-2 dependent asymmetry of root hair initiation on primary roots under water stress was shown here in 9 different experiments at different times over 2 years and under varying conditions (FIG. 6C, D-G, I, J, K). This asymmetry occurred consistently in additional experiments. Completely bare epidermal surfaces were cleanly observed in independent trials (e.g. FIG. 6C, D-F, I, J) where root hairs did not grow or bend to the opposite side of the root cylinder. The water-stress induced asymmetry was not observed in lateral roots. Images in (C, D-G, I, and J) represent independent trials conducted at different times over 2 years.. All images were from maize hybrid DKC38-55RIB, except (FIG. 6A-G), which are from inbred PHRE1. Abbreviations: LN, low nitrogen; HN, high nitrogen; LP, low phosphate; LW, low water.

[0137] These results demonstrate that application of effective amounts of Bv4E2b onto corn plants under water limitation causes it to switch the symmetry of its own colonization and dramatically alter the symmetry of root hair initiation and symmetry of mucilage / rhizosheath development on maize roots: i.e. the rhizosheath and associated root hairs only form on one side of roots. Such root asymmetry has never before been observed in any plant species in nature. This asymmetry presumptively creates an osmotic gradient to drive water uptake under water limitation (from low to high), which would also facilitate uptake of dissolved mineral nutrients40US_ACTI VE\132140149W-1(fertilizers) by maintaining a continuous zone along the root that is low in water (i.e. with no water-rich rhizosheath).Example 7: Application of Bv4E2b onto corn plants results in the development of proto- nodule-like spheres (rhizonests) constructed from root hairs and mucilage.

[0138] The following example demonstrates that effective amounts of Bv4E2b induce the development of proto-nodule-like spheres (rhizonests) constructed from root hairs and mucilage, which provide a habitat for beneficial microbes. For example, Bv4E2-inoculated plants, under severe water limitation (30% PEG-6000), showing mucilaginous rhizosheath were observed (FIG. 7A). A closeup of FIG. 7A showed a root hair cross-stitch pattern (FIG. 7B). After seed coating with Bv4E2b, the rhizosheath could further develop into elaborate bird's nest-like structures from root hairs, referred to herein as a rhizonest (FIG. 7C and D). Under mild water limitation (10% PEG-6000), root hairs were typically straight, but a single rhizonest, consisting of bending root hairs, could develop (FIG. 7C-F). By contrast, there were 1-3 rhizonests under severe water limitation (data not shown). A rhizonest could occur with mucilage under severe water limitation (FIG. 7A and B) or without mucilage under mild water stress (FIG. 7C-F). The rhizonest could be extended (FIG. 7C andD) or short (FIG. 7E-F). Rhizonests were typically associated with longer root hairs distal to the root tip (FIG 7A-G) but occasionally they could form close to the root tip. The rhizonests were colonized by Bv4E2-GFP (FIG. 7G) which could be ubiquitous (FIG. 7G) or discrete: this colonization remained asymmetric with respect to the primary root axis (FIG. 7G). Later in development (4 weeks after seed coating), rhizonests and associated Bv4E2-GFP colonization were also observed at the tip of post-embryonic crown roots. These results demonstrate that application of effective amounts of Bv4E2b can induce the formation of specialized habitats on the root surface, each composed of a nest of root hairs.

[0139] Normally, root hairs grow straight without Bv4E2b. However, to form its specialized rhizonest, Bv4E2b appeared to twist individual root hairs and bring them together in the early stage of nest formation (FIG. 8A). Here, Bv4E2b colonized individual root hairs at sites associated with bending (FIG. 8A), suggestive of an active process. In early stages, individual root hairs were sometimes observed to be completely colonized with Bv4E2b, while in later stages, individual root hairs were observed to be twisted at multiple sites. Under water stress, Bv4E2b stimulated individual root hairs to grow asymmetrically, with the orientation of tip growth alternating direction in a recurring pattern, resulting in helix-like structure (FIG. 8B, C, D). The root hair growth asymmetries resulted in complex patterns when multiple hairs41US_ACTI VE\132140149W-1interacted (FIG. 8E). When root hair bending occurred at right angles, and multiple times within individual root hairs, perpendicular cross-stitch patterns were formed (FIG. 8E). When the root hair bending was circular, the cross-stitch resulted in knots (FIG. 8F); when alternating, multiple root hairs formed complex helices. These cross-stitch patterns were also observed in field soil. All images were from maize hybrid DKC38-55RIB, except as noted.

[0140] Under water or nitrogen limitation, at specific locations on roots, application of effective amounts of Bv4E2b onto com plants under can cause asymmetric growth of individual root hairs, resulting in elaborate nest-like cross-stitch patterns including fascinating structures described herein as ‘rhizoncsts’. These rhizoncsts arc presumptive, proto-nodules that create a protective mucilage rich habitat for beneficial microbes on root surfaces (including as shown later, nitrogen-fixing bacteria), to protect them against competition from soil predators, with the walls being intertwined root hairs that elaborate cross-stich patterns. Such rhizonest structures induced by Bv4E2b are remarkable and are not believed to be observed in any plant species previously.Example 8: Induction of rhizosheath and rhizonests structures by Bv4E2b provides habitats for companion nitrogen-fixing bacteria including Klebsiella variicola

[0141] It was originally believed that Bv4E2b encodes genes for nitrogen fixation. However, despite repeated whole genome sequencing, no canonical open reading frames encoding the structural components of nitrogenase were discovered. Interestingly, it was also found that although the developing rhizosheaths and rhizonests contain abundant Bv4E2-GFP, the mucilage in mature structures appeared to be largely devoid of this microbe . It was hypothesized the chemical properties of mucilage itself may have optically prevented fluorescence detection. To determine the extent to which the mucilage was colonized by Bv4E2b, com seeds were inoculated with Bv4E2-GFP and the mucilage was dissected away from the root system. Microbiome profiling was then carried out using PacBio full-length 16S rRNA-based high throughput metagenomic sequencing. As a control, under non-stress conditions, in which no detectable mucilage is observed, the root surface tissue was scraped, enriched in tissues where colonization by Bv4E2b was visually observed, including the rhizoplane in particular and root hairs. Consistent with the confocal findings, in all 3 biological replicates, the maize root surface consisted almost completely of the inoculant, B. velezensis (78-99%). However, surprisingly, the mucilage induced by Bv4E2b under the low nitrogen conditions, was inhabited by bacteria, but not B. velezensis (only 0-0.5% relative abundance);42US_ACTI VE\132140149W-1rather this mucilage was inhabited almost entirely of two other bacteria, Klebsiella variicola (12%, 21%, 99% relative abundance in replicates) and Stenotrophomonas maltophilia (79%, 87%, 0.5% relative abundance in replicates) which appeared to be competing with one another. Similarly, under water limitation, the mucilage microbiome was dominated by K. variicola (37-85% relative abundance) and .S'. maltophilia (11-42% relative abundance), though significant B. velezensis was observed (3-20%), though nevertheless, the non-B. velezensis fraction was dominant. Other suspected plant beneficial bacteria were also observed as minor members of the mucilage microbiome including Stenotrophomonas pavanii, Bacillus nakamurai, and Pantoea ananatis. To verify this result, bacteria were directly cultured from the mucilage and the taxonomic identities of retrieved colonies were verified by 16S sequencing; consistent with the microbiome data, only 3 taxa of cultured bacteria were identified, namely B. velezensis, K. variicola and .S', maltophilia. K. variicola and S. maltophilia are well known nitrogen fixing bacteria in the literature; our own whole genome sequencing of K. variicola from maize showed the presence of genes encoding nitrogenase. Thus, under nitrogen or water limitation, application of effective amounts of Bv4E2b onto corn plants, results in a mucilage-rich, underground protective habitat on root surfaces for companion nitrogen-fixing bacteria including Klebsiella variicola.Example 9: The rhizosheath / rhizonests induced by the application of effective amounts of Bv4E2b contain sugars known to feed nitrogen-fixing bacteria.

[0142] Monosaccharides can support the growth of nitrogen-fixing bacteria. The monosaccharide composition of the underground root surface mucilage induced by application of effective amounts of Bv4E2b onto corn plants was investigated. Plants were grown in 4 L jars containing Phytagel (see Example 1 for media and growth conditions). As no stress does not produce mucilage, there were two treatments, Bv4E2b low nitrogen and Bv4E2b low water. There were 3 replicates per treatment, with one replicate representing the pooled mucilage of 3 plants in one jar. The treatments were randomized. To the roots, absolute ethanol was added to a final concentration of approximately 80% followed by a short period of vigorous shaking, done manually. Residual root / root hair tissue inside the mucilage was removed manually, using forceps, and the remaining suspension was kept at -80°C overnight. The mucilage was centrifuged at 4700 x g for 30 minutes at 4°C. The supernatant was discarded, and the mucilage was washed twice with 2 mL of 80% ethanol. The mucilage pellet was dissolved in 35 mL dH2O and frozen at -80°C, followed by lyophilization. Replicate mucilage samples: Bv4E2 / low43US_ACTI VE\132140149W-1N (Rl: 1.11 mg, R2: 0.77 mg, R3: 7.81 mg), and Bv4E2 / low water (Rl: 1.46 mg, R2: 1.98 mg, R3: 0.15 mg) where R=replicate, were prepared for high-pH anion-exchange chromatography (HPAEC) as follows: each lyophilized mucilage sample was dissolved in 500 uL of 6 M HC1. Samples were acid hydrolyzed at 96 °C for 1 h. Acid was removed in vacuo with heat, and then 500 uL of dH2O was added to each sample to create a stock solution. High-pH anion-exchange chromatography (HPAEC): For HPEAC analysis of carbohydrate content, a Dionex CarboPacTM PA20 analytical column (ThermoFisher, USA) was used combined with a Dionex CarboPacTM PA20 guard column (ThermoFisher, USA). The column compartment was set at ambient temperature. For HPAEC analyses, the column was equilibrated in 10 mM NaOH at a flow rate of 0.5 niL / min. Samples (10 uL) were injected into the column, and the 10 mM NaOH eluent was maintained for 20 minutes to separate neutral and amino sugars. This was followed by applying a 5 min gradient to 100 mM NaOH and then a further 5 min gradient to 100 mM NaOH + 150 mM sodium acetate to separate acidic sugars. Following 5 min in this final condition, the eluent was returned to 10 mM NaOH over 10 min, and the column was allowed to re-equilibrate for 15 min between sample injections. Undiluted and 10-fold dilutions of stock mucilage samples were analyzed. Select monosaccharides were chosen to spike the samples to confirm analyses based on previous identifications in maize mucilage with the exception of glucosamine. These sugars were added within a range from 0.1 - 10 nmoles. High-pH anion-exchange chromatography (HPAEC) based analysis showed that the sugar composition of the Bv4E2-induced mucilage, under the Phytagel media conditions used, after hydrolysis, was similar under water or nitrogen limitation. Both types were dominated by glucose (nitrogen limitation, 60% w / w; water limitation, 72%), followed by either galactose (8-20%) or galacturonic acid (11-13%), and then fucose (3-5%) . Additional monosaccharides were also present in lower concentrations (<2%) including rhamnose, glucuronic acid, glucosinamine, xylose and arabinose.

[0143] These results demonstrate that applying effective amounts of Bv4E2b onto plants induces a root surface mucilage that is rich in polysaccharides which when hydrolyzed result in simple sugars, and that these sugars are those known to support beneficial microbes, in particular, but not limited to, nitrogen-fixing bacteria and the biochemical nitrogen-fixation reaction itself.Example 10: Synergistic combinations of Bacillus velezensis (Bv4E2b) and Klebsiella variicola.44US_ACTI VE\132140149W-1

[0144] Bv4E2b and a strain of Klebsiella variicola (Kv3Hl) were seed inoculated together under water limitation, in replicated experiments, the dual inoculation promoted improved growth of root hairs which are the scaffolds for rhizosheath and rhizonest mucilage, with the mucilage not produced given the experimental conditions used (FIG. 9). Maize primary roots after inoculation with: (FIG. 9A) Bv4E2b under low nitrogen (control, FIG. 9A); Bv4E2b + Kv3Hl under low nitrogen (control, FIG. 9B); Bv4E2b under water limitation, showing asymmetric growth of root hairs (one side) (FIG. 9C); Bv4E2b + Kv3Hl under water limitation, showing improved, denser root hair growth compared to either inoculant alone (FIG. 9D). The images shown in (FIG. 9A-D) arc representative of 3 biological replicates, tested in independent baby jars. As such, Bv4E2b and Klebsiella variicola co-inoculation provide synergistic effects on plant traits.Example 11: Co-application of Bv4E2b and auxin stimulating compounds onto corn plants enlarges the rhizonest habitats for nitrogen fixing bacteria.

[0145] Bv4E2b was found to be positive for the standard colorimetric test for bacterial auxin secretion which detects indole containing compounds but at a relatively low concentration (FIG.10A; left tube = buffer; right tube = Bv4E2b. Auxin is known to stimulate root elongation and root hair growth. Based on the results described herein, the ability of Bv4E2b phenotypes to be promoted or altered by co-application with exogenous auxin was tested. In the absence of exogenous auxin (indole acetic acid, IAA) Bv4E2b caused root hair asymmetry along the primary root under water limitation (10% PEG-6000) (FIG. 10B). However, in the presence of auxin in the media, root hair growth symmetry was gained along the primary root (FIG. 10C). Moreover, in independent replicates, Bv4E2b + IAA improved root hair growth (FIG. 10C andE) and resulted in large rhizosheath / rhizonest at the root tip based on confocal microscopy where the green are the GFP-labelled Bv4E2b cells, and the red are the root tip of maize (FIG.10D). Dramatically, the co-application of Bv4E2b + IAA promoted rhizonest root hairs to grow symmetrically (straight) and longer, resulting in dramatically enlarged rhizonests amongst independent replicates (FIG. 10F and G) which is noteworthy since rhizonests are the habitat for nitrogen-fixing bacteria.

[0146] These results demonstrate that co-application of Bv4E2b and an auxin stimulating compounds onto plants results in remarkably elongated root hairs (and presumably associated rhizosheath) and remarkably large rhizonest habitats for nitrogen fixing bacteria (and45US_ACTI VE\132140149W-1presumably other beneficial microbes). These unusual, elaborate, large structures constructed out of cross-stitched root hairs are not known to develop in nature.Example 12: Bv4E2b inoculation promotes maize seedling growth under low nitrogen conditions.

[0147] The results presented in FIG. 11 demonstrate that under low nitrogen (LN) conditions, Bv4E2b seed inoculation improved seedling shoot and root growth of maize in diverse genotypes (inbred PHRE1, FIG. 11 A; commercial hybrid DKC3855RIB, FIG. 1 IB). As such, Bv4E2b inoculation, and its rhizosheath / rhizonest benefits, promote maize seedling growth under low nitrogen (LN).Example 13: Bv4E2b survives water limitation and its application onto corn seeds protects seedlings against severe water limitation.

[0148] As demonstrated herein, Bv4E2b shows excellent tolerance to polyethylene glycol (PEG-6000), which when added to agar, causes water limitation. Bv4E2b in vitro was demonstrated to grow at PEG6000 concentrations as high as 40% (w / v), much better than a control bacterial strain 3D9 (FIG. 12A). When coated onto corn seeds, Bv4E2b conferred tolerance to moderate and severe water limitation, mimicked by adding PEG6000 (FIG. 12B-D). This trait has not been reported before for this strain. In this figure, LW=low water, while WW= well watered. This example and the results presented in FIG. 12 demonstrate that the rhizosheath stimulation by Bv4E2b application onto com seeds can protect seedlings against severe water limitation under controlled conditions.Example 14: Application of Bv4E2b onto corn plants under dual nutrient and water limitation results in an enlarged rhizosheath and enhanced delivery of nitrogen to host plants.

[0149] The thickness of the mucilage induced by Bv4E2b application on the maize primary root surface under nitrogen limitation was variable but generally moderate as a ratio of the root width, compared to water limitation where the mucilage was thicker. Under water limitation, however, the mucilage formed asymmetrically, along only one half of the primary root axis. When the stresses were combined, the mucilage was on both sides, generally thicker as a ratio of root width and more consistent, especially in field soil. Since the mucilage under either nitrogen or water limitation contained similar carbon-rich metabolites and companion nitrogen-fixing bacteria, it was hypothesized that the increased mucilage of the dual stress would support an overall increased microbial habitat for nitrogen fixation, compared to either stress alone. To test46US_ACTI VE\132140149W-1this hypothesis, replicated growth room trials were conducted with maize (hybrid DKC3855RIB) in large pots with 50:50 peat:sand substrate containing only starter fertilizer, and hence all the plants were grown under low nutrients (LN) (FIG. 13A-G). Plants were either well watered (WW) or under water limitation (low water, LW) (n=4 plants per treatment per trial with each replicate in a separate pot). These were large pot maize trials. Pots (20 cm diameter) were sterilized with 70% alcohol, then washed with distilled water five times, and then filled with sterilized peat:sand (50:50 w / w); the peat was BM6HP (Berger, Canada, containing peat moss, perlite, limestone, and starter fertilizer. Three surface sterilized maize seeds (DKC38-55RIB hybrid) were planted into each pot, that had been coated with Bv4E2b in LB or an LB buffer control (see Example 1 for seed coating treatment protocol). Briefly, Bv4E2b (ODeoo = 0.95) was cultured for 30 h in a 250 mL flask on a shaker 225 rpm / 37°C; then seeds were coated from this stock using (10% v / v PVP) and placed on a shaker at 180 rpm for 2 h and dried on filter paper before planting. There were two trials. Trial 1 was in an indoor growth room and Trial 2 was in a plant growth chamber. For Trial 1, the conditions were: 25° C day, 20°C night, RH 70 %, with a 16 h light:8 h dark photoperiod. For Trial 2, plants were grown in a Conviron chamber at 22° C day, 22°C night, RH 60%, with a 16 h light:8 h dark photoperiod. All treatments contained low nutrients (LN), with only starter fertilizer (including nitrogen) in the commercial peat mix, and no additional fertilizer added during the trials. There were 4 treatments under this condition: Bv4E2b + low water (LW), Buffer + low water (LW), Bv4E2b + well watered (WW), Buffer + well watered (WW). Low water was defined as watering once per week, while well-watered was defined as watering three times per week, with each watering consisting of 500 ml per pot. However, in the first week, all plants were watered daily (500 ml per pot) to promote germination. There were 3 biological replicates per treatment in Trial 1, and 4 replicates in Trial 2. One replicate was defined as a single pot with 3 plants. Pots were randomized, and rotated continuously every 3 days throughout the experiment. Plants were harvested at 5 weeks from inoculation, and each pot was watered 5 h before the roots were harvested to facilitate excavation. Both root and shoot fresh weights were weighed and recorded, with each replicate representing a pool of 3 plants within a pot. For root biomass, after excavation, roots were gently shaken to remove the peat: sand, then washed with tap water, and then placed on paper towels to dry for 15 min, prior to weighing. Nitrogen analysis was only conducted in Trial 2. For nitrogen analysis, leaf #5 (counting from the base) was used for measuring the total N, while for roots, the entire root system was used. Per replicate, 3 leaf47US_ACTI VE\132140149W-1samples or 1 intact root sample per replicate were sent to the University of Guelph Food Laboratory for total N content quantification with the Dumas combustion method using a Leco CN828 nitrogen determinator (LECOCorp., St Joseph, MI, USA) which uses catalytic combustion (950 °C).

[0150] By five weeks, the shoots and root systems of Bv4E2b seed-inoculated plants were visually larger than the buffer controls (FIG. 13A and 13B). With one exception, the corresponding root and shoot fresh weights were statistically different (at p<0.05), consistent with the visual findings (FIG. 13C-F). Notably, inoculated plants grown under the dual stress (LN+LW), despite facing two significant stresses simultaneously, were visually much greener than the single stressed plants (LN+WW) (FIG. 13A). When quantified, the total percentage nitrogen content of the shoots of inoculated double-stress plants was, surprisingly, double that of any other treatment and significantly different (at p<0.01) (FIG. 13G), though the tissue nitrogen was only measured in Trial 2. For the graphs shown in FIG. 13, the means (n=4) were compared using one way ANOVA followed by a Tukey’s adjustment for pairwise comparisons; the lines above the histograms show the significance of pairwise comparisons at the following thresholds: *=P<().()5, **=P<0.01, ***=P<0.001; ns, p>0.05. The error bars represent the standard deviation (SD). These results suggest that application of Bv4E2b onto com plants under dual nutrient and water limitation results in its remarkable ability to deliver more nitrogen to its host plants, compared to either stress alone, by building an enlarged (thicker), sugar-rich rhizosheath habitat for companion bacteria including nitrogen-fixers.Example 15: Grain yield impact of Bv4E2b as a foliar spray under low N-fertilizer, without irrigation.

[0151] The rhizosheath and rhizonest benefits of applying effective amounts of Bv4E2b were tested as a foliar spray on a commercial maize hybrid under field conditions when N fertilizer was kept very low. In these trials there was no irrigation. The com variety used was Dekalb C33-78RIB which was not pre-coated with other biologicals. The trial was conducted in 2022 at the Elora Field Station, University of Guelph, Canada. Zero nitrogen fertilizer was applied, while other fertilizers (e.g. P and K) were not limiting. The trial was conducted as 4 field blocks, in an RCBD design. Plots were 4 m long, each consisting of 4 rows, with 4 m borders. In this first experiment, there were two foliar applications of Bv4E2b at approximately V3-V4 stage and again at the V8-V10 stage (in LB buffer with Silwet surfactant). There were 5 negative controls including the Bv4E2b buffer, and 3 positive controls which consisted of 45 kg N / ha (25% total48US_ACTI VE\132140149W-1N rate) of nitrogen applied early, late or split during the growing season (FIG. 14). Bv4E2b foliar applications under very low nitrogen fertilization conditions and without irrigation resulted in an -10% com grain yield increase under field conditions, comparable to 45 kg / ha of nitrogen fertilizer (FIG. 14).Example 16: Grain yield impact of Bv4E2b as a foliar spray with 75% N-fertilizer, without irrigation.

[0152] The rhizosheath and rhizonest benefits of Bv4E2b as a foliar spray were also tested on multiple commercial maize hybrids under field conditions when N fertilizer was raised to the >75% optimal rate (equivalent to 160 kg N / ha) for all treatments (FIG. 15). There were 4 commercial com hybrids used (from Pioneer, Dekalb and NK), which were coated or not seed coated with other biologicals. The trial was conducted in 2023 at the Elora Field Station, University of Guelph, Elora, Ontario, Canada. There was no irrigation. In total 160 kg / ha urea was applied (50% at planting, and 50% late season). At planting granular 6-20-20 was applied @150 kg / ha (achieving 100% optimal rate for K and P), with the remaining added as a granular top-dress. This split fertilization was used to match the dual application of Bv4E2b. Planting was at a density of 34,000 seeds / ha. There were two foliar applications of Bv4E2b or the mock control at approximately V2-V3 stage and again at the V8-V9 stage (in Tris buffer with Silwet surfactant). The trial was conducted as 6 large field blocks, in an RCBD design. The field was cultipacked prior to planting. Roundup and Acuron were used as recommended. Compared to the buffer-only control, with 75% N added, Bv4E2b increased grain yield from 3.5-9.5% in two varieties (significant at P=0.068 in the Pioneer 88-59 variety), with no improvement in the remaining 2 varieties (FIG. 15). The results demonstrate that under field conditions, Bv4E2b as a foliar spray can improve maize grain yield by up to nearly 10% in conjunction with a 75% optimal N fertilizer rate and without irrigation, in specific maize commercial varieties.Example 17: Grain yield impact of Bv4E2b as a seed coating agent with 75% N-fertilizer, without irrigation.

[0153] The rhizosheath and rhizonest benefits of Bv4E2b as a seed coating agent were also tested on multiple commercial maize hybrids under field conditions in conjunction with N fertilizer at the >75% optimal rate (equivalent to 160 kg N / ha) for all treatments (FIG. 16). There were 4 commercial com hybrids used (from Pioneer, Dekalb and NK), which were coated or not seed coated with other biologicals. The trial was conducted in 2023 at the Elora Field Station, University of Guelph, Elora, Ontario, Canada. There was no irrigation. In total 16049US_ACTI VE\132140149W-1kg / ha urea was applied, entirely at planting as granular 6-20-20 (achieving 100% optimal rate for K and P): this fertilization was used to match the sole initial application of Bv4E2b. Planting was at a density of 34,000 seeds / ha. Bv4E2h with PVP as an adhesive was coated onto seeds, while the mock control was coated with the buffer only. The trial was conducted as 6 large field blocks, in an RCBD design. The field was cultipacked prior to planting. Roundup and Acuron were used as recommended. Compared to the buffer-only control, with 75% N added, Bv4E2b increased grain yield from 3-6% in two varieties, with the greatest numerical increase occurring in the only variety not already coated with biologicals (Dekalb), with no improvement in the remaining 2 varieties (FIG. 16). The results demonstrate that under field conditions, Bv4E2b as a seed coat can improve maize grain yield in conjunction with a 75% optimal N fertilizer rate and lack of irrigation.Example 18: Effect of non-Bv4E2b Bacillus velezensis strain on grain yield under low nitrogen conditions without irrigation.

[0154] Another isolated endophytic Bacillus velezensis strain (BvLG51) was also tested as a foliar spray on a commercial corn hybrid to test whether B. velezensis could similarly improve maize yields under low nitrogen (and hence might stimulate rhizosheath and rhizonest formation). The trial was conducted in 2021 at the Elora Field Station, University of Guelph. The trial was conducted as 4 field blocks, in an RCBD design. Plots were 4 m long, each consisting of 4 rows, with 4 m borders. Zero nitrogen fertilizer was applied pre-plant, while other fertilizers (e.g. P and K) were not limiting. There was no irrigation. There were two foliar applications of BvLG51 at approximately V3 stage and again at the reproductive / silking stage (in LB buffer with Silwet surfactant). There were 5 negative controls including the BvLG51buffer, and 3 positive controls which consisted of 45 kg N / ha (25% total N rate) of nitrogen applied as an early side-dress (FIG. 17). Compared to the negative controls, BvLG51 resulted in an -20% grain yield decrease. This result demonstrates that the beneficial technical effects yielded by Bv4E2b is not common to every Bacillus velezensis strain suggesting that Bv4E2b and its mutants are unique.Example 19: Winter Wheat grain yield impact of Bv4E2b as a foliar spray under low N- fertilizer, without irrigation.

[0155] This following example investigated whether the rhizosheath and rhizonest stimulating benefits of Bv4E2b could be beneficial in other crops in addition to com. Bv4E2b as a foliar spray was tested on 8 commercial winter wheat varieties under field conditions (FIG. 18). The50US_ACTI VE\132140149W-1trial was conducted in 2022 at the Elora Field Station, University of Guelph, Canada. Zero nitrogen fertilizer was applied at planting, while other fertilizers (e.g. P and K) were not limiting. There was no irrigation. The trial was conducted as 3 field blocks, in an RCBD design. Plots were 1 m long, each consisting of 6 rows, with 0.5 m borders. Plants were sown in the Fall of 2021, and Bv4E2b was applied in June 2022. There was a single foliar application of Bv4E2b at a late vegetative stage (in LB buffer or with Silwet surfactant). There were 3 negative controls including the Bv4E2b buffer, and 4 positive controls which consisted of 20 kg N / ha (25% of optimal N rate) (FIG. 18). Compared to the negative controls, averaging results across all 8 varieties, the results demonstrate that Bv4E2b application onto diverse winter wheat varieties, grown without nitrogen fertilizer and without irrigation, results in an ~10%-15% grain yield increase. This yield increase is comparable to 20 kg / ha of nitrogen fertilizer (FIG. 18).Example 20: Spring Wheat grain yield impact of Bv4E2b at different dosages as a foliar spray under low N-fertilizer and without irrigation.

[0156] A field trial was undertaken to determine whether the rhizosheath and rhizonest stimulating benefits of Bv4E2b could be transferred to spring wheat. Bv4E2b as a foliar spray was tested on commercial variety OAC Wilken under field conditions (FIG. 19 and FIG. 20). The trials were conducted in 2022 at the Elora Field Station, University of Guelph, Canada. Zero nitrogen fertilizer was applied at planting, while other fertilizers were not limiting (e.g. 20 kg / ha P and K). There was no irrigation. The trial was conducted as 6 field blocks, in an RCBD design. Plots were 1 m long, each consisting of 7 rows, with 1 m borders. Plants were sown in the spring of 2022, and Bv4E2b was applied in the spring or summer, with Silwet. The negative controls were a mock buffer treatment, while the positive controls consisted of 20 kg N / ha (25% of optimal N rate). In the first experiment, increasing concentrations of a single Bv4E2b application at a vegetative stage was shown to linearly increase grain yield compared to the mock buffer controls (FIG. 19). In the second experiment, two applications of Bv4E2b at different vegetative stages increased grain yield by -20% compared to the mock buffer controls (FIG. 20). This result demonstrates that Bv4E2b application improves grain yield of spring wheat under low fertilizer, non-irrigated field conditions in a dosage-dependent manner.Example 21: Mutants of Bv4E2b for improved growth and tolerance to cooler soil temperatures.

[0157] At the time of rhizosheath and root microbiome establishment in May-early June in the Northern Hemisphere, at the 25 cm depth that corn roots rapidly grow towards, the soil51US_ACTI VE\132140149W-1temperature is only 13-18°C. Our prior results demonstrated that Bv4E2b grows robustly at warmer temperatures, e.g. 28-37 C. This means that the strain may be outcompeted by competitor bacteria on the root surface, resulting in poor rhizosheath and rhizonest formation. There have been no prior efforts to improve the cold tolerance of Bv4E2b which was our objective. Bacterial colony-based laboratory directed evolution was undertaken, consisting of multiple rounds of selection for spontaneous genetic mutations that increased strain survival at progressively decreasing temperatures. The strategy consisted of four phases (I-IV) :

[0158] Phase I, the environmental conditions for the evolution were designed. It was determined that the evolution should be undertaken in Petri dishes on semi-solid, nutrient-rich agar media (rich in sugar, nitrogen, and other minerals) to mimic the root surface-rhizosphere interface which is enriched in carbon exudates and mineral nutrients.

[0159] Phase II, the starting lower temperature tolerance limit and growth kinetics of the strain were mapped using this media: the strain was grown at different low temperatures until no growth was observed; in parallel, time courses were conducted to determine the number of hours until the first appearance of bacterial colonies at these low temperatures. Tiny colonies were observable at 15°C which was determined to be the minimal viability temperature (MVT) and starting point for the evolution.

[0160] Phase III was the evolution itself, consisting of 5 cycles of selection during which the temperature was progressively decreased to the target temperature of 13"C. At the start of each cycle, a single colony was selected, diluted in sterile water, and then 10-fold serial dilutions were plated onto Petri dishes containing the nutrient-rich agar; plates that had distinct, observable colonies were used in the screens. Petri dishes were placed at the desired temperature, then screened every few hours in the pre-mapped growth interval: the first colonies that appeared were marked and then individually tracked over a continuing time course consisting of several days. At the endpoint, the largest colony amongst the early growers was selected: this approach simultaneously selected for aggressive growth and cold-temperature tolerance. The first 2 cycles focused on improving Bv4E2b growth at the 15°C starter temperature.

[0161] Phase IV was to measure the impact of the evolution. A quantitative side-by-side comparison was undertaken of the starter strain (Bv4E2b) and the cold-adapted strain (Bv4E2c, where c=cold). In a replicated trial, each strain was grown to the same concentration (OD600), similarly serially diluted and then plated onto 2 sets of agar plates which were paired: one52US_ACTI VE\132140149W-1intended for incubation at 13°C and the other at the pre-evolutionary optimal temperature of 30°C. The results were as follows: first, the cell viability of Bv4E2c at 13°C, measured as colony forming units (CFUs), had been improved by an average of 6.9-fold, measured as the ratio of CFUs that formed at 13°C versus 30°C (FIG. 21). Second, across 25-30 replicates, the growth rate of Bv4E2c at 13°C was measured to have increased by an average of 24% compared to Bv4E2b; this was measured as the surface area of any surviving colony (measured using ImageJ software) within a given time point, and hence was a measurement of aggressive growth (FIG.22). It should be noted in this comparison that there were 7-times more Bv4E2c colonies that survived at 13 °C compared to Bv4E2b colonics. Finally, using the same technique, across 22-26 replicates, the growth rate of Bv4E2c at 30°C was measured to have increased by an average of 75% compared to Bv4E2b (FIG. 23). Though not the intention, this result demonstrated the evolved Bv4E2c strain had dramatically more aggressive growth than the starter strain at warmer temperatures, which means that when applied as a seed coat or foliar, it is more likely to outcompete competitor microbes at the warmer temperatures of the soil surface or above ground, which is important for establishment in the plant. Strain Bv4E2c has been deposited with the International Depository Authority of Canada (IDAC), and assigned IDAC Accession No.100125-01.

[0162] Combined these results demonstrate quantitatively that laboratory evolved, mutant strain Bv4E2c has 7-fold improved viability than Bv4E2b at lower temperatures, and more aggressive growth at both low and optimal temperatures. Additional cycles and modifications of this strategy can be used to screen for further mutants that improve the efficacy of Bv4E2b and Bv4Ec. When applied onto plants, evolved strain Bv4E2c is predicted to cause improved plant growth, perhaps associated with more robust rhizosheath / rhizonest formation, including at cooler temperatures.Example 22. Prophetic genetic improvement of Bv4E2 / Bv4E2c strain lineages.

[0163] Analogous to the efforts described in Example 21 to genetically improve Bv4E2b, human directed evolution to further increase the effectiveness of the strain will be carried out. For example, experiments will be carried using Bv4E2b / Bv4E2c to select for mutants exhibiting more consistent mucilage production and / or increased rhizosheath or rhizonest formation. Examples of such methods are outlined as follows:

[0164] Method A. Additional cycles of laboratory directed evolution may be employed, using the same or similar methodology as in Example 21, to select for more aggressive strain growth53US_ACTI VE\132140149W-1and improved tolerance to adverse environmental conditions. In place of progressively cooler temperatures, the selection conditions may include, but are not limited to, progressively more extreme water limitation, extreme temperature, altered pH, limiting or excess mineral nutrients, toxic soil compounds, the presence of competitor microbes, and / or antibiotics predicted to be secreted by competitors in the plant / soil microbiome.

[0165] Method B. Additional cycles of laboratory directed evolution may be employed to genetically improve mucilaginous biofilm secretion from Bv4E2 / Bv4E2c lineages. The strategy is a modification of that demonstrated in Example 21. Briefly, on solid semi-agar plates, large numbers of potential genetic variants of Bv4E2 / Bv4E2c colonics would be visually screened for thicker, more robust mucilaginous biofilm. At each evolutionary cycle, the colony with the greatest mucilaginous biofilm would be selected, then multiplied to obtain potential new genetic variants, and then re-plated for additional screening cycles. To mimic the rhizosphere, the selection conditions may include, but are not limited to, progressively increased water limitation, extreme temperature, altered pH, and / or increasing nitrogen. Alternatively or in addition, gene editing may also be carried out, including but not limited to, editing of bacterial mucilage biosynthesis and secretion genes, or regulators thereof, using methods such as those described for Bacillus amyloliquefaciens, an evolutionary close relative of Bv4E2 (e.g. Xin et al., 2022).

[0166] Method C. Additional cycles of human directed evolution may be employed in planta, in different plant varieties (e.g. corn, wheat), to genetically improve Bv4E2 / Bv4E2c strain efficacy in diverse plant genotypes (e.g. to improve rhizosheath production) and / or under environmental conditions in which strain activity is less robust (e.g. specific field conditions). The strategy will employ the GlnLux biosensor technology (US 9,279,139; CA 2,839,976) to measure biological nitrogen fixation and / or improved soil nitrogen uptake. Such methods are known in the art (Tessaro, Soliman and Raizada, 2012; Goron et al., 2017, Thilakarathna, Moroz and Raizada, 2017). For example, large numbers of plants will be inoculated with Bv4E2, then leaf punches will be taken, ground and co-incubated with GlnLux biosensor cells to quantify free plant Gin, as a proxy for enhanced rhizosheath / rhizonest habitat for nitrogen-fixing bacteria such as Klebsiella variicola, or improved nitrogen uptake from the soil. Root mucilage from the best plant will then be collected, and Bv4E2 colonies recovered using selection methods such as Bacillus selective media (e.g. Bacillus Differentiation Agar, HiCrome Bacillus Agar, or Bacillus ChromoSelect Agar) (from Sigma and HIMedia). Bv4E2 will then be multiplied to create new genetic variants, and then re-applied onto new plants in further evolutionary selection cycles.54US_ACTI VE\132140149W-1For the GlnLux assay, leaf tissue disks (6.35 mm in diameter) will be sampled with a hole-punch tool (Fiskars Brands Inc., USA) along the midrib at several growth stages. Leaf disks will be flash frozen or immediately placed in 200 pL of 0.1 % chilled protease inhibition cocktail (PIC) (P9599-lml, Sigma-Aldrich, USA) and homogenized with an automated tissue disruptor (e.g. Bead Ruptor 12, Omni International, Kennesaw, GA, USA). The homogenate will then be diluted six-fold in 0.1% chilled PIC and centrifuged (4 °C, 20 min, 13,000 rpm). The resulting plant tissue extract supernatant will be further diluted ten-fold in 0.1% chilled PIC and stored at -20 °C until analysis. To analyze the extract using the GlnLux whole -cell biosensor, GlnLux E. coli cells will be co-incubatcd with the plant tissue extracts, and then luminescence outputs from 96-well plates will be measured using a luminometer (e.g. MicroLumatPlus, Berthold Technologies, Germany) resulting in relative measurements of plant tissue Gin (“GlnLux glutamine”). The GlnLux Bacterial Growth Media composition has been previously described (Thilakarathna, Moroz and Raizada, 2017). GlnLux cells will be prepared as previously described (Tessaro et al., 2012). Luminometer measurements will be undertaken as previously described using Gin standards (Tessaro et al., 2012; Thilakarathna, Moroz and Raizada, 2017). Alternatively, or in addition, plants will be screened for enhanced chlorophyll using a SPAD meter as described in Goron et al. (2017) or in a high-throughput manner for increased greenness or tolerance to drought using the Phenospex traitfinder, which uses multispectral imaging to measure Normalized Pigment Chlorophyll ratio index (NPCI), Green Leaf Index (GLI), Normalized difference vegetation index (NDVI) and drought traits using the methodology described by Lazarevic et al. (2021, 2022).Example 23: Prophetic optimization of the Bv4E2 / Bv4E2c inoculant formulation.

[0167] Additional efforts to further enhance the inoculant formulation for improved rhizosheath / rhizonest / mucilage formation on plant roots, or to improve nitrogen fixation in those habitats will be carried out by modifying methods already described in Example 10 (coinoculation of Bv4E2 / Bv4E2 with Klebsiella variicola or other beneficial microbes)(FIG. 9) and Example 11 (co-inoculation of Bv4E2 / Bv4E2 with auxin-altering chemical compounds)(FIG.10). These strategies include the following:

[0168] METHOD A. Rhizosheath / rhizonest mucilage habitat constructed by application of Bv4E2 contains beneficial bacteria, including but not limited to nitrogen-fixing Klebsiella variicola and Stenotrophomonas maltophilia. FIG. 9 further demonstrates that including K. variicola in the Bv4E2 inoculant formulation improved plant traits. The K. variicola clade has55US_ACTI VE\132140149W-1been known for 50 years as an effective nitrogen fixer in maize, starting with K. pneumonia 342 (Kp342) (Fonts el al., 2008). More recently, there have been commercial efforts to use gene editing to improve K. variicola as a biofertilizer in maize (Wen et al., 2021). Different combinations of native and improved strains of K. variicola, S. maltophilia as well as other microbial species will be evaluated to further optimize the formulations described herein.

[0169] METHOD B. FIG. 10 demonstrates that co-application of Bv4E2 with an auxin altering compound (z.e. IAA) could dramatically enhance rhizonest formation. Thus, gene editing of Bv4E2 or its mutants may be carried out, including but not limited to, editing of auxin biosynthesis genes using methods such as those described for Bacillus amyloliquefaciens, an evolutionary close relative of Bv4E2 (e.g. Xin et al., 2022). Alternatively, other compounds that alter auxin activity will be tested to further enhance the synergistic effects of the Bv4E2 compositions and formulations described herein. Examples of such compounds are provided in Table 2 below. The methodology for auxin compound testing will be as follows: Maize or wheat seeds will be germinated in baby jars containing sterile modified Yi MS medium supplemented with compounds at concentrations that cause water or nutrient limitation. The jars will be kept in a dark drawer for one week to promote germination and then transferred to light shelves. Half of the jars will be supplemented with each auxin inhibitor or synthetic auxin listed in Table 2 and the other half will be the respective buffer control. After harvest, the primary roots of the plants post-harvest will be cut and stained with 1.0 mg / mL propidium iodide solution (#P4864-10ML, Sigma- Aldrich, USA) for 10 min. After staining, the roots will be washed using sterile ddH2O. The root samples will undergo light microscopy to screen for enhanced rhizonest formation. TABLE 2US_ACTI VE\132140149W-1

[0170] In view of the above, it will be seen that the several objects of the invention are achieved, and other advantageous results attained.

[0171] As various changes could be made in the above products and methods without departing from the scope of the invention, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense.US_ACTI VE\132140149W-1

Claims

WHAT IS CLAIMED IS:

1. A method for inducing a rhizosheath, wherein said method comprises applying a composition comprising an effective amount of Bacillus velezensis strain 4E2b (ID AC Accession No. 100125-01) to a plant, a plant seed, a plant growth medium, or an area surrounding a plant or plant seed.

2. The method of claim 1, wherein the method further comprises inducing a rhizonest.

3. The method of claim 1, wherein the plant comprises increased nutrient use efficiency as compared to a control plant lacking said application.

4. The method of claim 1, wherein the plant comprises increased drought resistance as compared to a control plant lacking said application.

5. The method of claim 1 , wherein the composition is applied to the plant under drought conditions; or under nitrogen limited conditions.

6. The method of claim 1, wherein the plant’s root hairs or rhizosheath develops asymmetrically.

7. The method of claim 1, wherein applying the composition comprises coating the plant seed with the composition; or applying the composition comprises a foliar spray.

8. The method of claim 1 , wherein the composition is applied at a concentration in excess of 101CFU / seed (colony forming units per seed) or 103CFU / plant (colony forming units per plant).

9. A method according to claim 1, wherein said plant is a com plant, a wheat plant, a barley plant, a sorghum plant a rice plant, an oat plant, or a turfgrass plant.58US_ACTI VE\132140149W-110. A composition for application to plants, plant seeds, or a plant growth medium, wherein the composition comprises:an effective amount of Bacillus velezensis strain 4E2b (ID AC Accession No. 100125-01); andat least one species of nitrogen fixing bacteria: orat least one plant growth regulator:in a synergistically effective amount.

11. The composition of claim 10, wherein the nitrogen fixing bacteria is a Klebsiella bacterium, a Stenotrophomonas bacterium, a Pantoea bacterium, a Bacillus bacterium, a Paenibacillus bacterium, an Azotobacter bacterium, a Kosakonia bacterium, an Herbaspirillum bacterium, a Gluconacetobacter bacterium, an Azospirillum bacterium, a Methylobacterium, a Curtobacterium bacterium, a member of the Rhizobiaceae family, or a combination of any thereof.

12. The composition of claim 10, wherein the nitrogen fixing bacterium comprises Klebsiella variicola.

13. The composition of claim 10, wherein the plant growth regulator comprises a plant hormone, an herbicide, or a bioactive metabolite.

14. The composition of claim 13, wherein the plant growth regulator comprises indole-3-acetic acid (IAA), 4- chloroindole-3-acetic acid (4-Me-IAA), 4-methylindole-3-acetic acid (4-Cl-IAA), a-Naphthalene acetic acid, 1 -naphthaleneacetic acid, DICAMB A, 2, 4-dichlorophenoxyacetic acid (2,4-D), 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB), 4-chloro-2-methylphenoxyacetic acid (MCPA), diflufenzopyr, halauxifen, aminopyralid, clopyralid, fluroxypyr, triclopyr, quinclorac, diflufenzopyr, picloram, ethrel or any combination of any thereof.

15. The composition of claim 10, wherein said composition is prepared as a formulation selected from the group consisting of an emulsion, a colloid, a dust, a granule, a pellet, a powder, a spray, an emulsion, and a solution.59US_ACTI VE\132140149W-116. A composition for application to plants, plant seeds, or a plant growth medium, wherein the composition comprises a mutant of Bacillus velezensis strain 4E2b (IDAC Accession No.100125-01), wherein the mutant is a drought-tolerant mutant, a cold-tolerant mutant, a biofilm / mucilage-secreting mutant, a fungicide tolerant mutant, an herbicide tolerant mutant, a plant host compatibility mutant, a plant microbiome compatible mutant, a soil microbiome compatible mutant, an auxin mutant, or a mutant with a combination of mutations to impart drought tolerance, cold tolerance, biofilm / mucilage-secreting ability, fungicide tolerance, herbicide tolerance, plant host compatibility, plant microbiomc compatibility, soil microbiomc compatibility, auxin activity and retains the ability to induce a rhizosheath or rhizonest or asymmetry on a plant root.

17. The composition of claim 16, wherein the mutant is Bacillus velezensis strain 4E2c (IDAC Accession No. 150125-01).

18. The composition of claim 16, wherein the composition further comprises:at least one species of nitrogen fixing bacteria; orat least one plant growth regulator;in a synergistically effective amount.

19. The composition of claim 10 or 16, wherein the composition comprises an agriculturally acceptable carrier.

20. A plant seed coated with the composition of claim 10 or 16.

21. The plant seed of claim 20, wherein the seed is a monocotyledon seed or a dicotyledon seed.

22. A method for stimulating plant growth, wherein the method comprises applying the composition of claim 10 or 16 to a plant, a plant seed, a plant growth medium, or an area surrounding a plant or plant seed.60US_ACTI VE\132140149W-123. The method of claim 22, wherein said method comprises applying the composition to the plant growth medium prior to, concurrently with, or after planting of seeds, seedlings, cuttings, bulbs, or plants in the plant growth medium.

24. The method of claim 22, wherein said method comprises applying the composition to plant leaves, roots, or stems.

25. The method of claim 22, wherein said method comprises applying the composition to plant seeds.

26. The method of claim 1 or 22, wherein the plant exhibits an improved agronomic characteristic selected from the group consisting of: increased average grain yield and shoot biomass, wherein the average grain yield or shoot biomass is increased by at least about 1%, 3%, 5%, 10%, 15%, 20%, or 25% as compared to the height of a control plant lacking application of the composition.

27. The method of claim 1 or 22, wherein the plant exhibits:increased root biomass by at least about 5%, 10%, 15%, 20%, or 25%;increased average primary root length by at least about 5%, 10%, 15%, 20%, or 25%; increased average root hair density by at least about 5%, 10%, 15%, 20%, or 25%; or increased average root hair length by at least about 5%, 10%, 15%, 20%, or 25%, as compared to the height of a control plant lacking application of the composition.

28. A method for increasing drought tolerance in a plant, wherein said method comprises applying a composition comprising an effective amount of Bacillus velezensis strain 4E2b (IDAC Accession No. 100125-01) to a plant, a plant seed, a plant growth medium, or an area surrounding a plant or plant seed.

29. The method of claim 28, wherein applying the composition comprises coating the plant seed with the composition; or applying the composition comprises a foliar spray.

30. The method of claim 28, wherein the plant exhibits:61US_ACTI VE\132140149W-1increased yield by at least about 5%, 10%, 15%, 20%, or 25%;as compared to the height of a control plant lacking application of the composition. US_ACTI VE\132140149W-1

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