Agricultural beneficial microorganisms, microbial compositions and consortia

Abstract

The present disclosure relates to isolated microorganisms, novel strains including microorganisms, microbial consortia, and agricultural compositions comprising the same. In addition, the present disclosure teaches methods of utilizing the microorganisms, microbial consortia, and agricultural compositions comprising the same in methods of conferring beneficial properties to target plant species. In particular aspects, the disclosure provides methods of enhancing desired plant traits in agronomically important crop species.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 312,322, filed on February 21, 2022, under 35 U.S.C. 119(e), and all provisional patent applications are incorporated herein by reference in their entirety.

[0003] Reference to a sequence listing submitted electronically

[0004] An official copy of the sequence listing is submitted electronically as an XML formatted sequence listing and is submitted simultaneously with this specification, the file name of the sequence listing being 22042-WO-PCT.xml, created on January 18, 2023, and having a size of 34,662 bytes. The sequence listing contained in this XML formatted file is part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure relates generally to the field of biology, and in particular to microorganisms and microbial compositions for plant improvement. Background Art

[0006] According to the United Nations World Food Program, there are nearly 900 million undernourished people worldwide. The malnutrition epidemic is particularly acute in developing countries around the world, where one in six children is underweight. The lack of available food can be attributed to a variety of socioeconomic factors; however, regardless of the ultimate cause, the fact remains that there is a shortage of food available to feed a growing world population that is expected to reach 9 billion people by 2050. The United Nations estimates that agricultural production must increase by 70% to 100% in 2050 to feed the projected global population.

[0007] These alarming world population and malnutrition figures highlight the importance of agricultural efficiency and productivity in sustaining a growing worldwide population. The technological advances achieved through modern row crop agriculture, which have resulted in unprecedented crop yields, are impressive. However, despite advances made through technological innovations such as genetically engineered crops and new insecticidal and herbicidal compounds, improved crop performance is still needed to meet the needs of an exponentially increasing global population.

[0008] Scientists estimate that if the global agricultural "yield gap" (i.e., the difference between the best observed yield and what is achieved elsewhere) could be closed, then worldwide crop production could rise by 45% to 70%. In other words, if all farmers (regardless of their location in the world) could achieve the highest achievable yields expected for their respective regions, much of the shortfall in world food production could be addressed. However, addressing the question of how to achieve higher yields in a heterogeneous global landscape is difficult.

[0009] Typically, yield gaps are explained by insufficient water, substandard farming practices, insufficient fertilizers, and the inability to use herbicides and pesticides. However, a significant increase in the use of water, fertilizers, herbicides, and pesticides worldwide would not only be economically unfeasible for large parts of the world, but would also have adverse environmental consequences.

[0010] Nematodes are a particular concern for agriculture, causing an estimated 12.3% or $157 billion in yield losses worldwide.

[0011] Therefore, it is simply not feasible to meet global agricultural output expectations by simply scaling up the current high-input agricultural systems used in most developed countries.

[0012] Therefore, there is a pressing need in the art for improved methods of enhancing crop performance and imparting beneficial traits to desirable plant species, particularly methods and compositions for addressing nematode damage in commercial crops. Summary of the invention

[0013] The present invention includes isolated and biologically pure microorganisms that have applications, particularly in agriculture. The disclosed microorganisms can be used in their isolated and biologically pure states or formulated into agriculturally acceptable compositions. Also provided are agriculturally beneficial microbial consortia (which comprise at least two members of the disclosed microorganisms) and methods of utilizing the consortia in agricultural applications. In some aspects, genome modification of microorganisms (individuals, consortia, and / or colonies) is contemplated to improve microbial traits and improve plants associated with microorganisms.

[0014] The present disclosure addresses the important question of how to improve crop performance, thereby closing yield gaps worldwide, while also providing a means of imparting other beneficial traits to plant species.

[0015] The solution for improving crop performance and increasing yield provided by the present disclosure is not harmful to the earth's resources because it does not rely on increasing water consumption or increasing the input of synthetic chemicals in the system. Instead, the present disclosure utilizes microorganisms to impart beneficial traits to desired plants, including increased yield.

[0016] Thus, the present disclosure provides an environmentally sustainable solution that allows farmers to increase yields of important crops that does not rely on increasing the use of synthetic herbicides and pesticides.

[0017] In embodiments, the present disclosure provides efficient and broadly applicable agricultural platforms that utilize microorganisms and microbial consortia (multiple microorganisms, in some aspects, multiple microorganisms that improve the health or desired phenotype (such as agronomic traits) of the plant with which they are associated) that promote one or more desired plant characteristics.

[0018] Microorganisms disclosed herein improve the performance of plants (such as crop plants) by direct and indirect mechanisms. In some respects, microorganisms form a symbiotic relationship with plants. In some respects, microorganisms produce compounds (for example, metabolites, toxins, proteins, lipopeptides or other compositions) that give benefits to plants or plants can be used to improve properties. In some respects, microorganisms improve the solubility of one or more compositions (such as nutrients), thereby benefiting plants. In some respects, microorganisms give tolerance to exogenous substances (such as herbicides or pesticides) to plants. In some respects, microorganisms produce compositions that are harmful to plant pests (such as insects). In some respects, microorganisms fix nitrogen, thereby improving the nutritional status of plants. Other aspects other than the exemplary non-limiting aspects listed above are contemplated.

[0019] In some embodiments, a single microorganism is utilized. In some aspects, the single microorganism is isolated and purified. In some aspects, the single microorganism is a classified species of bacteria. In some aspects, the single microorganism is an identifiable strain of a classified species of bacteria. In some aspects, the single microorganism is a new, recently discovered strain of a classified species of bacteria.

[0020] In some aspects, the single microorganism (whether a taxonomically identifiable species or strain) is combined with one or more other microorganisms of a different species or strain. In certain aspects, the combination of two or more microorganisms forms a consortia or consortium. The terms consortia and consortium are used interchangeably.

[0021] In certain aspects, the present disclosure provides the development of highly functional microbial consortia that help promote the development and expression of desired phenotypic or genotypic plant traits. In some embodiments, the consortia of the present disclosure possess functional attributes that do not exist in nature when the individual microorganisms live alone. That is, in various embodiments, specific microbial species are combined into a consortium such that the microbial combination possesses functional attributes that any individual member of the consortium does not possess when considered alone.

[0022] In some embodiments, the functional attribute possessed by the microbial consortium is the ability to confer one or more beneficial characteristics to a plant species, such as: increased growth, increased yield, increased nutrient utilization (e.g., nitrogen, phosphate, etc.), increased nitrogen use efficiency, increased stress tolerance, increased drought tolerance, increased photosynthetic rate, increased water use efficiency, increased pathogen resistance, modification of plant architecture (which does not necessarily affect plant yield, but addresses plant function), etc. Beneficial characteristics of pest resistance and / or tolerance are also contemplated, including adverse effects on nematodes, insects or other pests.

[0023] In some embodiments, individual microorganisms do not possess the ability to impart these beneficial properties to plants when they exist in nature. Instead, in some embodiments, these microorganisms are manually combined into consortia by humans to form functional compositions that possess attributes and functional properties that do not exist in nature. In some embodiments, the consortia may include microorganisms that have been genetically edited, altered, or modified by modifying genetic material (including DNA, RNA, protein, and / or combinations thereof) through techniques known to those of ordinary skill in the art.

[0024] However, in other embodiments, the present disclosure provides isolated and biologically pure individual microorganisms that are capable of conferring beneficial traits to desired plant species without the need to combine the microorganisms into a consortium.

[0025] In some embodiments, the microorganism is a strain of Paenibacillus cellulosilyticus and / or Priestia megaterium having nematicidal activity. In some aspects, the strain also comprises toxins active against other pests such as Coleoptera and Lepidoptera.

[0026] In some embodiments, the strain presented herein is strain 101136, deposited under the Budapest Treaty as NRRL B-68087, with a deposit date of January 26, 2022. Strain 101136 is Priesteria gigantea (formerly known as Bacillus megaterium) (16S similarity relative to NCBI GenBank methods), which was confirmed by pacbio sequencing.

[0027] In some embodiments, the strain presented herein is strain 101166, deposited under the Budapest Treaty as NRRL B-68088, with a deposit date of January 26, 2022. Strain 101166 is Paenibacillus cellulolyticus (16S similarity relative to NCBI GenBank methodology).

[0028] Any strain disclosed herein can be further combined with one or more other microorganisms to form a microbial consortium. A microbial consortium can be any combination of one or more individual microorganisms. In certain embodiments, the microbial consortium comprises two microorganisms or three microorganisms or four microorganisms or five microorganisms or six microorganisms or seven microorganisms or eight microorganisms or nine microorganisms or 10 microorganisms or more than 10 microorganisms.

[0029] Another object of the present disclosure relates to the use of isolated microorganisms and microbial consortia as plant growth promoters. In other aspects, isolated microorganisms and microbial consortia act as growth regulators, which can, for example, resist normal senescence, thereby causing an increase in biomass.

[0030] Yet another object of the present disclosure relates to the use of isolated microorganisms and microbial consortia as soil health enhancers and plant health enhancers. In other aspects, the isolated microorganisms and microbial consortia act as biostimulants.

[0031] Additional objects of the present disclosure relate to the use of isolated microorganisms and microbial consortia as insecticides. In other aspects, isolated microorganisms and microbial consortia act as biofungicides. In other aspects, isolated microorganisms and microbial consortia act as bionematicides.

[0032] Another object of the present disclosure is to design a microbial consortium that is capable of performing a common multidimensional activity. In some aspects, the microorganisms that comprise the consortium act synergistically. In various aspects, the effect of the microbial consortium on a certain plant characteristic is greater than the effect that would be observed when any individual microbial member of the consortium is used singly. That is, in some aspects, the consortium exhibits a greater than additive effect on a desired plant characteristic compared to the effect that would be found when any individual member of the consortium is used alone.

[0033] In some aspects, the consortium leads to the establishment of other plant-microbe interactions, for example by serving as primary colonizers or founder populations that set the trajectory of future microbiome development.

[0034] In embodiments, the present disclosure relates to synergistic combinations (or mixtures) of microbial isolates.

[0035] In some aspects, the consortia taught herein provide a wide range of agricultural applications, including: increasing the yield of grains, fruits and flowers; increasing the growth of plant parts; increasing the ability to utilize nutrients (e.g., nitrogen, phosphates, etc.), increasing disease resistance; bio-insecticide effects, including increasing resistance to fungi, insects and nematodes; increasing survival in extreme climates; and improving other desired plant phenotypic characteristics. Obviously, these benefits to plants and / or adverse effects on target pests and / or pathogens can be obtained without any harmful side effects to the environment.

[0036] In some aspects, individual microorganisms of the present disclosure or consortia comprising the same can be combined into agriculturally acceptable compositions.

[0037] In some embodiments, the agricultural compositions of the present disclosure include, but are not limited to, wetting agents, extenders, defoamers, detergents, chelating agents, drift reducers, neutralizers, buffers, corrosion inhibitors, dyes, odorants, extenders, penetration aids, stickers, binders, dispersants, thickeners, stabilizers, emulsifiers, freezing point depressants, antimicrobial agents, fertilizers, pesticides, herbicides, inert carriers, polymers, and the like.

[0038] In one embodiment of the present disclosure, microorganisms (including isolated single species or strains, consortia, or their compositions, such as metabolites) are supplied in the form of seed coatings or other seed applications. In embodiments, seed coatings can be applied to bare and untreated seeds. In other embodiments, seed coatings can be applied to previously treated seeds. Therefore, in some embodiments, the present disclosure teaches a method for treating seeds, which includes applying isolated bacterial strains or microbial consortia to seeds. In certain embodiments, isolated bacterial strains or microbial consortia are applied in the form of agricultural compositions comprising agriculturally acceptable carriers. In some embodiments, agricultural compositions can be formulated as: soil drenches, foliar sprays, immersion treatments, furrow treatments, soil conditioners, granules, broadcast treatments, post-harvest disease control treatments, or seed treatments. In some embodiments, agricultural compositions can be applied alone or in a rotary spraying procedure with other agricultural products. In some embodiments, agricultural compositions can be compatible with tank mixing. In some embodiments, agricultural compositions can be compatible with tank mixing with other agricultural products. In some embodiments, the agricultural compositions are compatible with equipment for ground, aerial, and irrigation applications.

[0039] In some embodiments, the applied microorganism may become endogenous and thus may be present in the treated growing plant and its progeny. In other embodiments, the microorganism may be applied simultaneously with the seed treatment in a co-treatment.

[0040] In one embodiment of the present disclosure, the microorganism is supplied in the form of a granule or plug or soil drench applied to a plant growth medium. In other embodiments, the microorganism is supplied in the form of a foliar application, such as a foliar spray or a liquid composition. The foliar spray or liquid application can be applied to a growing plant or a growth medium, such as soil.

[0041] In other embodiments, microorganisms (including separated single species or strains or consortia or their compositions, such as metabolites) are supplied in the form of fertilizers, pesticides or other amendments that can be applied to soil. In some embodiments, microorganisms are supplied in the form of fertilizers, pesticides or other amendments that are applied to soil before planting. In some embodiments, microorganisms are supplied in the form of fertilizers, pesticides or other amendments that are applied to soil while planting. In some embodiments, microorganisms are supplied in the form of fertilizers, pesticides or other amendments that are applied to soil after planting.

[0042] In other embodiments of the present disclosure, microorganisms (including isolated single species or strains or consortia) and / or compositions thereof (e.g., metabolites) are supplied in the form of a post-harvest disease control application.

[0043] In embodiments, the agricultural compositions of the present disclosure may be formulated as: (1) solutions; (2) wettable powders; (3) dusting powders; (4) soluble powders; (5) emulsion or suspension concentrates; (6) seed dressings, (7) tablets; (8) water-dispersible granules; (9) water-soluble granules (slow release or quick release); (10) microencapsulated granules or suspensions; (11) as irrigation components, and (12) components of fertilizers, pesticides and other compatible improvers, etc. In certain aspects, the composition may be diluted in an aqueous medium prior to conventional spray application. The compositions of the present disclosure may be applied to soil, plants, seeds, rhizosphere, rhizosheaths, or other areas where application of the microbial composition would be beneficial.

[0044] Another object of the present disclosure relates to agricultural compositions, which are formulated to provide high colony forming unit (CFU) bacterial colonies or consortia. In some aspects, agricultural compositions have adjuvants that provide relevant shelf life. In embodiments, the CFU concentration of the agricultural compositions taught is higher than the concentration at which the microorganisms will naturally occur outside the disclosed method. In another embodiment, the agricultural compositions include microbial cells at a concentration of 10^2-10^12 CFU / gram carrier or 10^5-10^9 CFU / gram carrier. In one aspect, microbial cells are directly applied to seeds as seed coatings at a concentration of 10^5-10^9 CFU. In other aspects, microbial cells are applied to the top of another sub-coating as seed outer coatings at a concentration of 10^5-10^9 CFU. In other aspects, microbial cells are applied to the top of another sub-coating with a concentration of 10^5-10^9 CFU together with another sub-treatment agent as a co-treatment agent at a ratio of 10^5-10^9 CFU.

[0045] In various aspects, the present disclosure relates to agricultural microbial preparations that promote plant growth. In various aspects, the present disclosure provides isolated microorganisms and consortia comprising them as taught to be formulated as agricultural biological inoculants. The taught biological inoculants can be applied to plants, seeds or soil, or combined with fertilizers, pesticides and other compatible modifiers. Suitable examples of preparing biological inoculants comprising isolated microorganisms can be found in U.S. Patent No. 7,097,830, which is incorporated herein by reference.

[0046] The disclosed microbial formulations can: reduce the need for nitrogen-containing fertilizers, solubilize minerals, provide biopesticide protection to plants, protect plants from pathogens (e.g., fungi, insects and nematodes), and make valuable nutrients such as nitrogen and / or phosphates available to plants, thereby reducing and eliminating the need to use chemical pesticides and chemical fertilizers.

[0047] In some embodiments, the isolated and biologically pure microorganisms of the present disclosure can be utilized in methods of conferring one or more beneficial characteristics or traits to a desired plant species.

[0048] In some embodiments, agriculturally acceptable compositions comprising an isolated and biologically pure microorganism of the present disclosure can be utilized in methods of imparting one or more beneficial characteristics or traits to a desired plant species.

[0049] In some embodiments, the consortia of the present disclosure may be utilized in methods of conferring one or more beneficial characteristics or traits to a desired plant species.

[0050] In some embodiments, agriculturally acceptable compositions comprising the consortia of the present disclosure may be utilized in methods of imparting one or more beneficial characteristics or traits to a desired plant species.

[0051] In some aspects, the isolated and biologically pure microorganisms of the present disclosure and / or the consortia of the present disclosure are derived from an accelerated microbial selection process ("AMS" process). The AMS process utilized in some aspects of the present disclosure is described, for example, in the following documents: (1) International Patent Application No. PCT / NZ2012 / 000041, published as International Publication No. WO 2012125050 A1 on September 20, 2012, and (2) International Patent Application No. PCT / NZ2013 / 000171, published as International Publication No. WO 2014046553 A1 on March 27, 2014, each of which is incorporated herein by reference in its entirety for all purposes.

[0052] However, in other embodiments, the microorganism of the present disclosure is not derived from an accelerated microorganism selection process. In some aspects, the microorganism utilized in the embodiments of the present disclosure is selected from the microorganism members present in the database. In specific aspects, the microorganism utilized in the embodiments of the present disclosure is selected from the microorganism present in the database based on the specific characteristics of the microorganism.

[0053] The present disclosure provides that plant components or plant parts can be effectively enhanced by coating the plant components or plant parts with an isolated microorganism or consortium of microorganisms in an amount not normally found on the plant components or plant parts.

[0054] Some embodiments described herein are methods for preparing agricultural seed compositions or seed coatings, comprising: contacting the surface of the seed with a preparation comprising a purified microbial colony, the purified microbial colony comprising at least one isolated microorganism that is heterologous to the seed or that rarely exists on the seed. Other embodiments require the preparation of an agricultural plant composition, comprising: contacting the surface of the plant with a preparation comprising a purified microbial colony, the purified microbial colony comprising at least one isolated microorganism that is heterologous to the plant. In other aspects, the preparation or microorganism is introduced into the interior of the seed, for example, into cotyledon or plumule other seed tissues.

[0055] In some aspects, applying the isolated microorganisms, microbial consortia, exudates, metabolites and / or agricultural compositions of the present disclosure to seeds or plants can modulate agronomically important traits. Agronomically important traits can be, for example, disease resistance, drought tolerance, heat tolerance, cold tolerance, salt tolerance, metal tolerance, herbicide tolerance, chemical tolerance, improved water use efficiency, improved nitrogen utilization, improved nitrogen stress resistance, improved nitrogen fixation, improved nutrient (e.g., phosphate, potassium, etc.) utilization, insect resistance, herbivore resistance, pathogen resistance, reduced pathogen levels (e.g., via secretion of metabolites that affect pathogen survival), increased yield, increased yield under water-limited conditions, enhanced health, increased vigor, improved growth, increased photosynthetic capacity, enhanced nutrition, altered protein content, altered oil content, increased biomass, increased bud length, increased root length, improved root configuration, increased seed weight, faster seed germination, altered seed carbohydrate composition, altered seed oil composition, number of pods, delayed aging, stay-green, and altered seed protein composition. In some aspects, at least 2, 3, 4 or more agronomically important traits are modulated. In some aspects, the modulation is a positive effect on one of the aforementioned agronomic traits.

[0056] In some aspects, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure can be applied to plants to modulate or alter plant characteristics, such as altered oil content, altered protein content, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, chemical tolerance, cold tolerance, delayed senescence, disease resistance, drought tolerance, ear weight, improved growth, enhanced health, heat tolerance, herbicide tolerance, herbivore resistance, increased nitrogen fixation, improved nitrogen utilization efficiency, improved root architecture, improved water use efficiency, increased biomass, reduced biomass, increased root length, reduced root length, increased seed weight, increased shoot length, reduced shoot length, yield relative to a reference plant. increase, increased yield under water-limiting conditions, kernel mass, kernel moisture content, metal tolerance, number of ears, number of kernels per ear, number of pods, nutritional enhancement, pathogen resistance, insect resistance, improved photosynthetic capacity, salt tolerance, stay-green, improved vigour, increased dry weight of mature seeds, increased fresh weight of mature seeds, increased number of mature seeds per plant, increased chlorophyll content, increased number of pods per plant, increased pod length per plant, reduced number of wilted leaves per plant, reduced number of severely wilted leaves per plant and increased number of non-wilted leaves per plant, detectable modulation at the level of metabolites, detectable modulation at the level of transcripts, and detectable modulation at the level of the proteome.

[0057] In some embodiments, the agricultural formulations taught herein comprise at least one member selected from the group consisting of an agriculturally compatible carrier, a tackifier, a microbial stabilizer, a fungicide, an antibacterial agent, an herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, and a nutrient.

[0058] The methods described herein can include contacting a seed or plant with at least 100 CFU or spores, at least 300 CFU or spores, at least 1,000 CFU or spores, at least 3,000 CFU or spores, at least 10,000 CFU or spores, at least 30,000 CFU or spores, at least 100,000 CFU or spores, at least 300,000 CFU or spores, at least 1,000,000 CFU or spores, or more of a microorganism taught herein.

[0059] The methods described herein may include contacting a seed or plant with a composition comprising a metabolite produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method includes contacting a seed or plant with a composition comprising at least 1 mg of metabolites produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method includes contacting a seed or plant with a composition comprising at least 10 mg of metabolites produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method includes contacting a seed or plant with a composition comprising at least 100 mg of metabolites produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method includes contacting a seed or plant with a composition comprising at least 1 g of metabolites produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method includes contacting a seed or plant with a composition comprising at least 10 g of metabolites produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method includes contacting a seed or plant with a composition comprising at least 100 g of metabolites produced by a single microorganism or microbial consortium disclosed herein. In some aspects, the method comprises contacting a seed or plant with a composition comprising at least 1 kg of a metabolite produced by a single microorganism or a consortium of microorganisms disclosed herein. In some aspects, the method comprises contacting a seed or plant with a composition comprising greater than 1 kg of a metabolite produced by a single microorganism or a consortium of microorganisms disclosed herein.

[0060] In some embodiments of the methods described herein, the isolated microorganisms of the present disclosure are present in the formulation in an amount effectively detectable in and / or on a target tissue of an agricultural plant. For example, at least 100 CFU or spores, at least 300 CFU or spores, at least 1,000 CFU or spores, at least 3,000 CFU or spores, at least 10,000 CFU or spores, at least 30,000 CFU or spores, at least 100,000 CFU or spores, at least 300,000 CFU or spores, at least 1,000,000 CFU or spores, or more of the microorganisms are detected in and / or on a target tissue of a plant. Alternatively or additionally, the microorganisms of the present disclosure may be present in a formulation in an amount effective to increase the biomass and / or yield of a plant to which the formulation is applied by at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more, when compared to a reference agricultural plant to which the formulation of the present disclosure has not been applied. Alternatively or additionally, the microorganisms of the present disclosure may be present in a formulation in an amount effective to detectably modulate an agronomic trait of interest of a plant to which the formulation is applied by at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more when compared to a reference agricultural plant to which the formulation of the present disclosure has not been applied.

[0061] In some embodiments of the methods described herein, one or more metabolites isolated from the microorganisms or consortia of the present disclosure are present in the formulation in an amount that can be effectively detected within and / or on the target tissue of the agricultural plant. For example, metabolites of at least 1 mg, at least 10 mg, at least 50 mg, at least 100 mg, at least 200 mg, at least 400 mg, at least 600 mg, at least 800 mg, at least 1 g or more are detected in and / or on the target tissue of the plant. Alternatively or additionally, metabolites isolated from the microorganisms and consortia of the present disclosure can be present in the formulation in an amount that effectively increases the biomass and / or yield of the plant to which the formulation is applied by at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more when compared to a reference agricultural plant to which the formulation of the present disclosure is not applied. Alternatively or additionally, metabolites isolated from the microorganisms and consortia of the present disclosure may be present in a formulation in an amount effective to detectably modulate an agronomic trait of interest in a plant to which the formulation of the present disclosure is applied by at least 1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more when compared to a reference agricultural plant to which the formulation of the present disclosure has not been applied.

[0062] In some embodiments, the agricultural composition taught herein is shelf stable. In some aspects, the microorganism taught herein is freeze dried. In some aspects, the microorganism taught herein is spray dried. In some aspects, the microorganism taught herein is placed in a liquid formulation. In some aspects, the microorganism taught herein is present on a particle.

[0063] Also described herein are a plurality of isolated microorganisms enclosed within an article selected from the group consisting of a bottle, a jar, an ampoule, a package, a vessel, a bag, a box, a storage box, an envelope, a carton, a container, a silo, a shipping container, a carriage, and a case.

[0064] In some aspects, the selected plant species is combined with the disclosed microorganisms (operational taxonomic units (OTUs), strains, or compositions comprising any of the foregoing) to result in increased crop yields and the generation of their products. Therefore, in one aspect, the present disclosure provides a synthetic combination of a seed of a first plant and a microbial preparation coated on the surface of the seed of the first plant, such that the microorganism is present on the surface of the seed at a higher level than that present on the surface of an uncoated reference seed. In another aspect, the present disclosure provides a synthetic combination of a portion of a first plant and a microbial preparation coated on the surface of the portion of the first plant, such that the microorganism is present on the surface of the portion of the first plant at a higher level than that present on the surface of an uncoated reference plant portion. The above methods can be used alone or simultaneously with plant breeding and transgenic techniques.

[0065] In some embodiments, the isolated bacterial strain has a morphology and physiological characteristics substantially similar to the isolated bacterial strain of the present disclosure. In some embodiments, the isolated bacterial strain has a genetic characteristic substantially similar to the isolated bacterial strain of the present disclosure. In some embodiments, the isolated bacterial strain is a naturally occurring or artificial mutant of the isolated bacterial strain of the present disclosure. In some embodiments, the isolated bacterial strain is a bacterial strain that has been genetically edited, changed or modified. In some embodiments, the isolated bacterial strain of the present disclosure is located in a substantially pure culture. In some embodiments, the isolated bacterial strain of the present disclosure is located in a pure culture. In some embodiments, the isolated bacterial strain of the present disclosure is located in a cell fraction, an extract or a supernatant.

[0066] In some embodiments, progeny and / or mutants of the isolated bacterial strains of the present disclosure are contemplated. In some embodiments, progeny, mutants and / or genetically modified forms of the isolated bacterial strains of the present disclosure are contemplated. In some embodiments, the isolated bacterial strains of the present disclosure comprise a polynucleotide sequence that shares at least 97% sequence identity with a 16S gene or protein-coding gene contained within a strain that is the same as NRRL Deposit No. B-68087 or NRRL Deposit No. B-68088 or has the same taxonomy.

[0067] In some embodiments, cell-free or inactivated preparations of isolated bacterial strains of the present disclosure, or mutants of the isolated bacterial strains are contemplated. In some embodiments, cell-free or inactivated preparations of isolated bacterial strains of the present disclosure, or mutants of the isolated bacterial strains, or genetically edited, altered, or modified variants are contemplated. In some embodiments, metabolites produced by isolated bacterial strains of the present disclosure, or mutants of the isolated bacterial strains are contemplated. In some embodiments, metabolites produced by isolated bacterial strains of the present disclosure, or mutants of the isolated bacterial strains, or genetically modified variants are contemplated.

[0068] In some embodiments, the agricultural composition comprises an isolated bacterial strain and an agriculturally acceptable carrier. The isolated bacterial strain may be present in the composition at 1×10^2 to 1×10^12 CFU / gram. The agricultural composition may be formulated as a seed coating.

[0069] In some embodiments, the method of conferring at least one beneficial trait to a plant species comprises applying an isolated bacterial strain to a plant or a growth medium in which the plant is located. In some embodiments, the method of conferring at least one beneficial trait to a plant species comprises applying an agricultural composition of the present disclosure to the plant or a growth medium in which the plant is located.

[0070] In some embodiments, the present disclosure teaches a method of growing a plant having at least one beneficial trait. In some embodiments, the method comprises applying an isolated bacterial strain or microbial consortium to a seed of a plant; sowing or planting the seed; and growing the plant. In certain embodiments, the isolated bacterial strain or microbial consortium is applied in the form of an agricultural composition that also comprises an agriculturally acceptable carrier.

[0071] In some embodiments, the microbial consortium has a morphology and physiological properties substantially similar to the microbial consortium of the present disclosure. In some embodiments, the microbial consortium has genetic properties substantially similar to the microbial consortium of the present disclosure. In some embodiments, the microbial consortium is located in a substantially pure culture. In some embodiments, subsequent generations of any microorganism of the microbial consortium are envisioned. In some embodiments, mutants of any microorganism of the microbial consortium are envisioned. In some embodiments, variants of any microorganism of the microbial consortium are envisioned that are genetically edited, altered or modified. In some embodiments, a cell-free or inactivated preparation of a microbial consortium or a mutant of any microorganism in the microbial consortium or a variant that is genetically edited, altered or modified is envisioned. In some embodiments, metabolites produced by a mutant of any microorganism in the microbial consortium or a variant that is genetically edited, altered or modified is envisioned.

[0072] In some embodiments, the agricultural composition comprises a microbial consortium and an agriculturally acceptable carrier. The microbial consortium of the agricultural composition may be present in the composition at 1×10^3 to 1×10^12 bacterial cells / gram. In some embodiments, the agricultural composition is formulated as a seed coating. In some embodiments, a method of imparting at least one beneficial trait to a plant species comprises applying a microbial consortium to the plant or a growth medium in which the plant is located. In some embodiments, a method of imparting at least one beneficial trait to a plant species comprises applying an agricultural composition to a plant or a growth medium in which the plant is located.

[0073] In any of the methods, the microorganism can comprise a 16S rRNA nucleic acid sequence having at least 97% sequence identity to a 16S rRNA nucleic acid sequence of a bacterium selected from the genus or species provided in Table 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] The present disclosure can be more fully understood from the following detailed description, the accompanying drawings and the Sequence Listing which constitute a part of this application.

[0075] The sequence description and the accompanying sequence listing comply with the rules governing disclosure of nucleotide and amino acid sequences in patent applications as set forth in 37 CFR §§ 1.821 and 1.825. The microorganisms described in this application are deposited with the Agricultural Research Type Culture Collection (NRRL), an international depository located at 1815 North University Street, Peoria, IL 61604, USA.

[0076] These deposits were made under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0077] These deposits were made in accordance with and meet the standards set forth in 37 CFR §§1.801-1.809 and Manual of Patent Examining Procedure §§2402-2411.05.

[0078] Table 1: Sequences

[0079]

[0080] DETAILED DESCRIPTION

[0081] Although the following terms are considered to be well understood by those of ordinary skill in the art, they are set forth to facilitate explanation of the presently disclosed subject matter.

[0082] The term "a" or "an" refers to one or more of the entity, i.e., multiple referents. Thus, the terms "a" or "an", "one (kind) or more (kinds)" and "at least one (kind)" are used interchangeably herein. In addition, reference to "an element" by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that there is one and only one element.

[0083] As used herein, the term "microorganism" or "microorganism" should be interpreted in a broad sense. These terms are used interchangeably and include, but are not limited to, two prokaryotic domains (i.e., bacteria and archaea) as well as eukaryotic fungi and protists. In some embodiments, the present disclosure relates to the "microorganisms" of Table 1, or the "microorganisms" of various other tables or paragraphs present in the present disclosure. This characterization can refer not only to the classified bacterial genus identified in the table, but also to the classified species identified, as well as various novel and newly identified bacterial strains in the table.

[0084] As used herein, the term "microorganism" or "microorganism" refers to any species or taxon of a microorganism, including but not limited to archaea, bacteria, microalgae, fungi (including mold and yeast species), mycoplasmas, microspores, nanobacteria, oomycetes, and protozoa. In some embodiments, a microorganism or microorganism encompasses a single cell (e.g., a unicellular microorganism) or more than one cell (e.g., a multicellular microorganism). Thus, a "microorganism population" may refer to multiple cells of a single microorganism, wherein the cells share a common genetic derivation.

[0085] As used herein, the term "bacteria" generally refers to any prokaryotic organism, and may relate to organisms from the Eubacteria (bacteria), Archaea (archaea), or both. In some cases, bacterial genera or other taxonomic classifications have been reassigned for other reasons (such as, but not limited to, the evolutionary field of whole genome sequencing), and it should be understood that such naming is reassigned within the scope of any claimed taxonomy. For example, some species of the genus Erwinia have been described in the literature as belonging to the genus Pantoea (Zhang, Y., Qiu, S. Examining phylogenetic relationships of Erwinia and Pantoea species using whole genome sequence data. Antonie van Leeuwenhoek 108, 1037-1046 (2015)).

[0086] The term "16S" refers to the DNA sequence of the 16S ribosomal RNA (rRNA) sequence of bacteria. 16S rRNA gene sequencing is an established method for studying the phylogeny and taxonomy of bacteria.

[00166] As used herein, the term "fungus" generally refers to any organism from the kingdom Fungi. Historically, fungi have been taxonomically classified based on morphological manifestations. Since the mid-nineteenth century, it has been recognized that some fungi have a polymorphic life cycle, and different nomenclature names are used for different forms of the same fungus. In 1981, the Sydney Congress of the International Mycological Association established the rule that fungi are named asexual, sexual or holotype according to their state (Taylor, JW One Fungus = One Name: DNA and fungal nomenclature twenty years after PCR. IMA Fungus 2, 113-120 (2011)). With the development of genome sequencing, it is clear that taxonomic classification based on molecular phylogenetics does not match the nomenclature based on morphology (Shenoy, BD, Jeewon, R. and Hyde, KD (2007). Impact of DNA sequence-data on the taxonomy of anamorphic fungi. Fungal Diversity 26: 1-54). Therefore, in 2011, the International Botanical Congress passed a resolution approving the International Code of Nomenclature for algae, fungi and plants (Melbourne Code) (2012), wherein the result is the designation of "one fungus = one name" (Hawksworth, DL Managing and coping with names of pleomorphic fungi in a period of transition. IMA Fungus 3, 15-24 (2012)).

[0087] The term "internal transcribed spacer" ("ITS") refers to the spacer DNA (non-coding DNA) between the small subunit ribosomal RNA (rRNA) and large subunit (LSU) rRNA genes in the corresponding transcribed regions in the chromosome or in the polycistronic rRNA precursor transcript. ITS gene sequencing is an established method for studying the phylogeny and taxonomy of fungi. In some cases, the "large subunit" ("LSU") sequence is used to identify fungi. LSU gene sequencing is an established method for studying the phylogeny and taxonomy of fungi. Some fungal microorganisms of the present invention can be described by ITS sequences, and some fungal microorganisms can be described by LSU sequences. It should be understood that both are equally descriptive and accurate for determining taxonomy.

[0088] The term "microbial consortia" or "microbial consortium" refers to a subset of a microbial community of individual microbial species, or strains of species, that can be described as performing a common function, or can be described as participating in or causing or being associated with an identifiable parameter or plant phenotypic trait. The community can include one or more species, or strains of species, of microorganisms. In some cases, these microorganisms coexist in a symbiotic manner within the community.

[0089] The term "microbial community" means a group of microorganisms comprising two or more species or strains. Unlike a microbial consortium, a microbial community does not necessarily perform a common function or participate in or cause or correlate with an identifiable parameter or plant phenotypic trait.

[0090] The term "accelerated microbial selection" or "AMS" is used interchangeably with the term "directed microbial selection" or "DMS" and refers to an iterative selection method that is used in some embodiments of the present disclosure to obtain the claimed microbial species or consortia of such species.

[0091] As used herein, "isolate," "isolated," "isolated microorganism," and similar terms are intended to mean that one or more microorganisms have been separated from at least one material with which they were associated in a particular environment (e.g., soil, water, plant tissue).

[0092] Thus, an "isolated microorganism" does not exist in its naturally occurring environment; rather, the microorganism is removed from its natural environment and placed in a non-naturally occurring state by the various techniques described herein. Thus, an isolated strain can exist, for example, in the form of a biologically pure culture or spores (or other forms of strains) associated with an agricultural carrier.

[0093] In certain aspects of the present disclosure, an isolated microorganism is present in the form of an isolated and biologically pure culture. It will be understood by those skilled in the art that an isolated and biologically pure culture of a particular microorganism means that the culture is substantially free of (within scientific reason) other living organisms and contains only the individual microorganism in question. The culture may contain varying concentrations of the microorganism. The present disclosure indicates that an isolated and biologically pure microorganism is generally "certainly distinct from less pure or impure material." See, for example, In re Bergstrom, 427F.2d 1394, (CCPA 1970) (discussing purified prostaglandins), also see In re Bergy, 596F.2d 952 (CCPA 1979) (discussing purified microorganisms), also see Parke-Davis & Co. v HK Mulford & Co., 189F.95 (SDNY 1911) (Learned Hand, discussing purified epinephrine), partially maintained, partially revoked, 196F.496 (2d Cir. 1912), each of which is incorporated herein by reference. In addition, in some aspects, the present disclosure provides certain quantitative measurements of concentrations or purity limits that will be found in isolated and biologically pure microbial cultures. In certain embodiments, the presence of these purity values ​​is another attribute that distinguishes microorganisms disclosed in the present invention from those microorganisms that exist in a natural state. See, e.g., Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4th Cir. 1958) (discussing the purity limits of vitamin B12 produced by microorganisms), which is incorporated herein by reference.

[0094] As used herein, "individual isolate" should be considered to mean a composition or culture that primarily contains a single genus, species, or strain of a microorganism, after separation from one or more other microorganisms. The phrase should not be considered to indicate the degree of separation or purification of a microorganism. However, an "individual isolate" may contain essentially only one genus, species, or strain of a microorganism.

[0095] As used herein, the term "growth medium" is any medium suitable for supporting plant growth. As examples, the medium can be natural or artificial, including but not limited to: soil, potting mix, bark, vermiculite, hydroponic solutions alone and applied to a solid plant support system, and tissue culture gel. It should be understood that the medium can be used alone or in combination with one or more other mediums. It can also be used with or without the addition of exogenous nutrients and physical support systems for roots and leaves.

[0096] In one embodiment, the growth medium is a naturally occurring medium, such as soil, sand, mud, clay, humus, topsoil, rock or water. In another embodiment, the growth medium is artificial. Such artificial growth medium can be constructed to simulate the conditions of a naturally occurring medium; however, this is not required. The artificial growth medium can be made of one or more of any number and combination of materials, including sand, minerals, glass, rock, water, metals, salts, nutrients, water. In one embodiment, the growth medium is sterile. In another embodiment, the growth medium is not sterile.

[0097] The culture medium may be modified or enriched with additional compounds or components, such as components that may facilitate the interaction and / or selection of a particular population of microorganisms with the plant and with each other. For example, antibiotics (such as penicillin) or sterilizing agents (e.g., quaternary ammonium salts and oxidizing agents) may be present, and / or physical conditions (such as salinity, plant nutrients (e.g., organic and inorganic minerals (such as phosphorus, nitrogen salts, ammonia, potassium and micronutrients, such as cobalt and magnesium), pH and / or temperature) may be modified.

[0098] The term "plant" generally includes whole plant, plant organ, plant tissue, seed, plant cell, seed and its offspring. Plant cell includes but is not limited to cells from seed, suspension culture, plumule, meristem area, callus, leaf, root, bud, gametophyte, sporophyte, pollen and microspore. "Plant component" is intended to refer to whole plant or plant component, which may include differentiation and / or undifferentiated tissue, such as but not limited to plant tissue, part and cell type. In one embodiment, plant component is one of the following: whole plant, seedling, meristem, basic tissue, vascular tissue, skin tissue, seed, leaf, root, bud, stem, flower, fruit, runner, bulb, tuber, corm, high bud, bud, bract, tumor tissue and various forms of cell and culture (for example, unicellular, protoplast, plumule, callus). The term "plant organ" refers to plant tissue or tissue group constituting the plant part different in form and function. As used herein, "plant part" is synonymous with a "part" of a plant and refers to any part of a plant, and may include different tissues and / or organs, and is used interchangeably with the term "tissue" throughout.

[0099] "Progeny" includes any subsequent generation of an organism produced by sexual or asexual reproduction.

[0100] As used herein, the term "plant parts" refers to plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant callus, plant masses and plant cells intact in plants or plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, grains, ears, cobs, rinds, stalks, roots, root tips, anthers, etc. and parts of themselves. Cereals are intended to mean mature seeds produced by commercial growers for purposes other than growing or breeding species. Offspring, variants and mutants of regenerated plants are also included within the scope of the present invention, provided that these parts contain introduced polynucleotides.

[0101] Similarly, "plant reproductive component" is intended to refer generally to any part of a plant that is capable of starting other plants through sexual or asexual reproduction of the plant, such as, but not limited to, seeds, seedlings, roots, buds, cuttings, scions, grafts, runners, bulbs, tubers, corms, high buds or bracts. Plant components can be located in plants or in plant organs, tissue cultures or cell cultures.

[0102] The term "monocotyledonous" or "monocotyledonous plant" refers to the subclass of angiosperms, also known as the "monocots," whose seeds typically contain only one embryonic leaf or cotyledon. The term includes reference to whole plants, plant parts, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny thereof.

[0103] The term "dicotyledonous" or "dicotyledonous plant" refers to the subclass of angiosperms, also known as the "dicots," whose seeds typically contain two embryonic leaves or cotyledons. The term includes reference to whole plants, plant parts, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny thereof.

[0104] As used herein, the term "cultivar" refers to a plant variety, line or race that has been produced by horticultural or agronomic techniques and that is not normally found in wild-type populations.

[0105] As used herein, "improved" should be interpreted broadly to encompass improvements in the properties of a plant compared to a control plant or compared to a known average quantity associated with the property in question. For example, "improved" plant biomass associated with the application of the beneficial microorganisms or consortia of the present disclosure can be demonstrated by comparing the biomass of a plant treated with the microorganisms taught herein with the biomass of an untreated control plant. Alternatively, the biomass of a plant treated with the microorganisms taught herein can be compared with the average biomass typically achieved by a given plant (as indicated in scientific or agricultural publications known to those skilled in the art). In the present disclosure, "improved" does not necessarily require that the data be statistically significant (e.g., p<0.05); rather, any quantifiable difference indicating that one value (e.g., average treatment value) is different from another value (e.g., average control value) can be raised to the extent of "improved".

[0106] As used herein, "inhibit and repress" and similar terms should not be construed as requiring complete inhibition or repression, although this may be desired in some embodiments.

[0107] As used herein, the term "genotype" refers to the genetic constitution of an individual cell, cell culture, tissue, organism (eg, plant), or population of organisms.

[0108] The compositions and methods herein can provide plants with improved "agronomic traits" or "agronomically important traits" or "agronomically interesting traits" which may include, but are not limited to, disease resistance, drought tolerance, heat tolerance, cold tolerance, salt tolerance, metal tolerance, herbicide tolerance, improved water use efficiency, improved nitrogen use efficiency, improved nitrogen fixation, insect resistance, herbivore resistance, pathogen resistance, increased yield, enhanced health, improved vigor, improved growth, increased photosynthetic capacity, enhanced nutrition, altered protein content, altered oil content, increased biomass, increased shoot length, increased root length, improved root architecture, metabolite modulation, proteome modulation, increased seed weight, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, altered seed nutrient composition, as compared to an isoline plant that does not contain the modifications derived from the methods or compositions herein.

[0109] "Agronomic potential" is intended to mean the ability of a plant component to express a phenotype (preferably an improved agronomic trait) at some point in its life cycle or to transmit the phenotype to another plant component with which it is associated in the same plant.

[0110] As used herein, the term "molecular marker", "marker" or "genetic marker" refers to an indicator used in a method for observing differences in nucleic acid sequence characteristics. Examples of such indicators are restriction fragment length polymorphism (RFLP) markers, amplified fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), insertion mutations, microsatellite markers (SSRs), sequence characterization amplified regions (SCARs), enzyme-cut amplified polymorphic sequences (CAPS) markers or isozyme markers or a combination of markers described herein (which define specific genes and chromosome positions). The positioning of molecular markers near alleles is a procedure that can be performed by an ordinary person with experience in molecular biotechnology.

[0111] As used herein, the term "trait" refers to a characteristic or phenotype. For example, in the context of some embodiments of the present disclosure, crop yields relate to the amount of marketable biomass (e.g., fruit, fiber, grain) produced by plants. Desired traits may also include other plant characteristics, including but not limited to: water use efficiency, nutrient use efficiency, productivity, mechanical harvestability, fruit maturity, shelf life, insect resistance / disease resistance, early plant maturity, stress tolerance, etc. Traits can be dominant or recessive, or inherited in a partially or incompletely dominant manner. Traits may be monogenic (i.e., determined by a single locus) or polygenic (i.e., determined by more than one locus) or may also be produced by the interaction of one or more genes with the environment.

[0112] As used herein, the term "phenotype" refers to an observable characteristic of an individual cell, cell culture, organism (eg, plant), or population of organisms that results from the interaction between the genetic makeup (ie, genotype) of the individual and the environment.

[0113] As used herein, a "synthetic nucleotide sequence" or "synthetic polynucleotide sequence" is a nucleotide sequence that is not known to occur in nature or that does not occur naturally. Generally, such a synthetic nucleotide sequence will contain at least one nucleotide difference when compared to any other naturally occurring nucleotide sequence.

[0114] As used herein, the term "nucleic acid" refers to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length or their analogs. The term refers to the primary structure of the molecule, and therefore includes double-stranded and single-stranded DNA, and double-stranded and single-stranded RNA. It also includes modified nucleic acids, such as methylated and / or capped nucleic acids, nucleic acids containing modified bases, backbone modifications, etc. The terms "nucleic acid" and "nucleotide sequence" are used interchangeably.

[0115] As used herein, the term "gene" refers to any DNA fragment associated with a biological function. Therefore, a gene includes, but is not limited to, a coding sequence and / or a regulatory sequence required for its expression. A gene may also include unexpressed DNA fragments that, for example, form recognition sequences for other proteins. A gene may be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.

[0116] As used herein, the term "homolog" or "homologue" or "ortholog" is known in the art and refers to related sequences that share a common ancestor or family member and are determined based on the degree of sequence identity. The terms "homology", "homolog", "substantially similar" and "substantially corresponding" are used interchangeably herein. They refer to nucleic acid fragments in which changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the present disclosure, such as the deletion or insertion of one or more nucleotides that do not substantially change the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. Therefore, it should be understood that the present disclosure encompasses more than specific exemplary sequences, as will be understood by those skilled in the art. These terms describe the relationship between a gene found in a species, subspecies, variant, cultivar or strain and a corresponding or equivalent gene in another species, subspecies, variant, cultivar or strain. For the purposes of the present disclosure, homologous sequences are compared. "Homologous sequences" or "homologues" or "orthologs" are considered, believed or known to be functionally related. Functional relationships can be indicated in any of a variety of ways, including but not limited to: (a) degree of sequence identity and / or (b) identical or similar biological functions. Preferably, both (a) and (b) are indicated. Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (ed. by FM Ausubel et al., 1987) Supplement 30, Section 7.718, Table 7.71. Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, UK), ALIGN Plus (Scientific and Educational Software, Pennsylvania), and AlignX (Vector NTI, Invitrogen, Carlsbad, CA). Another alignment program is Sequencher (Gene Codes, Ann Arbor, Michigan), which uses default parameters.

[0117] As used herein, the term "nucleotide changes" refers to, for example, nucleotide substitutions, deletions, insertions, chemical alterations, or any of the foregoing, as is well understood in the art.

[0118] As used herein, the term "protein modification" refers to, for example, amino acid substitutions, amino acid modifications, deletions and / or insertions, as are well understood in the art.

[0119] As used herein, the term "at least a portion" or "fragment" of a nucleic acid or polypeptide means a portion having the minimum size characteristic of such a sequence, or any larger fragment of a full-length molecule (up to and including the full-length molecule). Fragments of the polynucleotides of the present disclosure may encode biologically active portions of gene regulatory elements. The biologically active portion of a gene regulatory element may be prepared by isolating a portion of one of the polynucleotides of the present disclosure that contains the gene regulatory element and evaluating the activity as described herein. Similarly, a portion of a polypeptide may be 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, etc., up to a full-length polypeptide. The length of the portion to be used will depend on the specific application. The portion of a nucleic acid that can be used as a hybridization probe may be as short as 12 nucleotides; in some embodiments, it is 20 nucleotides. The portion of a polypeptide that can be used as an epitope may be as short as 4 amino acids. The portion of a polypeptide that functions as a full-length polypeptide will generally be longer than 4 amino acids.

[0120] As used herein, the term "primer" refers to an oligonucleotide that can anneal to an amplification target and allow a DNA polymerase to connect, thereby acting as a starting point for DNA synthesis under conditions that induce the synthesis of primer extension products (i.e., in the presence of nucleotides and reagents for polymerization such as DNA polymerase and at a suitable temperature and pH). (Amplification) primers are preferably single-stranded to obtain maximum amplification efficiency. Preferably, primers are oligodeoxyribonucleotides. Primers must be long enough to initiate the synthesis of extension products in the presence of reagents for polymerization. The exact length of a primer will depend on many factors, including the composition of temperature and primers (A / T vs. G / C content). A pair of bidirectional primers is composed of a forward primer and a reverse primer as commonly used in the field of DNA amplification (such as PCR amplification).

[0121] The term "stringency" or "stringent hybridization conditions" refers to hybridization conditions that affect the stability of the hybrid, such as temperature, salt concentration, pH, formamide concentration, etc. These conditions are optimized empirically to maximize the specific binding of the primer or probe to its target nucleic acid sequence and minimize non-specific binding. The term used includes reference to such conditions under which the probe or primer will hybridize to its target sequence to a detectably greater extent than other sequences (e.g., at least 2 times greater than the background). Stringent conditions are sequence-dependent and will vary in different situations. Longer sequences hybridize specifically at higher temperatures. In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) of the specific sequence at a defined ionic strength and pH. Tm is the temperature (under a defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a fully matched probe or primer. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na+ ions, typically about 0.01 to 1.0 M Na+ ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30°C for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes or primers (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved by the addition of destabilizing agents such as formamide. Exemplary low stringency conditions or "conditions of reduced stringency" include hybridization at 37°C with a buffer solution of 30% formamide, 1 M NaCl, 1% SDS and washing in 2×SSC at 40°C. Exemplary high stringency conditions include hybridization at 37°C in 50% formamide, 1 M NaCl, 1% SDS, and washing in 0.1×SSC at 60°C. Hybridization procedures are well known in the art and are described, for example, by Ausubel et al., 1998 and Sambrook et al., 2001. In some embodiments, stringent conditions are hybridization at 45°C in 0.25 M Na2HPO4 buffer (pH 7.2) containing 1 mM Na2EDTA, 0.5% to 20% sodium dodecyl sulfate (such as 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%), followed by washing in 5×SSC containing 0.1% (w / v) sodium dodecyl sulfate at 55°C to 65°C.

[0122] In some embodiments, the cell or organism has at least one heterologous trait. As used herein, the term "heterologous trait" refers to a phenotype conferred to a cell or organism by an exogenous molecule or other organism (e.g., a microorganism), a DNA fragment, a heterologous polynucleotide, or a heterologous nucleic acid.

[0123] Various changes in phenotype are of interest in the present disclosure, including, but not limited to, modifying fatty acid composition in a plant, altering the amino acid content of a plant, altering a plant's pathogen defense mechanism, increasing the yield of a plant with economically important traits (e.g., grain yield, forage yield, etc.), etc. These results can be achieved by providing expression of a heterologous product or increased expression of an endogenous product in a plant using the methods and compositions of the present disclosure.

[0124] A "synthetic combination" may include a combination of plants and microorganisms of the present disclosure. The combination may be achieved, for example, by coating the surface of a seed of a plant (such as an agricultural plant) or a host plant tissue (root, stem, leaf, etc.) with a microorganism of the present disclosure. In addition, a "synthetic combination" may include a combination of microorganisms of various strains or species. A synthetic combination has at least one variable that can distinguish the combination from any combination existing in nature. The variable may be, in particular, the concentration of microorganisms on seeds or plant tissues that would not naturally occur, or a combination of microorganisms and plants that would not naturally occur, or a combination of microorganisms or strains that would not naturally co-exist. In each of these cases, the synthetic combination shows traces of artificiality and has structural and / or functional attributes that do not exist when the individual elements in the combination are considered separately.

[0125] In some embodiments, a microorganism may be "endogenous" to a seed or plant. As used herein, a microorganism is considered "endogenous" to a plant or seed if it originates from a plant sample from which it is derived. That is, a situation in which the microorganism is found associated with the plant in nature. In embodiments in which endogenous microorganisms are applied to plants, the endogenous microorganisms are applied in amounts that differ from the levels found on the plant in nature. Therefore, if a microorganism endogenous to a given plant is present on the plant at a level that is not present in nature, the microorganism may still form a synthetic combination with the plant.

[0126] In some embodiments, a composition (such as a microorganism) may be "heterologous" (also referred to as "exogenous") to another composition (such as a seed or a plant), and in some aspects, is referred to herein as a "heterologous composition". As used herein, a microorganism is considered to be "heterologous" to a plant or seed if the microorganism is not derived from the plant sample from which it is derived. That is, a situation in which the microorganism is not found associated with the plant in nature. For example, a microorganism that is normally associated with the leaf tissue of a maize plant is considered to be exogenous to the leaf tissue of another maize plant that does not contain the microorganism in nature. In another example, a microorganism that is normally associated with a maize plant is considered to be exogenous to a wheat plant that does not contain the microorganism in nature.

[0127] A composition is "heterogeneously placed" when mechanically or manually applied, artificially inoculated, associated or placed on or in a plant component, seedling, plant, or on or in a plant growth medium or on or in a treatment formulation such that the treatment agent is present on or in the plant component, seedling, plant, plant growth medium or formulation in a manner not found in nature prior to application of the treatment agent, e.g., in that plant variety, at that stage of plant development, in that plant tissue, in that abundance, or in that growth environment (e.g., drought). In some embodiments, this manner is contemplated as being selected from the group consisting of: presence of microorganisms; presence of microorganisms in different cell numbers, concentrations, or amounts; presence of microorganisms in different plant components, tissues, cell types, or other physical locations in or on a plant; presence of microorganisms at different time periods, such as during the developmental period of a plant or plant component, time of day, time of season, and combinations thereof. In some embodiments, "placed heterogenously" means that the microorganism is applied to a tissue or cell type of a plant component that is different from the location where the microorganism naturally occurs. In some embodiments, "heterogeneously placed" means that a microorganism is applied to a certain developmental stage of a plant component, seedling or plant, and the microorganism is not associated in nature at this developmental stage, but may be associated at other stages. For example, if a microorganism is usually found in the flowering stage of a plant and not found in other stages, the microorganism applied at the seedling stage can be considered to be heterogenously placed. In some embodiments, if a microorganism is usually found in the root tissue of a plant component and not found in leaf tissue, and the microorganism is applied to the leaf, the microorganism is heterogenously placed. In another non-limiting example, if the microorganism is present in the mesophyll layer of leaf tissue in nature but is applied to the epithelial layer, the microorganism will be considered to be heterogenously placed. In some embodiments, "heterogeneously placed" means that a natural plant component, seedling or plant does not contain a detectable level of microorganisms in the same plant component, seedling or plant. In some embodiments, "being placed heterologously " means that compared with the concentration, quantity or amount present in described plant component, seedling or plant in nature, microorganism is applied to this plant component, seedling or plant with larger concentration, quantity or amount.For example, when compared with the concentration existing before placing microorganism with at least 1.5 times, between 1.5 times and 2 times, between 2 times, between 2 times and 3 times, between 3 times, between 3 times and 5 times, between 5 times, between 5 times and 7 times, between 7 times, between 7 times and 10 times, between 10 times or even more than 10 times quantity, amount or concentration exist, described microorganism is placed heterologously.In another non-limiting example, the microorganism present in cypress (cupressaceous tree) tissue in nature will be regarded as heterologous for the tissue of maize, wheat, cotton, soybean plant.In another example, a microorganism that occurs in nature in leaf tissue of a maize, spring wheat, cotton, or soybean plant is considered heterologous to leaf tissue of another maize, spring wheat, cotton, or soybean plant that does not contain the microorganism in nature or contains a different amount of the microorganism.

[0128] A microorganism may also be "heterologously placed" on a given plant tissue. This means that the microorganism is placed on a plant tissue on which it is not found in nature. For example, if a given microorganism only occurs naturally on the roots of a given plant, the microorganism may be exogenously applied to the plant's above-ground tissue and would thereby be "heterologously placed" on the plant tissue. Thus, a microorganism is considered to be heterologously placed when applied to a plant that does not naturally occur with the microorganism or does not naturally have the microorganism present in the amount applied.

[0129] The compositions and methods herein can provide a "modulated," "agronomic trait," or "agronomically important trait" to a host plant, which can include, but are not limited to, the following: altered oil content, altered protein content, altered seed carbohydrate composition, altered seed oil composition, and altered seed protein composition, chemical tolerance, cold tolerance, delayed senescence, disease resistance, drought tolerance, ear weight, improved growth, enhanced health, heat tolerance, herbicide tolerance, herbivore resistance, increased nitrogen fixation, improved nitrogen utilization efficiency, improved root architecture, improved water use efficiency, increased biomass, increased root length, increased seed weight, increased shoot length, compared to isotype plants grown from seeds without the seed treatment formulation , increased yield, increased yield under water-limited conditions, grain quality, grain moisture content, metal tolerance, number of ears, number of grains per ear, number of pods, nutritional enhancement, pathogen resistance, insect resistance, increased photosynthetic capacity, salt tolerance, stay-green, increased vigor, increased dry weight of mature seeds, increased fresh weight of mature seeds, increased number of mature seeds per plant, increased chlorophyll content, increased number of pods per plant, increased pod length per plant, reduced number of wilted leaves per plant, reduced number of severely wilted leaves per plant, and increased number of non-wilted leaves per plant, detectable modulation of metabolite levels, detectable modulation of transcript levels, and detectable modulation of the proteome. The term "modulated" is intended to refer to changes in agronomic traits that are altered by the presence of microorganisms, exudates, broths, metabolites, etc. In various aspects, the modulation provides for the conferment of beneficial traits.

[0130] Microorganisms and microorganisms

[0131] As used herein, the term "microorganism" should be interpreted in a broad sense. It includes, but is not limited to, prokaryotic bacteria and archaea as well as eukaryotic fungi and protists.

[0132] As an example, the microorganisms may include: Proteobacteria (such as Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azorh izobium), Azotobacter, Duganella, Delftia, Bradyrhizobium, Sinorhizobium, Variovorax, and Halomonas), Firmicutes (such as Bacillus, Paenibacillus, Lactobacillus, Mycoplasma, and Acetobacter), m)), Actinobacteria (such as Brevibacterium, Janibacter, Streptomyces, Rhodococcus, Microbacterium, Curtobacterium, Cellulomonas and Nocardioides) and fungi Ascomycota (such as Trichoderma, Ampelomycetes and Powdery Fungi) s), Coniothyrium, Paecoelomyces, Penicillium, Cladosporium, Hypocrea, Beauveria, Metarhizium, Verticullium, Cordyceps, Pichea and Candida), Basidiomycota (such as Coprinus),and Mucoromycota (such as Mucor and Mortierella); as well as Orbilia / Arthrobotrys, Lysinibacillus, Microbacterium, Talaromyces, Arthrobacter, Kosakonia, Masillia, Novosphingobium, and Tumebacillus.

[0133] In certain embodiments, the microorganism is an endophyte, or an epiphyte, or a microorganism that resides in the rhizosphere or rhizosheath of a plant. That is, the microorganism can be found in soil material attached to the roots of a plant or in the area immediately adjacent to the roots of a plant.

[0134] In one embodiment, the microorganism is an endophyte. Endophytes can benefit the host plant by preventing pathogenic organisms from colonizing it. Endophytes create a "barrier effect" to widespread colonization of plant tissues, where local endophytes outcompete and prevent pathogenic organisms from taking hold. Endophytes can also produce chemicals that inhibit the growth of competitors, including pathogenic organisms.

[0135] In certain embodiments, the microorganism is not culturable. This should be taken to mean that the microorganism is not known to be culturable or is difficult to culture using methods known to those skilled in the art.

[0136] The microbial organisms of the present disclosure may be collected or obtained from any source, or contained within and / or associated with material collected from any source.

[0137] In one embodiment, the microorganisms are obtained from any general terrestrial environment, including its soil, plants, fungi, animals (including invertebrates), and other biota, including sediments, water, and biota of lakes and rivers; from marine environments, their biota and sediments (e.g., seawater, marine mud, marine plants, marine invertebrates (e.g., sponges), marine vertebrates (e.g., fish)); terrestrial and marine lithosphere (topsoil and rocks, such as squeezed underground rocks, sands, and clays); cryosphere and its meltwater; atmosphere (e.g., filtered air dust, clouds, and raindrops); urban, industrial, and other man-made environments (e.g., organic and mineral deposits accumulated on concrete, roadside gutters, roof surfaces, road surfaces).

[0138] In another embodiment, microorganism is collected from the source that may be conducive to the selection of suitable microorganism.As an example, this source can be a specific environment that is suitable for other plant growth or is regarded as being associated with terroir.In another example, this source can be a plant with one or more desired traits, such as a plant that grows naturally in a specific environment or under certain conditions of interest.As an example, a certain plant can grow naturally in sandy soil or high salinity sand, or under extreme temperatures, or with very little water, or it can resist some pests or diseases in the environment, and may expect that cash crops grow in such conditions, particularly when they are only conditions in, for example, specific geographical locations.As another example, microorganism can be collected from the cash crops grown in such environments, or more specifically, the single crop plant that best shows the proterties of interest in the crop grown in any specific environment, such as the fastest growing plant in the crop grown in salt-restricted soil, or the plant with the lowest degree of damage in the crop exposed to severe insect damage or disease prevalence, or the plant with some metabolites and other compounds (including fiber content, oil content, etc.) of desired amount, or the plant showing desired color, taste or smell. The microorganisms can be collected from any material present in the plant of interest or the environment of interest, including fungi and other animal and plant biota, soil, water, sediment, and other elements of the environment as mentioned previously. In certain embodiments, the microorganisms are individual isolates isolated from different environments.

[0139] In one embodiment, the microorganism or combination of microorganisms used in the methods of the present disclosure can be selected from a pre-existing collection of individual microbial species or strains based on some knowledge of their possible or predicted benefits to the plant. For example, the microorganism can be predicted to: increase nitrogen fixation; release phosphate from soil organic matter; release phosphate from inorganic forms of phosphate (e.g., rock phosphate); "fix carbon" in root microspheres; live in the rhizosphere of the plant, thereby helping the plant absorb nutrients from the surrounding soil and then make these nutrients more easily available to the plant; increase the number of nodules on the plant's roots, thereby increasing the number of symbiotic nitrogen fixers (e.g., Rhizobium species) per plant and the amount of nitrogen fixed by the plant; trigger plant defense responses, such as ISR (induced systemic resistance) or SAR (systemic acquired resistance), which help plants resist invasion and spread of pathogenic microorganisms; compete with microorganisms that are harmful to plant growth or health through antagonism or competitive use of resources (such as nutrients or space); change the color of one or more parts of the plant, or change the chemical condition of the plant, its smell, taste, or one or more other qualities.

[0140] In one embodiment, the microorganism or combination of microorganisms is selected from a collection of pre-existing individual microbial species or strains whose possible or predicted benefits to plants are unknown. For example, a collection of unidentified microorganisms isolated from plant tissue without any knowledge of their ability to improve plant growth or health, or a collection of microorganisms collected to explore their potential for use in producing compounds that may lead to drug development.

[0141] In one embodiment, the microorganism is obtained from a source material (e.g., soil, rock, water, air, dust, plant or other organism) in which it naturally resides. Considering the intended use of the microorganism in the method of the present disclosure, it can be provided in any suitable form. However, as an example only, the microorganism can be provided in the form of an aqueous suspension, a gel, a homogenate, a granule, a powder, a slurry, a living organism or a dry material.

[0142] Microorganisms of the present disclosure can be separated in basically pure or mixed cultures. They can be concentrated, diluted or provided at the natural concentrations present in the source material. For example, microorganisms from salt deposits can be separated for use in the present disclosure in the following manner: the deposits are suspended in fresh water and the deposits are dropped to the bottom. Water containing most of the microorganisms can be removed by decantation after a suitable settling time period and directly applied to plant growth medium, or concentrated by filtration or centrifugation, diluted to a suitable concentration and applied to plant growth medium with most of the salt removed. As another example, microorganisms from mineralization or toxic sources can be similarly processed to recycle microorganisms to apply to plant growth materials, thereby minimizing the possibility of damaging plants.

[0143] In another embodiment, the microorganisms are used in crude form, where they are not separated from the source material in which they naturally reside. For example, the microorganisms are provided in combination with the source material in which they reside; for example, in soil form, or roots, seeds, or leaves of plants. In this embodiment, the source material may include one or more species of microorganisms.

[0144] In some embodiments, mixed populations of microorganisms are used in the methods of the present disclosure.

[0145] In embodiments of the present disclosure in which a microorganism is isolated from a source material (e.g., a material in which the microorganism naturally resides), any one or combination of a number of standard techniques that will be readily known to the skilled artisan may be used. However, as an example, these techniques generally employ processes that can be used to obtain solid or liquid cultures of a single microorganism in substantially pure form, typically by physical separation on the surface of a solid microbial growth medium or by volume dilution separation in a liquid microbial growth medium. These processes may include separation from a dry material, a liquid suspension, a slurry, or a homogenate (where the material is spread as a thin layer on an appropriate solid gel growth medium), or serial dilutions of the material in a sterile medium and inoculation into a liquid or solid medium.

[0146] Although not required, in one embodiment, the material containing microorganisms can be pre-treated prior to the separation process to multiply all microorganisms in the material, or to select a portion of the microbial population by enriching the material with microbial nutrients (e.g., by pasteurizing the sample to select microorganisms (e.g., bacilli) that are resistant to heat exposure, or exposing the sample to low concentrations of organic solvents or sterilizing agents (e.g., household bleach) to enhance the survival of spore-forming or solvent-resistant microorganisms. As described above, the microorganisms can then be isolated from the enriched material or the material treated to achieve selective survival.

[0147] In one embodiment of the present disclosure, endophytic or epiphytic plant microorganisms are separated from plant materials. Many standard techniques known in the art can be used and microorganisms can be separated from any suitable tissue (including, for example, roots, stems and leaves and plant reproductive tissue) in plants. As an example, conventional methods for separating from plants generally include aseptic excision of interested plant materials (e.g., roots or stem lengths, leaves), surface sterilization with suitable solutions (e.g., 2% sodium hypochlorite), followed by placement of plant materials on nutrient media for microbial growth (see, for example, Strobel G and Daisy B (2003) Microbiology and Molecular Biology Reviews 67 (4): 491-502; Zinniel DK et al., (2002) Applied and Environmental Microbiology 68 (5): 2198-2208).

[0148] In one embodiment of the present disclosure, the microorganisms are isolated from root tissue.Another method for isolating microorganisms from plant material is described in detail below.

[0149] In one embodiment, the microbial population is exposed to a selection pressure (either prior to the method or at any stage during the method). For example, exposing the microorganisms to pasteurization prior to their addition to the plant growth medium (preferably sterile) may enhance the probability that plants selected for desirable traits will be associated with spore-forming microorganisms that may more easily survive under adverse conditions, during commercial storage, or when applied to seeds as a coating in an adverse environment.

[0150] In certain embodiments, as mentioned above, microorganism can be used in crude form and need not be separated from plant or culture medium.For example, plant material or growth medium including microorganisms useful to selected plants can be obtained, and this plant material or growth medium is used as a crude microorganism source for the next round of methods, or is used as a crude microorganism source when the method ends.For example, complete plant material can be obtained and optionally processed, such as covered or broken.Alternatively, the single tissue or part (such as leaf, stem, root and seed) of the selected plant can be separated from the plant and optionally processed, such as covered or broken.In certain embodiments, one or more parts of the plant associated with the second group of one or more microorganisms can be removed from one or more selected plants, and in the case of any continuous repetition of the method, grafted on one or more plants used in any step of the plant breeding method.

[0151] Exemplary Microorganisms

[0152] In various aspects, the present disclosure provides isolated microorganisms, including novel strains of the identified microbial species presented in Table 1.

[0153] In other aspects, the present disclosure provides isolated whole microbial cultures of the species and strains identified in Table 1. These cultures can contain various concentrations of the microorganisms.

[0154] In various aspects, the present disclosure provides for utilizing a microorganism selected from Table 1 in agriculture.

[0155] In some embodiments, microorganisms from the genus Bacillus are utilized in agriculture to impart one or more beneficial traits to a plant species.

[0156] Additionally, the present disclosure relates to microorganisms having properties substantially similar to those of the microorganisms identified in Table 1.

[0157] The isolated microbial species and novel strains of said species identified in the present disclosure are capable of conferring beneficial characteristics or traits, such as agronomically important traits, to target plant species.

[0158] For example, the isolated microorganisms or consortia of said microorganisms described in Table 1 are capable of improving plant health and vigor. The improved plant health and vigor can be quantitatively measured, for example, by measuring the effect of the application of said microorganism on a phenotypic or genotypic trait of the plant.

[0159] Source of microorganisms

[0160] The microorganisms of the present disclosure were obtained in various areas of New Zealand and the United States, among other locations.

[0161] Isolation and cultivation of microorganisms

[0162] The microorganisms of Table 1 were identified by utilizing standard microscopy techniques to characterize the phenotype of the microorganism, which was then used to identify the microorganism as a taxonomically recognized species.

[0163] Isolation, identification and cultivation of the disclosed microorganisms can be achieved using standard microbiological techniques. Examples of such techniques are found in Gerhardt, P. (ed.) Methods for General and Molecular Microbiology. American Society for Microbiology, Washington, DC (1994) and Lennette, EH (ed.) Manual of Clinical Microbiology, 3rd edition, American Society for Microbiology, Washington, DC (1980), each of which is incorporated by reference.

[0164] Isolation can be achieved by streaking a sample on a solid medium (e.g., a nutrient agar plate) to obtain a single colony characterized by the phenotypic traits described above (e.g., Gram positive / negative, ability to sporulate aerobically / anaerobically, cell morphology, carbon source metabolism, acid / base production, enzyme secretion, metabolic secretion, etc.) and reducing the possibility of working with a culture that has been contaminated.

[0165] For example, for the isolated bacteria of the present disclosure, biologically pure isolates can be obtained by repeated subculture of biological samples, and streaking onto solid culture media after each subculture to obtain individual colonies. Methods for preparing freeze-dried bacteria, thawing them and growing them are well known, such as Gherna, RL and C Reddy. 2007. Culture Preservation, pages 1019-1033, in C Reddy, TJ Beveridge, J A Breznak, GA Marzluf, TMS Schmidt and LRS Nyder, eds., American Society for Microbiology, Washington, DC, page 1033; the document is incorporated herein by reference. Therefore, freeze-dried liquid preparations and cultures stored for a long time at -70°C in a solution containing glycerol are contemplated for use in providing the preparations of the present invention.

[0166] The bacteria of the present disclosure can be propagated in liquid culture medium under aerobic conditions. The culture medium for growing the bacterial strains of the present disclosure includes a carbon source, a nitrogen source and an inorganic salt, as well as special required substances such as vitamins, amino acids, nucleic acids, etc. Examples of suitable carbon sources that can be used to grow bacterial strains include, but are not limited to, starch, peptone, yeast extract, amino acids, sugars such as glucose, arabinose, mannose, glucosamine, maltose, etc.; salts of organic acids such as acetic acid, fumaric acid, adipic acid, propionic acid, citric acid, gluconic acid, malic acid, pyruvic acid, malonic acid, etc.; alcohols such as ethanol and glycerol; oils or fats such as soybean oil, rice bran oil, olive oil, corn oil, sesame oil. The amount of the added carbon source varies depending on the type of carbon source and is generally between 1 and 100 grams per liter of culture medium. Preferably, the culture medium contains 0.1% to 5% (W / V) concentrations of glucose, starch and / or peptone as the main carbon source. Examples of suitable nitrogen sources that can be used to grow the bacterial strains of the present invention include, but are not limited to, amino acids, yeast extract, tryptone, beef extract, peptone, potassium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonia, or a combination thereof. The amount of the nitrogen source varies depending on the type of nitrogen source, typically between 0.1 and 30 grams per liter of culture medium. Inorganic salts, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferrous sulfate, ferrous sulfate, ferric chloride, ferrous chloride, manganese sulfate, manganese chloride, zinc sulfate, zinc chloride, copper sulfate, calcium chloride, sodium chloride, calcium carbonate, sodium carbonate can be used alone or in combination. The amount of inorganic acid varies depending on the type of inorganic salt, typically between 0.001 and 10 grams per liter of culture medium. Examples of special required substances include, but are not limited to, vitamins, nucleic acids, yeast extracts, peptones, meat extracts, malt extracts, dry yeast, and a combination thereof. Cultivation can be achieved at a temperature that allows bacterial strains to grow (essentially between 20° C. and 46° C.). In some aspects, the temperature range is 30°C to 37°C. In order to achieve optimal growth, in some embodiments, the culture medium can be adjusted to pH 7.0 to 7.4. It should be understood that commercially available culture media can also be used to culture bacterial strains, such as Nutrient Broth or Nutrient Agar available from Difco, Detroit, MI. It should be understood that the culture time can vary depending on the type of culture medium used and the concentration of sugar as the main carbon source.

[0167] In various aspects, the culture lasts for 24 to 96 hours. The bacterial cells thus obtained are isolated using methods well known in the art. Examples include, but are not limited to, membrane filtration and centrifugation. The pH can be adjusted using sodium hydroxide or the like, and the culture can be dried using a freeze dryer until the water content becomes equal to 4% or less. Microbial co-cultures can be obtained by breeding each strain as described above. It should be understood that these microbial strains can be co-cultured when compatible culture conditions can be adopted.

[0168] Identification of microorganisms

[0169] Microorganisms can be distinguished as a genus based on polyphasic classification, which merges all available phenotypic and genotypic data into a consensus classification (Vandamme et al., 1996. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 1996, 60: 407-438). A recognized genotypic method for defining species is based on overall genome relatedness, so that under standard conditions, strains that share about 70% or more relatedness when using DNA-DNA hybridization at 5°C or lower ΔTm (difference in melting temperature between homologous and heterologous hybrids) are considered to be members of the same species. Therefore, groups that share more than the aforementioned 70% threshold can be considered to be variants of the same species.

[0170] For bacterial microorganisms, 16S rRNA sequences are often used to determine taxonomy and distinguish species because if a 16S rRNA sequence shares less than a specified % sequence identity with a reference sequence, then the two organisms from which the sequence was obtained are considered different species.

[0171] Thus, if microorganisms share at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity in 16S or 16S rRNA or rDNA sequences, these microorganisms can be considered to be the same species. In some aspects, microorganisms can be considered to be the same species only when they share at least 95% identity.

[0172] Furthermore, microbial strains of a species can be defined, such as strains that share at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity in the 16S rRNA sequence.

[0173] Comparisons can also be made to a reference sequence and a 23S rRNA sequence. In some aspects, microorganisms can be considered the same strain only if they share at least 95% identity. In some embodiments, "substantially similar genetic characteristics" means microorganisms that share at least 95% identity.

[0174] For fungal microorganisms, the ITS (internal transcribed sequence) is often used for taxonomic identification. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) has the highest probability of successful identification of the widest range of fungi, has the most clearly defined barcode gap between interspecific and intraspecific variation, and has been proposed as the official fungal identification sequence (Schoch et al., PNAS, April 17, 2012, 109 (16) 6241-6246).

[0175] In one embodiment, the microbial strains of the present disclosure include those comprising a polynucleotide sequence that shares at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any one of SEQ ID NOs: 1-14.

[0176] In one embodiment, the microorganisms of the present disclosure include those comprising a polynucleotide sequence that shares at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs: 1-14.

[0177] In one embodiment, a microbial consortium of the present disclosure includes two or more microorganisms comprising a polynucleotide sequence that shares at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs: 1-14.

[0178] In one embodiment, the microbial consortium of the present disclosure includes two or more microbial strains, wherein at least one of the microbial strains comprises a polynucleotide sequence that shares at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NOs: 1-14.

[0179] In one embodiment, the microbial consortium of the present disclosure comprises two or more microbial strains, wherein at least one of the microbial strains comprises a polynucleotide sequence that shares at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any one of SEQ ID NOs: 1-14, and wherein at least one of the microorganisms is optionally selected from Table 1.

[0180] Unculturable microorganisms usually cannot be assigned to a specific species without a confirmed phenotype; instead, they can be given a tentative name within the genus as long as their 16S rRNA sequence matches that of a known species.

[0181] A method is to observe the distribution of a large number of strains of closely related species in sequence space and identify the clustering of strains well separated from other clusters. The method has been developed by evaluating the clustering pattern using the tandem sequence of multi-core (housekeeping) genes, and has been referred to as multi-locus sequence analysis (MLSA) or multi-locus sequence phylogenetic analysis. MLSA has been successfully used to explore clustering patterns in a large number of strains assigned to extremely closely related species by current classification methods, to view the relationship between a few strains in a genus or in a more extensive classification group, and to solve specific classification problems. More generally speaking, the method can be used to explore whether bacterial species exist, that is, observe whether a large group of similar strains always belong to a well-separated cluster, or in some cases, whether there is no obvious separation into the gene continuity of each cluster.

[0182] In order to more accurately determine the genus, determination of phenotypic traits (such as morphology, biochemistry and physiological characteristics) is performed for comparison with reference genus prototypes. Colony morphology can include color, shape, pigmentation, mucus production, etc. Cells are characterized by shape, size, Gram reaction, extracellular material, presence of endospores, presence and position of flagella, motility and inclusion bodies. Biochemical and physiological characteristics describe the growth of organisms under different ranges of temperature, pH, salinity and atmospheric conditions, and growth in the presence of different sole carbon and nitrogen sources. One of ordinary skill in the art will reasonably be aware of the phenotypic traits that define the genera of the present disclosure. For example, species of the genus Rhizobium are identified using colony color, form and texture on a specific agar (e.g., YMA).

[0183] In one embodiment, the bacterial microorganisms taught herein are identified using 16S rRNA gene sequences. It is known in the art that 16S rRNA contains hypervariable regions, which can provide species / strain-specific characteristic sequences that can be used for bacterial identification. In the present disclosure, many microorganisms are identified via partial (500bp to 1200bp) 16S rRNA sequence features. In various aspects, each strain represents a pure colony isolate selected from an agar plate. Selection is based on any defining morphological characteristics of colonies on agar culture medium to represent the diversity of the organisms present. In embodiments, the culture medium used is R2A, PDA, nitrogen-free semi-solid culture medium or MRS agar. After 24 hours of growth, the colony description of each "picked" isolate is performed, and then the colony description is entered into our database. Sequence data for each isolate is subsequently obtained.

[0184] Phylogenetic analysis using the 16S rRNA gene was used to define "substantially similar" species belonging to a common genus, and also to define "substantially similar" strains of a given taxonomic species. In addition, we recorded the physiological and / or biochemical properties of the isolates, which can be used to highlight subtle and significant differences between strains that may lead to advantageous behaviors of the plant.

[0185] Microbial consortium

[0186] In various aspects, the present disclosure provides a microbial consortium comprising a combination of at least any two microorganisms, one of which is Priesteria gigantea strain 101136, or any strain comprising a polynucleotide sequence sharing at least 95% identity with one or more of SEQ ID NOs. 13-14.

[0187] In various aspects, the present disclosure provides a microbial consortium comprising a combination of at least any two microorganisms, one of which is Bacillus cellulolyticus strain 101166, or any strain comprising a polynucleotide sequence sharing at least 95% identity with one or more of SEQ ID NOs. 1-12.

[0188] In certain embodiments, the consortium of the present disclosure comprises two microorganisms or three microorganisms or four microorganisms or five microorganisms or six microorganisms or seven microorganisms or eight microorganisms or nine microorganisms or ten or more microorganisms. The microorganisms of the consortium are different microbial species, or different strains of microbial species.

[0189] Compositions produced by microorganisms

[0190] In some cases, the microorganisms of the present disclosure can produce one or more compounds and / or have one or more activities, such as one or more of the following: production of a metabolite, production of a plant hormone (such as an auxin), production of acetoin, production of an antimicrobial compound, production of a siderophore, production of a polyketide, production of a phenazine, production of a cellulase, production of a pectinase, production of a chitinase, production of a glucanase, production of a xylanase or a protease or an organic acid or a lipopeptide or a polynucleotide or a polypeptide, nitrogen fixation, mineral phosphate solubilization, or any combination and / or multiple of the foregoing.

[0191] For example, the microorganisms of the present disclosure can produce a plant hormone selected from the group consisting of auxins, cytokinins, gibberellins, ethylene, brassinolide, and abscisic acid.

[0192] Thus, "metabolites produced by a microorganism of the present disclosure" is intended to reflect any molecule (small molecule, vitamin, mineral, protein, nucleic acid, lipid, fat, carbohydrate, etc.) produced by a microorganism. In general, the precise mechanism of action by which a microorganism of the present disclosure confers a beneficial trait to a given plant species is unknown. It is speculated that in some cases, a microorganism produces a metabolite that is beneficial to a plant. Thus, in some aspects, a cell-free or inactivated preparation of a microorganism is beneficial to a plant, as the microorganism does not have to be alive to confer a beneficial trait to a given plant species, as long as the preparation contains a metabolite produced by the microorganism and that is beneficial to the plant.

[0193] In one embodiment, the microorganisms of the present disclosure can produce auxins (e.g., indole-3-acetic acid (IAA)). The production of auxins can be analyzed. Many of the microorganisms described herein are capable of producing the plant hormone auxin indole-3-acetic acid (IAA) when grown in culture. Auxins play a key role in altering the physiology of plants, including the extent of root growth.

[0194] Thus, in one embodiment, the microorganisms of the present disclosure are present as a population disposed on the surface or within the tissue of a given plant species. The microorganisms can produce a composition, such as a metabolite, in an amount effective to cause a detectable increase in the amount of the composition found on or within the plant when compared to a reference plant not treated with the microorganisms of the present disclosure or a cell-free or inactive preparation. The composition produced by the microbial population can be beneficial to the plant species.

[0195] Such microbial produced compositions may be present in the cell culture broth or culture medium in which the microorganism is grown, or may encompass exudates produced by the microorganism. As used herein, "exudate" refers to one or more compositions secreted by or extracted from one or more microbial cells. As used herein, "broth" refers to the common composition of the cell culture medium after the microbial cells are placed in the culture medium. The composition of the broth may change over time, changing during different stages of microbial growth and / or development. Broth and / or exudates may improve the traits of the plant with which it becomes associated.

[0196] Microbial-induced traits in plants

[0197] The present disclosure utilizes microorganisms to impart beneficial properties (or beneficial traits) to desired plant species, such as agronomic species of interest. In the present disclosure, the terms "beneficial properties" or "beneficial traits" are used interchangeably and refer to the modulation of a desired plant phenotype or genetic trait of interest by the application of a microorganism or microbial consortium as described herein. As previously mentioned, in some aspects, it may be desirable that the metabolites produced by a given microorganism are ultimately responsible for modulating or imparting a beneficial trait to a given plant.

[0198] There are many beneficial traits that can be modulated by applying the microorganisms of the present disclosure. For example, the microorganisms can have the ability to impart one or more beneficial characteristics to plant species, such as: increased growth, increased yield, increased nitrogen use efficiency, increased stress tolerance, increased drought tolerance, increased photosynthetic rate, increased water use efficiency, increased pathogen resistance, modification of plant architecture (which does not necessarily affect plant yield, but addresses plant function, causing the plant to increase production of metabolites of interest), etc.

[0199] In various aspects, the microorganisms taught herein provide a wide range of agricultural applications, including: increasing grain, fruit and flower yields; increasing the growth of plant parts; increasing the ability to utilize nutrients (e.g., nitrogen, phosphates, etc.); increasing disease resistance; bioinsecticide effects (including increasing resistance to fungi, insects and / or nematodes); increasing survival in extreme climates; and improving other desired plant phenotypic characteristics. In some aspects, nematodes include root-knot nematodes. In some aspects, the insect may belong to the genus Diabrotica.

[0200] In some aspects, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure can be applied to plants to modulate or alter plant characteristics, such as altered oil content, altered protein content, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, chemical tolerance, cold tolerance, delayed senescence, disease resistance, drought tolerance, ear weight, improved growth, enhanced health, heat tolerance, herbicide tolerance, herbivore resistance, increased nitrogen fixation, improved nitrogen utilization efficiency, improved nutrient (e.g., phosphate, potassium, etc.) utilization efficiency, improved root architecture, improved water use efficiency, increased biomass, increased root length, increased seed weight, increased shoot length, increased yield, yield under water-limiting conditions, etc., relative to a reference plant. The invention also provides for the invention of the invention a method for regulating the growth of plants and crops, for example, by regulating the growth of seeds, for example, by regulating the growth of seeds, for example, by regulating the growth of crops, for example, by regulating the growth of plants, for example, by regulating the growth of crops, for example, by regulating the growth of plants, for example, by regulating the growth of crops, for example, by regulating the growth of plants, for example, by regulating the growth of crops, for example, by regulating the growth of plants, for example, by regulating the growth of crops, for example, by regulating the growth of plants

[0201] In some aspects, the isolated microorganisms, consortia and / or agricultural compositions of the present disclosure can be applied to plants to regulate specific plant characteristics in a negative manner. For example, in some aspects, the microorganisms of the present disclosure can reduce phenotypic traits of interest because such functionality may be desirable in some applications. For example, the microorganisms of the present disclosure may have the ability to reduce root growth or reduce root length. Or the microorganisms may have the ability to reduce bud growth or reduce plant growth rate because these adjustments of plant traits may be desirable in certain applications.

[0202] In some embodiments, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure may be applied to plants to confer nematode stress tolerance to the plants.

[0203] In some embodiments, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure may be applied to plants to provide biological stimulation (biostimulant effect) to the plants. In some embodiments, the isolated microorganisms, consortia, and / or agricultural compositions of the present disclosure may be applied to plants to provide disease resistance to the plants.

[0204] Agricultural composition

[0205] In some embodiments, the microorganisms of the present disclosure are combined with agricultural compositions. Agricultural compositions generally refer to such organic and inorganic compounds, which may include compositions that promote the cultivation of microorganisms and / or plant components; compositions that participate in the preparation of microorganisms to be applied to plant components (such as but not limited to: wetting agents, extenders (also known as "compatibilizers"), defoamers, detergents, chelating agents, drift reducers, neutralizers and buffers, corrosion inhibitors, dyes, odorants, extenders (also known as "spreaders"), penetration aids (also known as "penetrants"), adhesives (also known as "adhesives" or "binders"), dispersants, thickeners (also known as "thickeners"), stabilizers, emulsifiers, freezing point depressants, antimicrobial agents, etc.); compositions that participate in imparting protection to plant components or plants (such as but not limited to: insecticides, nematicides, fungicides, bactericides, herbicides, etc.); and other compositions that may be of interest for specific applications.

[0206] In some embodiments, the agricultural composition of the present disclosure is solid. When using solid compositions, it may be desirable to include one or more carrier materials and isolated active microorganisms or consortia. In some embodiments, the present disclosure teaches the use of carriers, which include but are not limited to: mineral soils such as silicon dioxide, silica gel, silicates, talc, kaolin, activated clay, limestone, chalk, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, thiourea and urea, plant-derived products such as grain flour, bark powder, wood powder and husk powder, cellulose powder, attapulgite, montmorillonite, mica, vermiculite, synthetic silica and synthetic calcium silicate, or compositions of these substances.

[0207] Growth composition

[0208] In some embodiments, compositions that promote growth and development are provided to microorganisms and / or plant components. Exemplary compositions include liquids (such as broth, culture media) and / or solids (such as soil, nutrients). Various organic or inorganic compounds may be added to the growth composition alone or in combination with plant components to benefit the health of the microorganisms, such as, but not limited to: amino acids, vitamins, minerals, carbohydrates, monosaccharides, lipids.

[0209] Preparation composition

[0210] One or more compositions other than the microorganism or the composition produced by the microorganism may be combined for various application, stability, activity and / or storage reasons. The additional composition may be referred to as a "formulation component".

[0211] In some embodiments, the agricultural composition of the present disclosure is a liquid. Thus, in some embodiments, the present disclosure teaches that the agricultural composition disclosed herein may include a compound or salt, such as a monoethanolamine salt, sodium sulfate, potassium sulfate, sodium chloride, potassium chloride, sodium acetate, ammonium hydrogen sulfate, ammonium chloride, ammonium acetate, ammonium formate, ammonium oxalate, ammonium carbonate, ammonium hydrogen carbonate, ammonium thiosulfate, ammonium dihydrogen phosphate, ammonium monohydrogen phosphate, ammonium sodium hydrogen phosphate, ammonium thiocyanate, ammonium sulfamate, or ammonium carbamate.

[0212] In some embodiments, the present disclosure teaches that agricultural compositions may include binders such as: polyvinyl pyrrolidone, polyvinyl alcohol, partially hydrolyzed polyvinyl acetate, carboxymethyl cellulose, starch, vinyl pyrrolidone / vinyl acetate copolymers and polyvinyl acetate, or combinations thereof; lubricants such as magnesium stearate, sodium stearate, talc or polyethylene glycol, or combinations thereof; defoamers such as silicone emulsions, long chain alcohols, phosphate esters, acetylene glycols, fatty acids or organofluorine compounds, and complexing agents such as: salts of ethylenediaminetetraacetic acid (EDTA), salts of trinitrilotriacetic acid or salts of polyphosphoric acid, or combinations thereof.

[0213] In some embodiments, agricultural compositions include surfactants. In some embodiments, surfactants are added to liquid agricultural compositions. In other embodiments, surfactants are added to solid formulations, especially those designed to be diluted with a carrier before application. Therefore, in some embodiments, agricultural compositions include surfactants. Surfactants are sometimes used alone or with other additives (such as minerals or vegetable oils) as adjuvants for spray tank mixtures to improve the biological performance of microorganisms to targets. The type of surfactant used for bioenhancers generally depends on the properties and mode of action of the microorganism. The characteristics of surfactants can be anionic, cationic or nonionic, and can be used as emulsifiers, wetting agents, suspending agents or for other purposes. In some embodiments, surfactants are nonionic surfactants, such as: alkyl ethoxylates, linear fatty alcohol ethoxylates and fatty amine ethoxylates. Surfactants commonly used in the formulation art and which may also be used in the formulation of the present invention are described in McCutcheon's Detergents and Emulsifiers Annual, MC Publishing Corp., Ridgewood, NJ, 1998, and Encyclopedia of Surfactants, Vols. I-III, Chemical Publishing Co., New York, 1980-81. In some embodiments, the present disclosure teaches the use of surfactants, which include alkali metal, alkaline earth metal or ammonium salts of aromatic sulfonic acids (e.g., ligninsulfonic acid, phenolsulfonic acid, naphthalenesulfonic acid and dibutylnaphthalenesulfonic acid) and fatty acids of arylsulfonates, alkyl ethers, dodecyl ethers, fatty alcohol sulfates and fatty alcohol glycol ether sulfates; condensates of sulfonated naphthalene and its derivatives with formaldehyde; condensates of naphthalene or naphthalenesulfonic acid with phenol and formaldehyde; condensates of phenol or phenolsulfonic acid with formaldehyde; condensates of phenol with formaldehyde and sodium sulfite; polyoxyethylene octylphenyl ether; ethoxylated isooctylphenol, ethoxylated octylphenol or ethoxylated nonylphenol; tributylphenyl polyglycol ether; alkylaryl polyether alcohol; isotridecyl alcohol; ethoxylated castor oil; ethoxylated triarylphenol; salts of phosphorylated triarylphenol ethoxylates; dodecanol polyglycol ether acetate; sorbitan esters; lignin-sulfite waste liquor or methylcellulose, or combinations of these substances.

[0214] In some embodiments, the present disclosure teaches other suitable surfactants, including salts of alkyl sulfates, such as diethanolammonium dodecyl sulfate; alkylaryl sulfonates, such as calcium dodecylbenzene sulfonate; alkylphenol-alkylene oxide addition products, such as nonylphenol-C18 ethoxylate; ethanol-alkylene oxide addition products, such as tridecyl alcohol-C16 ethoxylate; soaps, such as sodium stearate; alkylnaphthalene-sulfonates, such as sodium dibutylnaphthalene sulfonate; dialkyl esters of sulfosuccinates, such as di(2-ethylhexyl)sulfonate; succinate; sorbitol esters, such as sorbitol oleate; quaternary amines, such as dodecyltrimethylammonium chloride; polyethylene glycol esters of fatty acids, such as polyethylene glycol stearate; block copolymers of ethylene oxide and propylene oxide; salts of monoalkyl and dialkyl phosphates; vegetable oils, such as soybean oil, rapeseed / canola oil, olive oil, castor oil, sunflower oil, coconut oil, corn oil, cottonseed oil, linseed oil, palm oil, peanut oil, safflower oil, sesame oil, tung oil, etc.; and esters of the above vegetable oils, especially methyl esters.

[0215] In some embodiments, the agricultural composition comprises a wetting agent. A wetting agent is a substance that, when added to a liquid, increases the spreading or penetration ability of a liquid by reducing the interfacial tension between the liquid and the surface on which the liquid is spread. Wetting agents are used in agricultural chemical formulations for two main functions: to increase the wettability of powders in water during processing and manufacturing to prepare soluble liquid concentrates or suspension concentrates; and to shorten the wetting time of wettable powders and increase the penetration rate of water into water-dispersible granules during mixing of the product with water in a spray tank or other container. In some embodiments, examples of wetting agents used in agricultural compositions of the present disclosure (including wettable powders, suspension concentrates, and water-dispersible granule formulations) are: sodium lauryl sulfate; sodium dioctyl sulfosuccinate; alkylphenol ethoxylates; and fatty alcohol ethoxylates.

[0216] In some embodiments, agricultural compositions of the present disclosure include dispersants. Dispersants are substances that are adsorbed on the surface of particles and help to maintain the dispersed state of particles and prevent them from re-aggregating. In some embodiments, dispersants are added to agricultural compositions of the present disclosure to promote dispersion and suspension during manufacture, and ensure that particles are redispersed in water in a spray tank. In some embodiments, dispersants are used in wettable powders, suspension concentrates, and water-dispersible granules. The surfactant used as a dispersant has the ability to be strongly adsorbed on the surface of particles and provide a charged or spatial barrier that prevents particles from re-aggregating. In some embodiments, the most commonly used surfactants are anionic, nonionic, or a mixture of these two types.

[0217] In some embodiments, for wettable powder formulations, the most commonly used dispersant is sodium lignin sulfonate. In some embodiments, suspension concentrates use polyelectrolytes (such as sodium naphthalene sulfonate formaldehyde condensates) to provide excellent adsorption and stabilization. In some embodiments, tristyrylphenol ethoxylate phosphate is also used. In some embodiments, such as alkyl aryl ethylene oxide condensates and EO-PO block copolymers are sometimes combined with anionic surfactants as dispersants for suspension concentrates.

[0218] In some embodiments, the agricultural compositions of the present disclosure include polymeric surfactants. In some embodiments, polymeric surfactants have extremely long hydrophobic "backbones" and a large number of ethylene oxide chains, which form the "teeth" of a "comb-like" surfactant. In some embodiments, these high molecular weight polymers can provide excellent long-term stability of suspension concentrates because the hydrophobic backbone has many points of anchoring to the surface of the particles. In some embodiments, examples of dispersants used in the agricultural compositions of the present disclosure are: sodium lignin sulfonate; sodium naphthalene sulfonate formaldehyde condensate; tristyrylphenol ethoxylate phosphate; fatty alcohol ethoxylate; alkyl ethoxylate; EO-PO block copolymer; and graft copolymer.

[0219] In some embodiments, agricultural compositions of the present disclosure include emulsifiers. Emulsifiers are substances that stabilize a suspension of droplets of a liquid phase in another liquid phase. In the absence of emulsifiers, the two liquids will separate into two immiscible liquid phases. In some embodiments, the most commonly used emulsifier blends include alkylphenols or fatty alcohols with 12 or more ethylene oxide units and oil-soluble calcium salts of dodecylbenzene sulfonic acid. Hydrophile-lipophile balance ("HLB") values ​​within the range of 8 to 18 will generally provide good stable emulsions. In some embodiments, emulsion stability can sometimes be improved by adding a small amount of EO-PO block copolymer surfactants.

[0220] In some embodiments, the agricultural compositions of the present disclosure include a solubilizing agent. A solubilizing agent is a surfactant that will form micelles in water at a concentration exceeding the critical micelle concentration. The micelles are then able to dissolve or solubilize water-insoluble materials within the hydrophobic portion of the micelle. The types of surfactants commonly used for dissolution are nonionic surfactants: sorbitan monooleate; sorbitan monooleate ethoxylate; and methyl oleate.

[0221] In some embodiments, the agricultural composition of the present disclosure comprises an organic solvent. Organic solvents are mainly used in the preparation of emulsifiable concentrates, ULV preparations, and to a lesser extent in granular preparations. Sometimes a mixture of solvents is used. In some embodiments, the present disclosure teaches the use of solvents, and the solvents include aliphatic paraffin oils, such as kerosene or refined paraffin. In other embodiments, the present disclosure teaches the use of aromatic solvents, such as the higher molecular weight fractions of xylene and C9 and C10 aromatic solvents. In some embodiments, chlorinated hydrocarbons can be used as cosolvents to prevent insecticide crystallization when the preparation is emulsified in water. Alcohol is sometimes used as a cosolvent to improve dissolving power.

[0222] In some embodiments, the agricultural composition comprises a gelling agent. Thickeners or gelling agents are mainly used in the formulation of suspension concentrates, emulsions and suspoemulsions to change the rheology or fluidity of the liquid and prevent the separation and sedimentation of dispersed particles or droplets. Thickeners, gelling agents and anti-settling agents generally belong to two categories, namely water-insoluble particles and water-soluble polymers. It is possible to use clay and silica to produce suspension concentrate formulations. In some embodiments, the agricultural composition comprises one or more thickening agents, including but not limited to: montmorillonite, such as bentonite; magnesium aluminum silicate; and attapulgite. In some embodiments, the present disclosure teaches the use of polysaccharides as thickening agents. The most commonly used types of polysaccharides are natural extracts of seeds and seaweed or synthetic derivatives of cellulose. Some embodiments utilize xanthan gum and some embodiments utilize cellulose. In some embodiments, the disclosure teaches the use of thickeners, including but not limited to: guar gum; locust bean gum; carrageenan; alginates; methylcellulose; sodium carboxymethylcellulose (SCMC); hydroxyethylcellulose (HEC). In some embodiments, the disclosure teaches the use of other types of anti-settling agents, such as modified starch, polyacrylates, polyvinyl alcohol, and polyethylene oxide. Another good anti-settling agent is xanthan gum.

[0223] In some embodiments, the presence of surfactants (which reduce interfacial tension) can cause water-based formulations to foam during the mixing operation in the preparation and application by spray cans. Therefore, in some embodiments, in order to reduce the foaming tendency, defoamers are usually added during the preparation stage or before filling into the bottle / spray can. Generally speaking, there are two types of defoamers, i.e. silicones and non-silicone defoamers. Silicones are usually aqueous emulsions of dimethylpolysiloxane, while non-silicone defoamers are water-insoluble oils, such as octanol and nonanol, or silicon dioxide. In both cases, the function of the defoamer is to transfer the surfactant from the air-water interface.

[0224] In some embodiments, the agricultural composition comprises a preservative.

[0225] In some embodiments, the agricultural composition can be formulated as a soil drench, foliar spray, drench treatment, in-furrow treatment, soil conditioner, granules, broadcast treatment, post-harvest disease control treatment, or seed treatment. In some embodiments, the agricultural composition can be applied alone or in combination with other agricultural products in a rotary spray procedure.

[0226] In some embodiments, the agricultural composition is compatible with tank mixing. In some embodiments, the agricultural composition is compatible with tank mixing with other agricultural products. In some embodiments, the agricultural composition is compatible with equipment for ground, aerial, and irrigation applications.

[0227] In some embodiments, the agricultural composition may be applied to genetically modified seeds or plants.

[0228] Protective composition

[0229] In addition, the individual microorganisms or microorganism consortia or microbial communities developed according to the disclosed method can be combined with known active agents available in the agricultural field, such as: pesticides, herbicides, bactericides, fungicides, insecticides, virucides, acaricides, nematicides, scabicides, plant growth regulators, rodenticides, anti-algae agents, biological control or beneficial agents. In addition, the microorganisms, microorganism consortia or microbial communities developed according to the disclosed method can be combined with known fertilizers. Such combinations can show synergistic properties. In addition, the individual microorganisms or microorganism consortia or microbial communities developed according to the disclosed method can be combined with inert ingredients. In addition, in some aspects, the disclosed microorganisms are combined with bioactive agents.

[0230] In some embodiments, a single microorganism or microbial consortium or microbial community developed according to the disclosed methods can be combined with a biopesticide that acts as a herbicide, bactericide, fungicide, insecticide, viricide, acaricide, nematicide, scabicide, rodenticide, and / or anti-algae agent. Such biopesticides can be, but are not limited to, macrobiological organisms (e.g., beneficial nematodes, etc.), microbial organisms (e.g., Serenade, Bt, etc.), plant extracts (e.g., Timorex Gold, etc.), biochemicals (e.g., insect pheromones, etc.), and / or minerals and oils (e.g., canola oil).

[0231] Pesticides and biopesticides

[0232] In some embodiments, the agricultural compositions of the present disclosure comprise a pesticide used in combination with the taught microorganisms. In some embodiments, the agricultural compositions of the present disclosure comprise a biopesticide used in combination with the taught microorganisms.

[0233] In some embodiments, individual microorganisms or microbial consortia or microbial communities developed according to the disclosed methods can be combined with known pesticides in the agricultural field, such as: pesticides that act as herbicides, bactericides, fungicides, insecticides, virucides, miticides, nematicides, scabicides, rodenticides and / or anti-algae agents.

[0234] In some embodiments, individual microorganisms or microbial consortia or microbial communities developed according to the disclosed methods can be combined with known biopesticides in the agricultural field, such as: biopesticides that act as herbicides, bactericides, fungicides, insecticides, viricides, miticides, nematicides, scabicides, rodenticides and / or anti-algae agents.

[0235] For example, in some embodiments, the present disclosure teaches agricultural compositions comprising one or more of the following active ingredients, including: macrobiological organisms (e.g., beneficial nematodes, etc.), microbial organisms (e.g., Serenade, Bt, etc.), plant extracts (e.g., Timorex Gold, etc.), biochemicals (e.g., insect pheromones, etc.), and / or minerals and oils (e.g., canola oil).

[0236] In some embodiments, individual microorganisms or microbial consortia or microbial communities developed according to the disclosed methods can be combined with a herbicide selected from the group consisting of: acetamides selected from the group consisting of: acetochlor, alachlor, butachlor, dimethachlor, dimethenamid, flufenacet, benzathine, isopropylamine, pyraclostrobin, naproxen, pethoxydim, pretilachlor, cypermethrin and dimethenamid; amino acid derivatives selected from the group consisting of: bialaphos, glufosinate and glufosinate; aryloxyphenoxypropionates selected from the group consisting of: clodinafop-butyl, cyhalofop-butyl, oxazolidinone, fluazifop-butyl, fluazifop-butyl, oxadiazol-butyl, quinazolin-butyl, quinazolin-butyl and quinazolin-butyl; quinazolin-butyl, quinazolin-butyl, quinazolin-butyl, quinazolin-butyl and quinazolin-butyl; quat and paraquat; (thio) carbamates selected from the group consisting of: cypermethrin, butadiene, carbamate, betamethrin, piperocarb, EPTC, cypermethrin, cypermethrin, cypermethrin, betamethrin, betamethrin, cypermethrin, cypermethrin and wild wheat cypermethrin; cyclohexanedione selected from the group consisting of: butadiene, clethodim, cypermethrin, cypermethrin, sethoxydim, pyraclostrobin and oxamethoxam; dinitroanilines selected from the group consisting of: fluazifop, ethylbutene, oryzalin, pendimethalin, aminofluralin and trifluralin; diphenyl ethers selected from the group consisting of: acifluorfen, benfibrate, cypermethrin, difluanid, chlorpyrifos, fomesulfuron, lactofen and oxyfluorfen ; Hydroxybenzonitriles selected from the group consisting of bromoxynil, dichlobenil and ioxynil; Imidazolinones selected from the group consisting of imazapic, imazapic, methyl imazapic, imazapic, imazapic and imazapic; Phenoxyacetic acids selected from the group consisting of chloranil, 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4-DB, dichlorprop, MCPA, MCPA-thioethyl, MCPB and 2-methyl-4-chloropropionic acid; Pyrazines selected from the group consisting of herbicide, flupyridazine, fluazifop-butyl, fluazifop-butyl, fluazifop-butyl and fluazifop-butyl; Pyridines selected from the group consisting of clomiphene carboxylic acid, clopyralid, difluanid, fluthiopyr, flupyralid, fluazifop-butyl, fluazifop-butyl, fluazifop-butyl, fluazifop-butyl , fluazifop and thiabendazole; a sulfonylurea selected from the group consisting of acylsulfuron-methyl, tetrazosulfuron-methyl, benzylsulfuron-methyl, chlorimuron-methyl, chlorsulfuron-methyl, ethoxysulfuron-methyl, flazasulfuron-methyl, flutosulfuron-methyl, flutosulfuron-methyl, foramsulfuron-methyl, chlorpyrisulfuron-methyl, pyrazosulfuron-methyl, iodosulfuron-methyl, mesosulfuron-methyl, metsulfuron-methyl, nicosulfuron, epoxysulfuron-methyl, primisulfuron-methyl, prosulfuron, pyrazosulfuron-methyl, sulfonesulfuron-methyl, sulfosulfuron-methyl, thifensulfuron-methyl, etherbensulfuron-methyl, bensulfuron-methyl, trifloxysulfuron, trifloxysulfuron, trifloxysulfuron and 14 (2-chloro-6-propyl-imidazo[1,2-b]pyridazin-3-yl)sulfonyl)-3-(4,6-dimethoxy-pyrimidin-2-yl)urea;A triazine selected from the group consisting of: ametryn, atrazine, cypermethrin, isopentanol, ethidium, hexazinone, metamitron, metribuzin, promethazine, simazine, terbuthion, benzamid and triazinon; a urea compound selected from the group consisting of: chlorotoluron, cypermethrin, diuron, flumethuron, isoproturon, linuron, methylbenzenesulfuron and tetrathion; an acetolactate synthase inhibitor selected from the group consisting of: sodium salt of bispyribac, chlorpyrifos, bispyribac, bispyribac, fluazifop, sulfamethoxam, sulfamethoxam, sulfamethoxam, penoxsulam, propoxysulfone, propylsulfuron ... oxazolidinone, pyrimidine oxime, pyrimidine oxime, pyrimidine oxime, pyriproxyfen, pyrimidine oxime, sulfamethoxazole and sulfamethoxazole; and a compound selected from the group consisting of: amitriptyline, aminotriazole, thiamethoxam, fluazifop, chloranil, bencarbazone, benfluresate, pyraclostrobin, bentazon, bicyclosulfuron, herbicide, bromobutyric acid, fluazifop-butyl, chloranil, fenpyroxil ... monoceras), grass algae, ethoxyfuroxan, ethoxybencloram, tetrazoline, flufenac, fluazifop, flufenac, fluchloridone, furochloridone, indole, isoxaflutamide, isoxaflutamide, cypermethrin, propanil, propargyl, quinclorac, clomiphenecarb, mesotrione, methylarsonic acid, oxadiazine, oxadiazine, oxadiazine, cyclopentadiazine, pinoxaden, bispyribac, pyraclostrobin, pyrasuloftole, benzaclostrobin, pyrazol, pyrazol, quinoline, sulfaquinoxaline, terbacil, furansulfuron, cypermethrin, thiamethoxam, 4-hydroxy-3-[2-(2-methoxy-ethoxymethyl)-6-trifluoromethyl-pyridine-3-carbonyl]-dimethoate Cyclo[3.2.1]oct-3-en-2-one, (3-[2-chloro-4-fluoro-5-(3-methyl-2,6-dioxo-4-trifluoromethyl-3,6-dihydro-2H-pyrimidin-1-yl)-phenoxy]-pyridin-2-yloxy)-acetic acid ethyl ester, 6-amino-5-chloro-2-cyclopropyl-pyrimidine-4-carboxylic acid methyl ester, 6-chloro-3-(2-cyclopropyl-6-methyl-phenoxy)-pyridazin-4-ol, 4-amino-3-chloro-6-(4-chloro-phenyl)-5-fluoro-pyridine-2-carboxylic acid, 4-amino-3-chloro-6-(4-chloro-2-fluoro-3-methoxy-phenyl)-pyridine-2-carboxylic acid methyl ester and 4-amino-3-chloro-6-(4-chloro-3-dimethylamino-2-fluoro-phenyl)-pyridine-2-carboxylic acid methyl ester. ;

[0237] In some embodiments, individual microorganisms or microbial consortia or microbial communities developed according to the disclosed methods can be combined with an insecticide selected from the group consisting of: an organo(thio)phosphate selected from the group consisting of acephate, methyl pyraclostrobin, azinphos-methyl, chlorpyrifos, methyl chlorpyrifos, chlorfenvinphos, diazinon, dichlorvos, dicrotophos, dimethoate, ethion, ethion, fenitrothion, fenthion, isoxazophos, malathion, methamidophos, methidathion, methyl parathion, mefenphos, monocrotophos, sulfone, paraoxon, parathion, fenthion, phosphamidon ... Phosphorus, triazophos and trichlorfon; carbamates selected from the group consisting of: aldicarb, bensulfuron, carbaryl, carbofuran, carbofuran, fenoxycarb, furamicarb, methiocarb, methomyl, oxamyl, pirimicarb, propoxur, thiodicarb and trichlorfon; pyrethroids selected from the group consisting of: allethrin, bifenthrin, cypermethrin, flucythrin, cypermethrin, alpha-cypermethrin, beta-cypermethrin, zeta-cypermethrin, deltamethrin, cyper ... Tefluthrin, tetramethrin, tralomethrin, transfluthrin, profluthrin and transfluthrin; an insect growth regulator selected from the group consisting of: a) a chitin synthesis inhibitor, wherein the chitin synthesis inhibitor is a benzoyl urea selected from the group consisting of: chlorpyrifos, cyramazin, diflubenzuron, flufenoxuron, flufenoxuron, fluflumuron, lufenuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron; buprofezin, benzathine, hexathiazox, etoxazole and clofentezin; b) an ecdysone antagonist selected from the group consisting of: chlorfenapyr, methoxyfenopyr, fenbufenozide and azadirachtin; c) a juvenile hormone analog selected from the group consisting of: pyriproxyfen, methoprene and phenoxy or d) a lipid biosynthesis inhibitor selected from the group consisting of spirodiclofen, spiromesifen and spirotetramat; a nicotinic receptor agonist / antagonist compound selected from the group consisting of clothianidin, dinotefuran, imidacloprid, thiamethoxam, nitenpyram, acetamiprid, thiamethoxam and 1-(2-chloro-thiazol-5-ylmethyl)-2-nitrylimino-3,5-dimethyl-[1,3,5]triazine; a GABA antagonist compound selected from the group consisting of endosulfan, ethiprole, fipronil, flupyrazone, pyrazine fipronil, pyraclostrobin and 5-amino-1-(2,6-dichloro-4-methyl-phenyl)-4-sulfonylaminoacyl-1H-pyrazole-3-thiocarboxamide;A macrolide insecticide selected from the group consisting of avermectin, emamectin, mibemycin, lepidomectin, spinosad and ethyl spinosad; a mitochondrial electron transport inhibitor (METI) I scabicide selected from the group consisting of fenazaquin, pyraclostrobin, tebufenpyrad, tolfenpyrad and pyrimidine; a METI selected from the group consisting of II and III compounds: acequinoxaline, fluacyprim and hydrazone; chlorfenapyr; oxidative phosphorylation inhibitors selected from the group consisting of tricyclam, diafenthiuron, fenbutatin and propargite; cryomazine; piperonyl butoxide; sodium channel blockers selected from the group consisting of indoxacarb and metaflumizone; and compounds selected from the group consisting of benclothiaz, bifenazate, batan, flonicamid, pyridalyl, pymetrozine, sulfur, flubendiamide, chlorfenapyr, cyantraniliprole (HGW86), cypermethrin, flufenapyr, fluazifop, flufenapyr, sulfamethoxam, imicyafos, bistrifluan and fluquintocet. ;

[0238] In some embodiments, the present invention teaches the synergistic use of the microorganisms or microbial consortia disclosed herein with known pesticides in the agricultural field, such as: pesticides that act as herbicides, bactericides, fungicides, insecticides, viricides, miticides, nematicides, scabicides, rodenticides and / or anti-algae agents.

[0239] In some embodiments, the present invention teaches the synergistic use of the microorganisms or microbial consortia disclosed herein with known biopesticides in the agricultural field, such as: biopesticides that act as herbicides, bactericides, fungicides, insecticides, viricides, miticides, nematicides, scabicides, rodenticides and / or anti-algae agents.

[0240] In some embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a pesticide, an additive effect on a plant phenotypic trait of interest is observed. In other embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a pesticide, a synergistic effect on a plant phenotypic trait of interest is observed.

[0241] In some embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a biopesticide, an additive effect on a plant phenotypic trait of interest is observed. In other embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a biopesticide, a synergistic effect on a plant phenotypic trait of interest is observed.

[0242] The synergistic effect obtained by the methods taught can be quantified according to the Colby formula (i.e., (E) = X + Y - (X * Y / 100)). See Colby, RS, "Calculating Synergistic and Antagonistic Responses of Herbicide Combinations," 1967 Weeds, Vol. 15, pp. 20-22, which is incorporated herein by reference in its entirety. Thus, the term "synergistic" is intended to be an amount that, when present, increases the desired effect beyond the additive amount.

[0243] The isolated microorganisms and consortia of the present disclosure can enhance the efficacy of agriculturally active pesticide compounds as well as agriculturally adjunct pesticide compounds in a synergistic manner.

[0244] The isolated microorganisms and consortia of the present disclosure can enhance the efficacy of agriculturally active biopesticide compounds as well as agriculturally auxiliary biopesticide compounds in a synergistic manner.

[0245] Plant growth regulators and biostimulants

[0246] In some embodiments, the agricultural compositions of the present disclosure include plant growth regulators and / or biostimulants used in combination with the taught microorganisms.

[0247] In some embodiments, individual microorganisms or microbial consortia or microbial communities developed according to the disclosed methods can be combined with known plant growth regulators in the agricultural field, such as: auxins, gibberellins, cytokinins, ethylene generators, growth inhibitors, and growth retardants.

[0248] For example, in some embodiments, the present disclosure teaches agricultural compositions comprising one or more of the following active ingredients, including: cyprodinil, butralin, alcohol, chlormequat, cytokinin, butyric acid, ethepohon, furamidol, gibberellic acid, gibberellic acid mixture, indole-3-butyric acid (IBA), maleic hydrazide, mefludide, mepiquat, mepiquat pentaborate, naphthaleneacetic acid (NAA), 1-naphthaleneacetamide (NAD), n-decanol, placlobutrazol, prohexadione-calcium, trinexapac-ethyl, uniconazole, salicylic acid, abscisic acid, ethylene, brassinolide, jasmonate, polyamines, nitric oxide, strigolactones or karrikins, etc.

[0249] In some embodiments, individual microorganisms or consortia or communities of microorganisms developed according to the disclosed methods can be combined with seed inoculants known in the agricultural arts, such as: In some embodiments, a Bradyrhizobium inoculum is used in combination with any single microorganism or microbial consortium disclosed herein. In certain aspects, when one of the aforementioned inoculants (e.g., or Bradyrhizobium) with a microorganism or a consortium of microorganisms as taught herein, a synergistic effect was observed.

[0250] In some embodiments, an agricultural composition of the present disclosure comprises a plant growth regulator comprising: kinetin, gibberellic acid, and indolebutyric acid and copper, manganese, and zinc.

[0251] In some embodiments, the present disclosure teaches agricultural compositions comprising one or more commercially available plant growth regulators, including but not limited to: Royaltac Early Boll Cotton Super Early Green PGR Sucker DipN Turf Armor and

[0252] In some embodiments, the present invention teaches the synergistic use of the disclosed microorganisms or microbial consortia with plant growth regulators and / or stimulants, such as plant hormones or chemicals that affect the production or destruction of plant growth regulators.

[0253] In some embodiments, the present invention teaches plant hormones which may include: auxins (e.g., indoleacetic acid, IAA), gibberellins, cytokinins (e.g., kinetin), abscisic acid, ethylene (and its production, as regulated by ACC synthetase and destroyed by ACC deaminase).

[0254] In some embodiments, a single microorganism or a consortium of microorganisms or a community of microorganisms developed according to the disclosed methods can be combined with a biostimulant. Such biostimulants can be, but are not limited to, microbial organisms, plant extracts, algae, acids, biochar, and the like.

[0255] In some embodiments, individual microorganisms or microbial consortia or microbial communities developed according to the disclosed methods can be combined with fertilizers, which can be organic (e.g., manure, blood meal, fish meal, etc.), nitrogen-based (e.g., nitrates, ammonium, urea, etc.), phosphate and potash fertilizers. Such fertilizers may also contain micronutrients, including but not limited to sulfur, iron, zinc, etc.

[0256] In some embodiments, the present invention teaches additional plant growth promoting chemicals that can act synergistically with the microorganisms and microbial consortia disclosed herein, such as: humic acid, fulvic acid, amino acids, polyphenols, and protein hydrolysates.

[0257] Thus, in some embodiments, the present disclosure provides for combining the taught microorganisms with In addition, the present disclosure provides for applying the taught microorganisms with The combination can be applied to any crop and using any method or application rate.

[0258] In some embodiments, the present disclosure teaches agricultural compositions having biostimulants.

[0259] As used herein, the term "biostimulant" refers to any substance used to stimulate the growth of microorganisms that may be present in soil or other plant growth media.

[0260] The level of microorganisms in the soil or growth medium is directly related to plant health. Microorganisms feed on biodegradable carbon sources, and therefore plant health is also related to the amount of organic matter in the soil. While fertilizers provide nutrients to feed and grow plants, in some embodiments, biostimulants provide biodegradable carbon (e.g., molasses, carbohydrates (e.g., sugars)) to feed and grow microorganisms. Unless otherwise expressly stated, a biostimulant may comprise a single ingredient, or a combination of several different ingredients that are capable of enhancing microbial activity or plant growth and development due to the effects of one or more of the ingredients, acting alone or in combination.

[0261] In some embodiments, biostimulants are compounds that produce non-nutritional plant growth responses. In some embodiments, many important benefits of biostimulants are based on their ability to influence hormonal activity. Hormones in plants (phytohormones) are chemical messengers that regulate normal plant development and responses to the environment. Root and shoot growth, as well as other growth responses, are regulated by phytohormones. In some embodiments, compounds in biostimulants can change the hormonal status of plants and have a significant impact on their growth and health. Therefore, in some embodiments, the present disclosure teaches kelp, humic acid, fulvic acid, and vitamin B as common components of biostimulants. In some embodiments, the biostimulants of the present disclosure enhance antioxidant activity, thereby enhancing the defense system of the plant. In some embodiments, vitamin C, vitamin E, and amino acids (such as glycine) are antioxidants contained in biostimulants.

[0262] In other embodiments, biostimulants can be used to stimulate the growth of microorganisms present in soil or other plant growth media. Previous studies have shown that when certain biostimulants comprising specific organic seed extracts (e.g., soybeans) are used in combination with a microbial inoculum, the biostimulants are able to stimulate the growth of microorganisms contained in the microbial inoculum. Thus, in some embodiments, the present disclosure teaches one or more biostimulants that, when used with a microbial inoculum, are able to enhance the population of native microorganisms and inoculum microorganisms. For a review of some popular uses of biostimulants, see Calvo et al., 2014, Plant Soil 383:3-41.

[0263] Combinations of plant parts, microorganisms and agricultural compositions

[0264] In some embodiments, the present disclosure teaches that a single microorganism or a microbial consortium or a microbial community or any combination of the foregoing (e.g., comprising any one or more microorganisms, including strain 101166 or 101136 or any strain comprising a polynucleotide sharing at least 95% identity with any of SEQ ID NOs. 1-14) can be applied to plant parts (optionally in combination with any agricultural composition) to improve plant phenotype.

[0265] Isolated microorganisms or communities or consortia (often interchangeably referred to as "microbes" or "microbes") can be applied to heterologous plant components to form a synthetic combination. A microorganism is considered heterologous to a plant component if it is not normally associated with the plant component in nature or, when present, is applied in an amount different from that found in nature. In some embodiments, a microorganism may be present in nature in one part of a plant and not in another part, and introduction of the microorganism into the other part of the plant is considered a heterologous association.

[0266] It is further contemplated that the microorganism, isolated or combined with a plant or plant component, can be further associated with one or more agricultural compositions, such as those described above.

[0267] Synthetic combinations of microorganisms and plant components, microorganisms and agricultural compositions, and microorganisms and plant components and agricultural compositions (often referred to as "synthetic compositions," ie, compositions comprising components not normally found associated in nature) are contemplated.

[0268] Plant component processing

[0269] In some embodiments, the present disclosure also relates to the discovery that treating plant parts with a combination of one or more of the disclosed microorganisms or agricultural compositions prior to sowing or planting can enhance desirable plant traits, such as plant growth, plant health, and / or plant insect resistance.

[0270] Thus, in some embodiments, the present disclosure teaches the use of one or more of a microorganism or microbial consortium as a plant component treatment agent. A plant component treatment agent can be a plant component coating applied directly to an untreated and "naked" plant component. However, a plant component treatment agent can be a plant component outer coating applied to a plant component that has been coated with one or more previous plant component coatings or plant component treatment agents. Previous plant component treatment agents can contain one or more active compounds (chemical or biological) and one or more inert ingredients.

[0271] The term "plant component treatment" generally refers to the application of materials to plant components prior to or during planting in the soil. Plant component treatments with the microorganisms and other agricultural compositions of the present disclosure have the advantage of delivering the treatment to areas where plant components are planted shortly before plant component germination and plant component emergence.

[0272] In other embodiments, the present disclosure also teaches that the use of plant component treatments can minimize the amount of microorganisms or agricultural compositions required to successfully treat plants and further limit worker exposure to microorganisms and compositions compared to application techniques such as spraying above the soil or over emerging plant components.

[0273] In addition, in some embodiments, the present disclosure teaches that the microorganisms disclosed herein are important for enhancing the early stages of plant life (e.g., within the first thirty days after emergence of plant components). Thus, in some embodiments, delivering the microorganisms and / or compositions of the present disclosure in the form of plant component treatments can place them in the area of ​​action at a time that is important for microbial activity.

[0274] In some embodiments, the microbial composition of the present disclosure is formulated as a plant component treatment agent. In some embodiments, it is envisioned that by using a treatment agent application equipment that is specifically designed and manufactured to accurately, safely and effectively apply plant component treatment products to plant components, one or more layers of microorganisms and / or agricultural compositions disclosed herein can be substantially uniformly applied to plant components using conventional mixing, spraying methods or their combinations. This type of equipment uses various types of coating techniques, such as a rotary coater, a drum coater, a fluidized bed technology, a fountain bed, a rotary spray or their combinations. Liquid plant component treatment agents (such as liquid plant component treatment agents of the present disclosure) can be applied by a spin "atomizer" disk or a spray nozzle, which evenly distributes the plant component treatment agent on the plant component when moving in a spray mode. In various aspects, the plant component is then mixed or tumbled for a period of time to realize additional treatment agent distribution and drying.

[0275] Prior to coating with the microbial composition, the plant components may be primed or unprimed to improve uniformity of germination and emergence. In an alternative embodiment, the dry powder formulation may be provided on the moving plant components by metering and allowed to mix until fully distributed.

[0276] In some embodiments, at least a portion of the surface area of ​​the plant component is coated with a microbial composition according to the present disclosure. In some embodiments, the plant component coating comprising the microbial composition is applied directly to the naked plant component. In some embodiments, the plant component outer coating comprising the microbial composition is applied to the plant component on which the plant component coating has been applied. In some aspects, the plant component may have a plant component coating comprising, for example, clothianidin and / or Bacillus firmus-I-1582, on top of which the present composition will be applied in the form of a plant component outer coating. In some aspects, the microbial composition taught is applied in the form of a plant component outer coating to a plant component that has been treated with PONCHO TM VOTiVO TM In some aspects, the plant component may have a plant component coating, which includes, for example, metalaxyl and / or clothianidin and / or Bacillus firmus-I-1582, on top of which the composition of the present invention is applied in the form of a plant component coating. In some aspects, the microbial composition taught is applied in the form of a plant component coating to a plant component that has been treated with ACCELERON TM Processed plant components.

[0277] In some embodiments, the plant components treated with microorganisms have a microbial spore concentration or a microbial cell concentration of about 10^2 to 10^12, 10^2 to 10^11, 10^2 to 10^10, 10^2 to 10^9, 10^2 to 10^8, 10^2 to 10^7, 10^2 to 10^6, 10^2 to 10^5, 10^2 to 10^4, or 10^2 to 10^3 per plant component.

[0278] In some embodiments, the plant components treated with microorganisms have a microbial spore concentration or a microbial cell concentration of about 10^3 to 10^12, 10^3 to 10^11, 10^3 to 10^10, 10^3 to 10^9, 10^3 to 10^8, 10^3 to 10^7, 10^3 to 10^6, 10^3 to 10^5, or 10^3 to 10^4 per plant component.

[0279] In some embodiments, the plant components treated with microorganisms have a microbial spore concentration or a microbial cell concentration of about 10^4 to 10^12, 10^4 to 10^11, 10^4 to 10^10, 10^4 to 10^9, 10^4 to 10^8, 10^4 to 10^7, 10^4 to 10^6, or 10^4 to 10^5 per plant component.

[0280] In some embodiments, the plant components treated with microorganisms have a microbial spore concentration or a microbial cell concentration of about 10^5 to 10^12, 10^5 to 10^11, 10^5 to 10^10, 10^5 to 10^9, 10^5 to 10^8, 10^5 to 10^7, or 10^5 to 10^6 per plant component.

[0281] In some embodiments, the plant components treated with microorganisms have a microbial spore concentration or a microbial cell concentration of about 10^5 to 10^9 per plant component.

[0282] In some embodiments, the plant components treated with microorganisms have a microbial spore concentration or microbial cell concentration of at least about 1×10^3 or 1×10^4 or 1×10^5 or 1×10^6 or 1×10^7 or 1×10^8 or 1×10^9 per plant component.

[0283] In some embodiments, the amount of one or more of the microorganisms and / or agricultural compositions applied to plant components depends on the final formulation, and the size or type of the plant or plant components utilized. In some embodiments, one or more of the microorganisms are present with about 2% w / w to about 80% w / w of the total formulation. In some embodiments, by weight, one or more of the microorganisms employed in the composition are about 5% w / w to about 65% w / w of the total formulation, or 10% w / w to about 60% w / w.

[0284] In some embodiments, the plant components may also have more spores or microbial cells per plant component, for example, about 10^2, 10^3, 10^4, 10^5, 10^6, 10^7, 10^8, 10^9, 10^10, 10^11, 10^12, 10^13, 10^14, 10^15, 10^16, or 10^17 spores or cells per plant component.

[0285] In some embodiments, the thickness of the coating of the plant component of the present disclosure can be at most 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280 μm, 290 μm, 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360 μm, 370 μm, 380 μm, 390 μm, 400 μm, 410 μm, 420 μm, 430 μm, 440 μm, 450 μm, 460 μm, 470 μm, 480 μm, 490 μm, 500 μm, 510 μm, 520 μm, 530 μm, 540 μm, 550 μm, 560 μm, 570 μm, 580 μm, 590 μm, 600 μm, 610 μm, 620 μm, 630 μm, 640 μm, 650 μm, 660 μm, 670 μm, 680 μm, 690 μm, 700 μm, 710 μm, 720 μm, 730 μm, 740 μm, 750 μm, 760 μm, 770 μm, 780 μm, 790 μm, 800 μm, 810 μm, 820 μm, 830 μm, 840 μm, 850 μm, 860 μm, 870 μm, 880 μm, 890 μm, 900 μm, 910 μm, 920 μm, 930 μm, 940 μm, 950 μm, 960 μm, 970 μm, 980 μm, 990 μm, 1000 μm, 1010 μm, 1020 μm, 1030 μm, 1040 μm, 1050 μm, 1060 μm, 1070 μm, 1080 μm, 1090 μm, 1100 μm, 1110 μm, 1120 μm, 1130 μm, 1140 μm, 1150 μm, 1160 μm, 1170 μm, 1180 μm, 1190 μm, 1200 μm, 1210 μm, 1220 μm, 1230 μm, 1240 μm, 1250 μm, 1260 μm, 1270 μm, 1280 μm, 1290 μm, 1300 μm, 1310 μm, 1320 μm, 1330 μm, 1340 μm, 1350 μm, 1360 μm, 1370 μm, 1380 μm, 1390 μm, 1400 μm, 1410 μm, 1420 μm, 1430 μm, 1440 μm, 1450 μm, 1460 μm, 1470 μm, 1480 μm, 1490 μm, 1500 μm, 1510 μm, 1520 μm, 1530 μm, 1540 μm,1550μm, 1560μm, 1570μm, 1580μm, 1590μm, 1600μm, 1610μm, 1620μm, 1630μm, 1640μm, 1650μm, 1660μm, 1670μm, 1680μm, 1690μm, 1700μm, 1710μm, 1720μm ,1730μm,1740μm,1750μm,1760μm,1770μm,1780μm,1790μm,1800μm,1810μm,1820μm,1830μm,1840μm,1850μm,1860μm,1870μm,1880μm,1890μm,1900μm m, 1910μm, 1920μm, 1930μm, 1940μm, 1950μm, 1960μm, 1970μm, 1980μm, 1990μm, 2000μm, 2010μm, 2020μm, 2030μm, 2040μm, 2050μm, 2060μm, 2070μm, 2080 μm, 2090μm, 2100μm, 2110μm, 2120μm, 2130μm, 2140μm, 2150μm, 2160μm, 2170μm, 2180μm, 2190μm, 2200μm, 2210μm, 2220μm, 2230μm, 2240μm, 2250μm, 226 0μm, 2270μm, 2280μm, 2290μm, 2300μm, 2310μm, 2320μm, 2330μm, 2340μm, 2350μm, 2360μm, 2370μm, 2380μm, 2390μm, 2400μm, 2410μm, 2420μm, 2430μm, 24 40μm, 2450μm, 2460μm, 2470μm, 2480μm, 2490μm, 2500μm, 2510μm, 2520μm, 2530μm, 2540μm, 2550μm, 2560μm, 2570μm, 2580μm, 2590μm, 2600μm, 2610μm, 2 620μm, 2630μm, 2640μm, 2650μm, 2660μm, 2670μm, 2680μm, 2690μm, 2700μm, 2710μm, 2720μm, 2730μm, 2740μm, 2750μm, 2760μm, 2770μm, 2780μm, 2790μm, 2800μm, 2810μm, 2820μm, 2830μm, 2840μm, 2850μm, 2860μm, 2870μm, 2880μm, 2890μm, 2900μm, 2910μm, 2920μm, 2930μm, 2940μm, 2950μm, 2960μm, 2970μm,2980μm, 2990μm or 3000μm.

[0286] In some embodiments, the thickness of the plant component coating of the present disclosure can be 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm.

[0287] In some embodiments, the coating of the plant components of the present disclosure can be at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24% by weight of the uncoated plant component. , 24.5%, 25%, 25.5%, 26%, 26.5%, 27%, 27.5%, 28%, 28.5%, 29%, 29.5%, 30%, 30.5%, 31%, 31.5%, 32%, 32.5%, 33%, 33.5%, 34%, 34.5%, 35%, 35.5%, 36%, 36.5%, 37% , 37.5%, 38%, 38.5%, 39%, 39.5%, 40%, 40.5%, 41%, 41.5%, 42%, 42.5%, 43%, 43.5%, 44%, 44.5%, 45%, 45.5%, 46%, 46.5%, 47%, 47.5%, 48%, 48.5%, 49%, 49.5% or 50%.

[0288] In some embodiments, microbial spores and / or cells may be freely applied to the plant component or they may be formulated in a liquid or solid composition prior to being applied to the plant component. For example, a solid composition comprising microorganisms may be prepared by mixing a solid carrier with a suspension of spores until the solid carrier is impregnated with the spore or cell suspension. The mixture may then be dried to obtain the desired particles.

[0289] In some other embodiments, it is contemplated that the solid or liquid microbial compositions of the present disclosure further contain functional agents, such as activated carbon, nutrients (fertilizers), and other agents capable of improving germination and quality of the product or a combination thereof.

[0290] Plant component coating methods and compositions known in the art may be particularly useful when they are modified by adding one of the embodiments of the present disclosure. Such coating methods and their application equipment are disclosed, for example, in U.S. Pat. Nos. 5,916,029, 5,918,413, 5,554,445, 5,389,399, 4,759,945, 4,465,017, and U.S. Patent Application No. 13 / 260,310, each of which is incorporated herein by reference.

[0291] Plant part coating compositions are disclosed, for example, in U.S. Pat. Nos. 5,939,356, 5,876,739, 5,849,320, 5,791,084, 5,661,103, 5,580,544, 5,328,942, 4,735,015, 4,634,587, 4,372,080, 4,339,456, and 4,245,432, each of which is incorporated herein by reference.

[0292] In some embodiments, a variety of additives can be added to the plant component treatment formulation comprising the composition of the present invention. A binder can be added, and the binder includes a binder consisting of a natural or synthetic adhesive polymer that has no phytotoxic effect on the applied plant component. The binder can be selected from polyvinyl acetate; polyvinyl acetate copolymer; ethylene-vinyl acetate (EVA) copolymer; polyvinyl alcohol; polyvinyl alcohol copolymer; cellulose, including ethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose and carboxymethyl cellulose; polyvinyl pyrrolidone; polysaccharides, including starch, modified starch, dextrin, maltodextrin, alginate and deacetylated chitosan; fat; oil; protein, including gelatin and zein; gum arabic; shellac; vinylidene chloride and vinylidene chloride copolymer; calcium lignin sulfonate; acrylic acid copolymer; polyvinyl acrylate; polyethylene oxide; acrylamide polymers and copolymers; polyhydroxyethyl acrylate, methacrylamide monomer; and polychloroprene.

[0293] Any of a variety of colorants may be employed, including organic chromophores, which are classified as nitroso; nitro; azo, including monoazo, disazo and polyazo; acridine, anthraquinone, azine, diphenylmethane, indamine, indophenol, methine, oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, xanthene. Other additives that may be added include trace nutrients, such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc.

[0294] Polymers or other dust control agents may be applied to retain the treatment on the surfaces of plant components.

[0295] In some embodiments, in addition to microbial cells or spores, the coating may also include an adhesive layer. The adhesive should be non-toxic, biodegradable and adhesive. Examples of such materials include, but are not limited to, polyvinyl acetate; polyvinyl acetate copolymers; polyvinyl alcohol; polyvinyl alcohol copolymers; cellulose, such as methylcellulose, hydroxymethylcellulose and hydroxymethylpropylcellulose; dextrin; alginate; sugar; molasses; polyvinyl pyrrolidone; polysaccharides; proteins; fats; oils; gum arabic; gelatin; syrups; and starch. More examples can be found, for example, in U.S. Pat. No. 7,213,367, which is incorporated herein by reference.

[0296] Various additives may also be included in the plant component treatment formulation, such as adhesives, dispersants, surfactants and nutrients and buffering components. Other conventional plant component treatment additives include, but are not limited to, coating agents, wetting agents, buffers and polysaccharides. At least one agriculturally acceptable carrier, such as water, solids or dry powders, may be added to the plant component treatment formulation. Dry powders may be derived from a variety of materials, such as calcium carbonate, gypsum, vermiculite, talc, humus, activated carbon and a variety of phosphorus compounds.

[0297] In some embodiments, the plant component coating composition may include at least one filler, which is an organic or inorganic, natural or synthetic component with which the active ingredients are combined to facilitate its application on the plant component. In various aspects, the filler is an inert solid such as clay, natural or synthetic silicates, silica, resins, waxes, solid fertilizers (e.g., ammonium salts), natural soil minerals (such as kaolin, clay, talc, lime, quartz, attapulgite, montmorillonite, bentonite or diatomaceous earth) or synthetic minerals (such as silica, alumina or silicates, especially aluminum silicate or magnesium silicate).

[0298] In some embodiments, the plant component treatment formulation may also include one or more of the following ingredients: other insecticides, including compounds that work only below the ground; fungicides, such as captan, thiram, metalaxyl, fludioxonil, oxadone, and isomers of each of these materials, etc.; herbicides, including compounds selected from glyphosate, carbamates, thiocarbamates, acetamides, triazines, dinitroanilines, glycerol ethers, pyridazinones, uracils, phenoxy groups, ureas, and benzoic acid; herbicide safeners , such as benzoxazines, benzhydryl derivatives, N,N-diallyl dichloroacetamide, various dihaloacetyl, oxazolidinyl and thiazolidinyl compounds, acetone, naphthalic anhydride compounds and oxime derivatives; chemical fertilizers; biological fertilizers; and biological control agents, such as other naturally occurring or recombinant bacteria and fungi from the genera Rhizobium, Bacillus, Pseudomonas, Serratia, Trichoderma, Glomus, Gliocladium and mycorrhizal fungi. These ingredients can be added as a separate layer on the plant component, or alternatively can be added as part of the plant component coating composition of the present disclosure.

[0299] In some embodiments, the formulations used in the present disclosure for treating plant parts may be in the form of suspensions; emulsions; slurries of particles in an aqueous medium (e.g., water); wettable powders; wettable granules (dry flowable); and dry granules. If formulated as a suspension or slurry, the concentration of the active ingredient in the formulation may be from about 0.5% to about 99% by weight (w / w), or 5% to 40% by weight, or otherwise formulated by one skilled in the art.

[0300] As mentioned above, other conventional inactive or inert ingredients may be incorporated into the formulation. Such inert ingredients include, but are not limited to, conventional adhesives; dispersants, such as methylcellulose, for example, acting as a combined dispersant / adhesive for plant component treatment agents; polyvinyl alcohol; lecithin, polymeric dispersants (e.g., polyvinyl pyrrolidone / vinyl acetate); thickeners (e.g., clay thickeners for increasing viscosity and reducing sedimentation of particle suspensions); emulsion stabilizers; surfactants; antifreeze compounds (e.g., urea), dyes, colorants, etc. Additional inert ingredients that may be used in the present disclosure may be found in McCutcheon's, Vol. 1, "Emulsifiers and Detergents," MC Publishing Company, Glen Rock, NJ, USA, 1996, which is incorporated herein by reference.

[0301] The plant component coating formulations of the present disclosure can be applied to the plant component by a variety of methods, including but not limited to: mixing in a container (e.g., a bottle or bag), mechanical application, tumbling, spraying, and immersion. A variety of active or inert materials can be used to contact the plant component with the microbial composition according to the present disclosure.

[0302] In some embodiments, the amount of microorganism or agricultural composition used to treat the plant component will vary depending on the type of plant component and the type of active ingredient, but treatment will include contacting the plant component with an agriculturally effective amount of a composition of the invention.

[0303] As discussed above, an effective amount means an amount of a composition of the invention sufficient to affect beneficial or desired results. An effective amount can be administered over the course of one or more administrations.

[0304] In some embodiments, in addition to the coating layer, the plant parts may be treated with one or more of the following ingredients: other pesticides, including fungicides and herbicides; herbicide safeners; fertilizers and / or biological control agents. These ingredients may be added as a separate layer, or alternatively may be added in the coating layer.

[0305] In some embodiments, the plant component coating formulations of the present disclosure can be applied to the plant component using a variety of techniques and machines, such as fluidized bed technology, roller mill methods, drum electrostatic plant component processors, and drum coaters. Other methods (such as spouted beds) may also be useful. The plant component can be pre-sized before coating. After coating, the plant component is typically dried and then transferred to a sizing machine for sizing. Such procedures are known in the art.

[0306] In some embodiments, the plant parts treated with microorganisms can also be wrapped with a film outer coating to protect the coating. Such outer coatings are known in the art and can be applied using fluidized bed and drum film coating techniques.

[0307] In other embodiments of the present disclosure, can cause by using solid matrix to be introduced on the plant component according to the composition of the present disclosure.For example, a certain amount of the present composition can be mixed with solid matrix material, and then can place plant component and make it contact with solid matrix material for a period of time to allow composition to be introduced into plant component.Then, can optionally plant component be separated from solid matrix material and store or use, or can directly store or plant the mixture that solid matrix material adds plant component.Solid matrix material that can be used for the present disclosure comprises polyacrylamide, starch, clay, silicon dioxide, aluminum oxide, soil, sand, polyurea, polyacrylate or any other material that can absorb or adsorb the present composition for a period of time and the composition is released in the plant component or is released on the plant component.It is very useful to ensure that the present composition and solid matrix material are compatible with each other.For example, solid matrix material should be selected so that it can be released at a reasonable rate (for example in the time period of a few minutes, a few hours or a few days).

[0308] In some embodiments, the present disclosure teaches that individual microorganisms or consortia of microorganisms or communities of microorganisms developed according to the disclosed methods can be combined with any plant biostimulant.

[0309] In some embodiments, the present disclosure teaches agricultural compositions comprising one or more commercially available biostimulants, including but not limited to: Diehard TM Diehard TM Fe, Diehard TM Soluble Kelp, Diehard TM Humate SP, Foliar Plus TM 、Plant Plus TM 、Accomplish Soil Builder TM 、Nutri Life、SoilSolution TM 、Seed Coat TM 、PercPlus TM 、Plant Thrust TM , and wait.

[0310] In some embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with an active chemical agent, an additive effect on a plant phenotypic trait of interest is observed. In other embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with an active chemical agent, a synergistic effect on a plant phenotypic trait of interest is observed.

[0311] In some embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a fertilizer, an additive effect on a plant phenotypic trait of interest is observed. In other embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a fertilizer, a synergistic effect on a plant phenotypic trait of interest is observed.

[0312] In some embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a plant growth regulator, an additive effect on a plant phenotypic trait of interest is observed. In some embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a plant growth regulator, a synergistic effect is observed. In some aspects, combining a microorganism of the present disclosure with a plant growth regulator Combine and observe synergistic effects on one or more phenotypic traits of interest.

[0313] In some embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a biostimulant, an additive effect on a plant phenotypic trait of interest is observed. In some embodiments, when a microorganism or microbial consortium identified according to the methods taught is combined with a biostimulant, a synergistic effect is observed.

[0314] The synergistic effect obtained by the methods taught can be quantified according to the Colby formula (i.e., (E) = X + Y - (X * Y / 100)). See Colby, RS, "Calculating Synergistic and Antagonistic Responses of Herbicide Combinations," 1967 Weeds, Vol. 15, pp. 20-22, which is incorporated herein by reference in its entirety. Thus, the term "synergistic" is intended to be an amount that, when present, increases the desired effect beyond the additive amount.

[0315] The isolated microorganisms and consortia of the present disclosure can enhance the efficacy of agriculturally active compounds as well as agricultural adjuvant compounds in a synergistic manner.

[0316] In other embodiments, a synergistic effect is observed when a microorganism or consortium of microorganisms identified according to the taught methods is combined with a fertilizer.

[0317] In addition, in certain embodiments, the present disclosure utilizes synergistic interactions to define microbial consortia. That is, in certain aspects, the present disclosure combines certain isolated microbial species that act synergistically together into a consortium, which imparts beneficial traits to plants, or is associated with enhancing beneficial plant traits.

[0318] Agricultural compositions developed according to the present disclosure can be formulated with certain adjuvants to increase the activity of known active agricultural compounds. This has the advantage that the amount of active ingredient in the formulation can be reduced while maintaining the efficacy of the active compound, thus keeping the cost as low as possible and complying with any official regulations. In individual cases, it is also possible to broaden the scope of action of the active compound, because plants (which are not successfully treated with a specific active ingredient without addition) can actually be successfully treated by adding certain adjuvants and the disclosed microbial isolates and consortia. In addition, when environmental conditions are unfavorable, the performance of the active substance can be increased in individual cases by suitable formulations.

[0319] Such auxiliary agents that can be used in agricultural compositions can be adjuvants. Usually, adjuvants are in the form of surface-active or salt compounds. According to their mode of action, they can be roughly classified as regulators, activators, fertilizers, pH buffers, etc. Regulators affect the wetting, adhesion and spreading characteristics of the preparation. Activators destroy the waxy surface layer of plants and improve the penetration of active ingredients into the surface layer (short-term (several minutes) and long-term (several hours)). Fertilizers (such as ammonium sulfate, ammonium nitrate or urea) improve the absorption and solubility of active ingredients and can reduce the antagonistic behavior of active ingredients. pH buffers are routinely used to adjust the preparation to the optimal pH.

[0320] For additional embodiments of the agricultural compositions of the present disclosure, see "Chemistry and Technology of Agrochemical Formulations," DA Knowles, ed., copyright 1998, Kluwer Academic Publishers, which is hereby incorporated by reference.

[0321] Botanical and Agronomic Benefits

[0322] A wide variety of plants, including those cultivated agriculturally, can benefit from the application of microorganisms, such as those described herein, including single microorganisms, consortia, and / or compositions produced therefrom, or including any of the foregoing. Many various plants, including mosses and lichens and algae, can be used in the methods of the present disclosure. In embodiments, plants have economic, social or environmental value. For example, plants can include those plants used as food crops, fiber crops, oil crops, for forestry, for pulp and paper industry, as raw materials for biofuel production, and as ornamental plants.

[0323] In other embodiments, the plant may be economically, socially or environmentally undesirable, such as a weed. The following is a list of non-limiting examples of plant types to which the methods of the present disclosure may be applied:

[0324] Food Crops

[0325] Cereals, such as maize, rice, wheat, barley, sorghum, millet, oats, rye, triticale, and buckwheat;

[0326] Leafy vegetables, such as cruciferous vegetables such as kale, broccoli, bok choy, and rocket; salad greens such as spinach, celery, and lettuce;

[0327] Fruiting and flowering vegetables such as avocado, sweet corn, fodder corn, artichokes; melons such as winter squash, cucumber, melon, courgette, pumpkin; nightshade vegetables / fruits such as tomatoes, eggplants and peppers;

[0328] Legume vegetables, such as groundnuts, peanuts, peas, soybeans, beans, lentils, chickpeas, and okra;

[0329] Bulb and stem vegetables, such as asparagus, celery, and Allium crops, such as garlic, onions, and leeks;

[0330] Root and tuber vegetables, such as carrots, beets, bamboo shoots, cassava, yams, ginger, Jerusalem artichokes, parsnips, radishes, potatoes, sweet potatoes, taro, turnips, and horseradish;

[0331] Sugar crops, including sugar beet (Beta vulgaris), sugar cane (Saccharum officinarum);

[0332] Growing crops used to prepare non-alcoholic beverages and stimulants, such as coffee, black, herbal and green teas, cocoa, and tobacco;

[0333] Fruit crops, such as true berry fruits (e.g. kiwi, grape, currant, gooseberry, guava, feijoa, pomegranate), citrus fruits (e.g. orange, lemon, lime, grapefruit), epiphytic fruits (e.g. banana, cranberry, blueberry), aggregate fruits (blackberry, raspberry, boysenberry), multiple fruits (e.g. pineapple, fig), stone fruit crops (e.g. apricot, peach, cherry, plum), pome fruits (e.g. apple, pear) and other fruits, such as strawberry, sunflower seeds;

[0334] Culinary and medicinal herbs, such as rosemary, basil, bay, cilantro, mint, dill, Hypericum, foxglove, aloe vera, and rose hips;

[0335] Plants that produce spices, such as black pepper, cumin, nutmeg, ginger, cloves, saffron, cardamom, mace, red pepper, masala, star anise;

[0336] Planting of crops for tree nuts, such as almonds and walnuts, Brazil nuts, cashews, coconuts, chestnuts, macadamia nuts, pistachios, peanuts, pecans;

[0337] growing crops such as grapes and hops for the preparation of beer, wine, and other alcoholic beverages;

[0338] Oilseed crops such as soybeans, peanuts, cotton, olives, sunflower, sesame, lupin species and Brassica crops (e.g. canola / rape); and edible fungi such as white button mushrooms, shiitake mushrooms and oyster mushrooms;

[0339] Plants for gardening

[0340] Leguminosae: Trifolium, Medicago and Lotus species; White clover (T. repens); Red clover (T. pratense); Caucasian clover (T. ambigum); Subterraneum (T. subterraneum); Medicago sativum; Annual alfalfa; Truncatula terrestris; Blue alfalfa; Onobrychis viciifolia; Lotus corniculatus; Lotus pedunculatus;

[0341] Seed legumes / dry beans, including peas (Pisum sativum), beans (Phaseolus vulgaris), broad beans (Vicia faba), mung beans (Vigna radiata), cowpeas (Vigna unguiculata), chickpeas (Cicerarietum), lupins (Lupinus species); cereals, including maize / corn (Zeamays), sorghum (Sorghum spp.), millet (Panicum miliaceum, P. sumatrense), rice (Oryza sativa indica, Oryza sativa japonica), wheat (Triticum aestivum), barley (Hordeum vulgare), rye (Secale cereale), triticale (Triticum X Secale), oats (Avena sativa);

[0342] Forage and woodland grasses: Temperate grasses such as Lolium species; Festuca species; Agrostis species, perennial ryegrass (Lolium perenne); hybrid ryegrass (Lolium hybridum); annual ryegrass (Lolium multiflorum); tall fescue (Festuca arundinacea); cowgrass (Festucapratensis); red fescue (Festuca rubra); fescue (Festuca ovina); fescue ryegrass (Festuloliums) (Lolium x Festuca crosses); orchardgrass (Dactylis glomerata); Kentucky bluegrass (Poa pratensis); marsh bluegrass (Poa palustris); woodland bluegrass (Poa nemoralis); common bluegrass (Poa trivialis; Poa compresa; Bromus species; Phalaris (Phleum species); Arrhenatherum elatius; Agropyron species; Avena strigosa; Setaria italic;

[0343] Tropical grasses, such as: Phalaris species; Brachiaria species; Eragrostis species; Panicum species; Paspalum notatum; Brachypodium species; and grasses used for biofuel production, such as switchgrass (Panicum virgatum) and Miscanthus species;

[0344] Fiber crops

[0345] Cotton, kapok, jute, coconut, sisal, flax (Linum species), New Zealand flax (Phormium species); plantations and natural forest species harvested for paper and engineered wood fiber products, such as coniferous and broadleaf forest species;

[0346] Planting tree and shrub species used in forestry and biofuel crops

[0347] Pine (Pinus species); fir (Pseudotsuga species); spruce (Picea species); cypress (Cupressus species); acacia (Acacia species); alder (Alnus species); oak (Quercus species); redwood (Sequoiadendron species); willow (Salix species); birch (Betula species); cedar (Cedurus species); ash (Fraxinus species); larch (Larix species); eucalyptus (Eucalyptus species); bamboo (Bambuseae species) and poplar (Populus species).

[0348] Planting for conversion into energy, biofuels or industrial products through extraction, biological, physical or biochemical processing Plants

[0349] Oil-producing plants, such as oil palm, jatropha, corn, soybean, cotton, linseed; latex-producing plants, such as Hevea brasiliensis and Castilla elastica); plants used as direct or indirect feedstock for the production of biofuels (i.e. after chemical, physical (e.g. thermal or catalytic) or biochemical (e.g. enzymatic pretreatment) or biological (e.g. microbial fermentation) conversion during the production of biofuels, industrial solvents or chemical products (e.g. ethanol or butanol, propylene glycol or other fuels or industrial materials), including sugar crops (e.g. sugar beets, sugar cane), starch-producing crops (e.g. C3 and C4 cereal crops and tuber crops), cellulosic crops such as forest trees (e.g. pine, eucalyptus) and grasses and grasses such as bamboo, switchgrass, miscanthus; crops used in energy, biofuel or industrial chemical production by gasification and / or microbial or catalytic conversion of gases into biofuels or other industrial feedstocks (such as solvents or plastics, with or without the production of biochar), for example biomass crops such as coniferous, eucalyptus, tropical or broadleaved forest trees, grasses and grasses such as bamboo, switchgrass, miscanthus, sugar cane or softwoods such as poplar, willow; and biomass crops used for the production of biochar;

[0350] Crops producing natural products useful in the pharmaceutical, agro-nutraceutical and cosmeceutical industries

[0351] Crops that produce pharmaceutical precursors or compounds or nutraceutical and cosmeceutical compounds and materials, such as star anise (shikimic acid), Japanese knotweed (resveratrol), kiwi fruit (soluble fiber, proteolytic enzymes);

[0352] Flowering, ornamental and landscaping plants grown for their aesthetic or environmental properties

[0353] Flowers, such as roses, tulips, and chrysanthemums;

[0354] Ornamental shrubs such as boxwood, heber, rose, azalea, and ivy;

[0355] Landscaping plants such as sycamore, Mexican orange, acanthus, euphorbia, and liverwort;

[0356] and / or moss, such as sphagnum moss.

[0357] Planting for bioremediation

[0358] In certain aspects, the microorganisms of the present disclosure are applied to hybrid plants to enhance the beneficial traits of the hybrids. In other aspects, the microorganisms of the present disclosure are applied to genetically modified plants to enhance the beneficial traits of the GM plants. The microorganisms taught herein can be applied to hybrids and GM plants, and thus can maximize the superior genetics and trait technologies of these plants.

[0359] It should be understood that plants can be provided in the form of seeds, seedlings, cuttings, propagules, or any other plant material or tissue capable of growth. In one embodiment, seeds can be surface sterilized with materials such as sodium hypochlorite or mercuric chloride to remove microorganisms that contaminate the surface. In one embodiment, propagules are grown in axenic cultures, such as in the form of sterile seedlings in tissue culture, before being placed in a plant growth medium.

[0360] Application method

[0361] The microorganisms may be applied to plants, seedlings, cuttings, propagules, etc. and / or to a growth medium containing the plants using any suitable technique known in the art.

[0362] However, as an example, the isolated microorganism, consortium, or compositions comprising and / or produced therefrom may be applied to plants, seedlings, cuttings, propagules, etc. by spraying, coating, spreading, or any other method known in the art.

[0363] In another embodiment, the isolated microorganism, consortium or composition comprising the same may be applied directly to plant seeds prior to sowing.

[0364] In another embodiment, the isolated microorganism, consortium or composition comprising the same may be applied directly to plant seeds in the form of a seed coating.

[0365] In one embodiment of the present disclosure, the isolated microorganism, consortium or composition comprising the same is supplied in the form of granules or a plug or soil drench for application to a plant growth medium.

[0366] In other embodiments, the isolated microorganism, consortium, or composition comprising the same is provided in the form of a foliar application, such as a foliar spray or a liquid composition. The foliar spray or liquid application can be applied to growing plants or a growing medium, such as soil.

[0367] In some embodiments, the isolated microorganism, consortium, or composition comprising the same is provided in a form selected from the group consisting of a soil drench, a foliar spray, a drench treatment, an in-furrow treatment, a soil conditioner, a granule, a broadcast treatment, a post-harvest disease control treatment, or a seed treatment. In some embodiments, the agricultural composition may be applied alone or in a rotary spray procedure.

[0368] In some embodiments, the isolated microorganisms, consortia, or compositions comprising the same are compatible with tank mixing. In some embodiments, the agricultural compositions are compatible with tank mixing with other agricultural products. In some embodiments, the agricultural compositions are compatible with equipment for ground, aerial, and irrigation applications.

[0369] In another embodiment, the isolated microorganism, consortium or composition comprising the same may be formulated into granules and applied next to the seeds during planting. Or the granules may be applied after planting. Or the granules may be applied before planting.

[0370] In some embodiments, the isolated microorganism, consortium or composition comprising the same is applied to plants or growth media in the form of topical application and / or drench application to improve crop growth, yield and quality. Topical application can be carried out by using a dry mixture or a powder or dusting composition or can be a liquid-based formulation.

[0371] In embodiments, the isolated microorganisms, consortia, or compositions comprising the same may be formulated as: (1) solutions; (2) wettable powders; (3) dusting powders; (4) soluble powders; (5) emulsions or suspension concentrates; (6) seed dressings or seed coatings, (7) tablets; (8) water-dispersible granules; (9) water-soluble granules (slow release or quick release); (10) microencapsulated granules or suspensions; (11) as irrigation components, and (12) as components of fertilizers, pesticides, and other compatible improvers, etc. In certain aspects, the composition may be diluted in an aqueous medium prior to conventional spray application. The compositions of the present disclosure may be applied to soil, plants, seeds, rhizosphere, rhizosheath, or other areas that would be conducive to the application of the microbial composition. In addition, impact methods may be used as a means for introducing endogenous microorganisms.

[0372] In various aspects, the composition is applied to the leaves of the plant. The composition can be applied to the leaves of the plant in the form of an emulsion or suspension concentrate, a liquid solution or a foliar spray. The application of the composition can be carried out in a laboratory, a growth chamber, a greenhouse or a field.

[0373] In another embodiment, the microorganisms can be inoculated into the plant by pruning the roots or stems and exposing the plant surface to the microorganisms by spraying, dipping or otherwise applying a liquid microorganism suspension or a gel or powder.

[0374] In another embodiment, the microorganisms can be injected directly into the foliage or root tissue, or otherwise inoculated directly into or onto the foliage or root prunings, or inoculated into isolated embryos or radicles or coleoptiles. These inoculated plants can then be further exposed to growth media containing additional microorganisms; however, this is not required.

[0375] In other embodiments, particularly in the case where the microorganism is not culturable, the microorganism can be transferred to the plant by any one or combination of the following means: grafting, insertion into an explant, suction, electroporation, wounding, root pruning, inducing stomatal opening, or any physical, chemical or biological treatment that provides an opportunity for the microorganism to enter the plant cells or intercellular spaces. Those skilled in the art will readily appreciate the many alternative techniques that can be used.

[0376] In one embodiment, the microorganism infiltrates a part of a plant such as a root, stem, leaf, and / or reproductive plant part (becoming endophytic), and / or grows on the surface of a root, stem, leaf, and / or reproductive plant part (becoming epiphytic), and / or grows in the rhizosphere of the plant. In one embodiment, the microorganism forms a symbiotic relationship with the plant.

[0377] Although the present invention is particularly shown and described in conjunction with preferred embodiments and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention. For example, although the following specific examples may use specific plants to illustrate the methods and embodiments described herein, the principles in these examples may be applied to any plant. Therefore, it should be understood that the scope of the present invention is encompassed by the embodiments described herein, rather than by only the specific examples illustrated below.

[0378] The present disclosure enables those skilled in the relevant art to make and use the invention provided herein according to multiple and varied embodiments. Various changes, modifications and improvements (including certain changes, modifications, substitutions and improvements) of the present disclosure that are easily expected by those skilled in the art are also a part of the present disclosure. Therefore, the above specific embodiments illustrate the discovery provided herein by way of example. In addition, the above specific embodiments and examples illustrate the present invention, rather than limiting it. Therefore, the scope of the present invention is set forth in the appended claims.

[0379] All cited patents, patent applications, and publications mentioned in this application are incorporated by reference in their entirety for all purposes to the same extent as if each cited patent and publication was individually and specifically indicated to be incorporated by reference.

[0380] Example

[0381] The methods and compositions presented herein (based on the use of the disclosed isolated microorganisms, consortia, and / or compositions comprising and / or produced by microorganisms or consortia or consortia) improve one or more characteristics of plants (e.g., agricultural crops).

[0382] Strain 101136 and strain 101166 are spore-forming bacteria that each impart plant growth promoting (PGP) properties to plants, reduce nematode populations on plants, and increase crop yields.

[0383] Example 1: Isolation and identification of microorganisms

[0384] The isolation of microorganisms can be performed according to any method known in the art. An illustrative, non-limiting example is given below.

[0385] Approximately 4 cm sections from a variety of plant component sections were prepared and placed in a plastic bag with a zip lock enclosure. For example, if corn was the plant and roots were the desired tissue, sections were taken from (1) primary roots, (2) seminal roots, (3) crown roots, and (4) buttress roots to ensure diversity of tissues to be isolated.

[0386] Then tissue is sterilized as follows.In 50mL centrifuge tube (falcon tube), use 25mL sterile reverse osmosis (RO) water to wash tissue by vortex to ensure that it does not contain soil.This can be washed multiple times and use tweezers to clean the soil on the root.Use tweezers, the root tissue is immersed in the 70% ethanol in the 50mL centrifuge tube for 10 seconds.Immediately root system is taken out from ethanol bath, throw off excess liquid, and be immersed in the 1.5%NaOCl in the 50mL centrifuge tube for 3 minutes.Root system is moved to sterile 50ml centrifuge tube and washed 6 times with sterile RO water by immersing plant material in clean sterile water 6 times.

[0387] Extraction of the microorganism associated with plant tissue can be performed by cultivating, subculture and selecting the supernatant from the previous step. Alternatively, sterilized and washed tissue is moved to a culture dish and sterile scissors are used to cut tiny fragments. Approximately 500 μ L of this tissue is moved to a sterile 2 ml tube and 100 μ L of culture medium is added and the rear end of a sterile utensil such as tweezers or an L-shaped spreader is used for dipping. These small fragments of root tissue can be added to the culture medium just poured out (warm, not hot) to be embedded in agar. Look for the bacterium colony growing from the "cutting" end of plant material. Every 500 μ L tissue is added to 1 ml of 10 mM sterile potassium phosphate buffer pH 7. The solution is fully mixed. Using 50 μ L of this solution in 450 μ L of phosphate buffer, 10x, 100x and 1000x dilutions are produced by serial dilution. Use a sterile L-shaped spreader that 100 to 150 μ L of this solution is coated on a suitable separation plate and incubated.

[0388] Isolation of spore-forming bacteria can be performed as follows. Take an aliquot of the undiluted solution from above and pipette it into a sterile Eppendorf tube. Seal the tube and heat in a 60°C water bath for 20 minutes to enrich for spore-forming bacteria. Using 50 μl of this solution in 450 μl of phosphate buffer, make 10x, 100x and 1000x dilutions by serial dilution. Use a sterile L-shaped spreader to spread 100 to 150 μl of this solution on an appropriate separation plate and incubate.

[0389] Sequencing preparation and long-term storage for microbial identification can be performed by:

[0390] Day 1:

[0391] Use a 10 μL sterile pipette tip to transfer the colonies from the plates to flasks containing the appropriate liquid growth medium. Place the isolates on a shaker at room temperature and incubate for 2 days.

[0392] Day 3:

[0393] After 2 days on the shaker, the tubes may be turbid. All samples will be analyzed by PCR. Vortex each tube, collect 50 μL of sample from each vortexed tube, and distribute it to a 96-well plate. Use a multichannel pipette to distribute 15 μL of the 50 μL sample to a new 96-well plate. A 96-well plate containing 35 μL of each sample will be used for phenotyping, and a 96-well plate containing 15 μL of each sample will be used for PCR analysis. 27F / 1492R primers are often used for 16S PCR analysis because they obtain better results than PB36 / 38. The plate should be accompanied by appropriate negative controls and these negative controls are analyzed by PCR. The plate is analyzed by PCR using an Eppendorf thermal cycler. Once PCR is completed, the gel is run using standard gel electrophoresis techniques. This is important because most isolates have grown to a sufficient degree and ideally, they should be stored for a long time on the 3rd day. Use PCR and gel electrophoresis analysis to confirm that these isolates contain bacteria, not other microorganisms. For isolates that did not pass PCR or had clarified broth, vortex the tube and streak it onto a petri dish using an inoculating loop. Check after a few days to see if anything has grown or if the tube is contaminated. For isolates that passed PCR, dispense 600 μl of 50% glycerol into a 2 ml screw cap tube and add 1200 μL of bacterial culture so that the broth is stored in 20% glycerol. Store the glycerol stock at -80°C and record images of the gel of the PCR samples.

[0394] On Day 4:

[0395] Check the culture plates for growth of the streaked isolates that failed PCR. (The 2 ml broth tube will remain on the shaker during this time.) Once there is growth on the plate and the colonies appear to have been successfully isolated, dispense 600 μl of the broth-glycerol mixture into small tubes and place the two tubes into their corresponding -80 boxes. The isolate may fail the PCR check for any of the following reasons: the primers may not be effective for all bacteria, the isolate is actually a fungus, the isolate has strong adherence and is therefore not homogenized in the broth, the isolate produces too much EPS and therefore needs to be diluted before the PCR setup, or the isolate grows very slowly. Continue to check the plate over the next few days to confirm that only a single bacterial species is isolated. If contamination is observed, prepare a new isolate. The viability of the prepared glycerol stock should be verified.

[0396] Sequencing and bioinformatics

[0397] Strain 101136 was sequenced using Pacbio and Illumina sequencing technologies. Illumina reads were trimmed with Trimmomatic and had a quality score of Q20. PacBio raw reads and trimmed Illumina reads were co-assembled using Unicycler with default parameters to balance the requirements for chromosome assembly and short circular plasmid assembly. To confirm assembly quality, trimmed Illumina reads were mapped back to the assembly and the assembly was manually checked for breaks / misassemblies and SNPs. The genome was annotated using Prokka and genes of interest were identified by annotation. 16S was confirmed by BLAST alignment against the NCBI database. Taxonomy was assigned by 16S.

[0398] Genomic analysis showed that strain 101136 contained genes involved in nematicidal / insecticide activity (8), proteases (39), secondary metabolites (10), PGP / colonization / siderophores (38), and glycosidases / glycosylases (48). Genes associated with insecticide / nematicide activity included: a putative D-aminopeptidase (aprA2), nematode protease (purL), two extracellular serine proteases, and two aspartate kinases. Genes associated with secondary metabolites included: Fengycin, FGAM synthase II, acetolactate synthase, extracellular alkaline serine protease, a putative D-aminopeptidase (aprA2), RsmE, tktA, aroB, aroD, aroE. Disease resistance genes included: acetoin biosynthesis, flagellin HAP1, flagellin HAP2, flagellin HAG, and uronic acid teichoic acid / lipopolysaccharide synthesis (tag). Proteases include: aminopeptidase 2, aminopeptidase, D-aminopeptidase, glutamyl aminopeptidase, peptidase T, putative aminopeptidase YadC, cysteine ​​protease StiP, EDTA-sensitive protease (aprA), germination protease, protease HtpX, regulator of σ-W protease RasP, stage IV sporulation protein FB, putative zinc protease, ATP-dependent Clp protease, Lon protease 1, Lon protease 2, Lon protease, nicotinic acid phosphoribosyltransferase, putative signal peptide peptidase SppA, signal peptidase IU, signal peptidase IV, signal peptidase I, signal peptidase IB, SpoIVB peptidase, putative CdtpA-like serine protease, putative peptidase. PGP / colonization / siderophore genes include: spore membrane-related, putative ymcA, 28 flagellar biosynthesis and regulatory genes, swarming motility, two tyrosine recombinases, two exopolysaccharide genes (eps), indoleacetic glucose 1-dehydrogenase and two indole-3-pyruvate decarboxylases. Glycosidases / glycosylation enzymes include: β-xylosidase, folic acid polyglutamate synthase, two lipid II, putative endo-β-N-acetylglucosaminidase.

[0399] AntiSMASH analysis of the biosynthetic gene clusters showed the presence of regions for the synthesis of phosphonates, type III PKS, bacillidin, NRPS-independent siderophores, and three terpenes. Strain 101166 was sequenced using Illumina sequencing technology. Illumina reads were trimmed using Trimmomatic with a quality score of Q20. Trimmed Illumina reads were assembled using Unicycler with default parameters to balance the requirements for chromosome assembly and short circular plasmid assembly. The genome was annotated using Prokka, and genes of interest were identified through annotation. 16S was confirmed by BLAST alignment against the NCBI database. Taxonomy was assigned by 16S.

[0400] Genomic analysis showed that strain 101166 contained genes involved in nematicidal / insecticide activity (15), proteases (33), secondary metabolites (18), PGP / colonization / siderophores (35), and glycosidases / glycosylases (110). Genes associated with insecticide / nematicide activity included: alkaline phosphatase, extracellular alkaline serine protease (BLG4), chitinase (CrChi1), 46 kDa chitinase, nematode protease (purL), cyanobacterin synthase, two aspartate kinases, and hemolysin (tlyA). Genes associated with secondary metabolites included: polyketide (mln), paenibacterin (pbt), fennexin (Fen), FGAM synthase II (purL), alkaline phosphatase, acetolactate synthase, 2-hydroxyphenazine F, pyruvate kinase, RsmE, tktA, aroB, aroE. Disease resistance genes include: two acetoin biosynthesis, flagellin HAP1, flagellin HAP2, flagellin HAG. Proteases include: three aminopeptidases 2, three aminopeptidases, three peptidases T, putative aminopeptidase YadC, sporulation σ-E factor processing, cysteine ​​protease StiP, germination protease, regulator of σ-W protease RasP, stage IV sporulation protein FB, four ATP-dependent Clp proteases, carboxyl terminal processing protease CtpA, carboxyl terminal processing protease CtpB, Lon protease 1, Lon protease, nicotinic acid phosphoribosyltransferase, signal peptidase IP, signal peptidase IT, signal peptidase I, signal peptidase IB, SpoIVB peptidase. PGP / colonization / iron carrier genes include: putative ymcA, 25 flagellar biosynthesis and regulation, swarming motility, tyrosine recombinase, IAA, glucose-1-dehydrogenase, iron binding protein, three indole-3-pyruvate decarboxylases.Glycosidases / glycosylases included: six cellulases, eight xylanases, α-L-arabinofuranosidase C, three α-galactosidases A, α-galactosidase AgaA, five α-galactosidases, anti-σ-I factor RsgI6, two arabinogalactan endo-β-1,4-galactanases, three β-galactosidases, two β-xylosidases, six endo-1,4-β-xylanases A, endoglucanase A, endoglucanase H, six endoglucanases, extracellular exo-α-L-arabinofuranosidase, folic acid synthase, two glucoamylases, two polyglutamate synthases. The 101166 strain was characterized by the presence of SMASH resistance in the biosynthetic gene clusters of paeninodin and lankacidin C, and the presence of α-L-arabinofuranosidase, α-L-arabinofuranosidase, α-L-arabinofuranosidase, α-L-arabinofuranosidase, α-L-arabinofuranosidase, α-L-arabinofuranosidase, α-L-arabinofuranosidase, α-L-arabinofuranosidase, α-L-arabinofuranosidase, α-L-N-acetylglucosaminidase, α-L-arabinofuranosid ...

[0401] Example 2: Preparation of microorganisms

[0402] Microorganisms identified according to previous embodiments can be formulated with additional components so as to be applied via methods such as, but not limited to, seed treatment, root washing, seedling soaking, foliar application, soil inoculum, furrow application, lateral application, soil pretreatment, wound inoculation, drip irrigation, vector-mediated via pollinators, injection, osmotic initiation, hydroponics, aquaponics, aeroponics. Preparations containing microorganisms are prepared in the form of liquid, solid or gaseous preparations for agricultural applications. Application to plants is achieved, for example, in the form of powders for surface deposition on plant leaves, sprays for whole plants or selected plant components, a portion of drip irrigation fluids for soil or roots, or coatings for plant components before planting. Such embodiments are intended to be illustrative and do not limit the scope of the present invention.

[0403] The components of the culture medium used for the exemplary microorganism preparation are shown in Table 2 below. All contents are added and 50% of the final water volume is required, and the solution is stirred at high temperature until dissolved. After all contents have dissolved, the solution is fixed to the final required volume using sterile RO water. 4x formulations are usually used for field trial preparation.

[0404] Table 2: Exemplary media components and concentrations of microbial preparations

[0405]

[0406] The procedure for mixing the TIX formulation is as follows: measure all dry ingredients into a 50 ml tube. Vortex the ingredients thoroughly to ensure that the xanthan gum is "separated" from the other carbon sources. Add about half of the total sterile RO water to the mixture and vortex. Use the long end of an L-shaped applicator to break up large chunks as much as possible. Heat some sterile RO water in a microwave to warm up the water bath temperature (45-50°C). Add the remaining sterile RO water to the mixture and vortex. Repeat step 4 as needed and vortex until a clear solution with no lumps is obtained. Spin down the foam formed during mixing by "quick centrifuging" the centrifuge tube for 5 to 10 seconds. Remember to balance the formulation (TIX) tube. Let the formulation cool to room temperature. 1. Mix in the microbial consortium. Vortex to ensure homogeneity. Ideally, a concentration of 10^9 CFU / ml of microorganisms is added to the formulation.

[0407] The formulations are applied to plants or plant parts for testing in field trials.

[0408] Example 3: Application of microorganisms to plant parts and cultivation thereof

[0409] A microbial composition (comprising a single strain of one or more isolated microorganisms, a consortium, a population, a combination, or any combination of the foregoing) is prepared according to the previous examples. The microbial composition comprises one or more microorganisms, optionally in combination with one or more additional microorganisms disclosed herein.

[0410] Microbial compositions for administration

[0411] In some methods, the microbial composition is dried and applied directly to the plant parts.

[0412] In some methods, the microbial composition is suspended in a liquid formulation for application to the plant parts.

[0413] In some methods, the microbial composition is combined with another composition, such as but not limited to: a carrier, a wetting agent, a stabilizer, a salt. In some methods, the other composition comprises a molecule that brings additional agricultural beneficial results to the plant to which the microbial composition is applied. The other composition includes, for example, but not limited to: herbicides, fungicides, bactericides, insecticides, insecticides, nematicides, biostimulants.

[0414] Application type

[0415] The microbial composition is applied to the plant components at a time during development appropriate for the desired result, for example: in a formulation applied in soil drench / in-furrow before planting; as a seed or other propagation component treatment; as a propagation component application after planting; as an in-furrow, drip irrigation or drench application after planting; as a direct application to plant components (e.g., roots, leaves, stems); as an application to harvested plant components (e.g., fruit or grain). Combinations of application types are also tested.

[0416] Application method

[0417] The microbial composition is applied to (inoculated) plants or plant parts or plant products (before planting, after planting, before harvesting or after harvesting). This can be accomplished, for example, by applying the agricultural composition to a hopper or spreader or tank containing the microbial composition and configured to spread the microbial composition.

[0418] The seed coating of the microbial composition is applied to one or more seeds of a crop plant. After application of the isolated microorganisms in the form of a seed coating, the seeds are planted and cultivated according to established customary practices for the crop.

[0419] Alternatively, the microbial composition is applied to the soil to benefit plants present in the soil. Methods of soil application include in-furrow treatment, drench and drip irrigation application.

[0420] Alternatively, the microbial composition is applied to the surface of the plant or plant part after germination.

[0421] Alternatively, the microbial composition is applied to material obtained from the plant after harvesting.

[0422] A control plot of plants was also planted to which the isolated microorganism was not applied.The plants associated with the microbial composition exhibit improved traits of interest.

[0423] The method of administration can be performed according to any protocol known in the art.

[0424] The plant parts, plants or growth medium (eg, soil) may further be inoculated with the disease or pest, depending on the purpose of the test.

[0425] An exemplary, non-limiting protocol for watering a tomato plant is given below:

[0426] 1. Ten days after planting, carefully separate the plants so that there are 6 replicates per treatment group. The plants are delicate and the leaves can be easily torn off. Make sure the size and general appearance of the plants are as uniform as possible (the goal of thinning is to continue to maintain a uniform plant population). If there are not enough plants per replicate, transplant. See step 3 for guidelines on transplanting.

[0427] 2. Begin thinning the pots so that there is one plant per pot. Remove smaller plants, i.e., plants that are unhealthy or misshapen in some way. If there are 2 or more healthy plants per pot, transplant the extra plants to another pot. Use leftover soil pre-treated from the initial planting or from pots where the seeds did not germinate.

[0428] 3. Transplanting: If some pots do not germinate, fill them with plants from another container. To do this, simply dig out the extra plants with a scoopula (try to dig out as much root mass as possible without disturbing other plants) and place in the hole dug in the empty pot. Press lightly with your fingers to compact the soil around the plants.

[0429] 4. Space the pots into 6 rows of pots (1 row of pots per treatment group), which will require 4 RL98 trays. Once completed, take a look at all treatment groups and consider doing some pot swaps to ensure that some treatment groups do not have all tall plants and other treatment groups have all tall plants.

[0430] 5. Change gloves if necessary. Label each pot with the Avery labels you prepared. Treatments should be labeled in rows of 6 replicates, i.e. 1-1, 1-2, 1-3 to 1-6, etc. This will make it easier to find all the replicates for each treatment.

[0431] 6. Two weeks after planting (approximately 4 days after thinning and marking), obtain treatments from the Microbiology team; place trays of prepared plants on the table. Collect combitips, repeaters, and RO water. (Note: plants should be lightly watered on the day of treatment)

[0432] 7. Mix the microbial solution by inverting the tube / container (microbial treatment) 2 to 3 times or gently shaking. Set the dispensing tip to dispense 2 ml. Collect the treatment fluid into the dispensing tip and dispense the first step back into the tube. Make sure the treatment you have corresponds to the row of plants to be treated. Once confirmed, gently dispense 2 ml of the treatment onto the surface soil of each pot, close to the stem but avoiding direct contact with the stem and leaves.

[0433] 8. Dispose of the dispensing tip and repeat step 6 for all treatments. For the inoculated controls (IC or InoCon) and the untreated controls (UTC), RO water was applied instead of the treatments.

[0434] Once all treatments were applied, plants were returned to the growth chamber for processing (optional inoculation), growth, and evaluation.

[0435] Visualization of microorganisms associated with plant components

[0436] Individual microorganisms can be labeled with fluorescent proteins according to methods known in the art.Microscopic image analysis confirmed that the microorganisms disclosed herein were found associated with various plant tissues.

[0437] Example 4: Improving crop yield and pest resistance

[0438] Increased plant / crop yields are achieved without the need for added chemical fertilizers, in some aspects by virtue of the association of plants with one or more microorganisms disclosed herein. In some aspects, the microorganisms act as "biostimulants," i.e., agents that promote plant health, growth, vigor, and / or production ("yield").

[0439] In some aspects, "yield" can be determined by, for example, but not limited to, biomass, seed size, seed weight, leaf composition, fiber production of one or more crop products.

[0440] In some aspects, the increase in yield is statistically significant. In other aspects, the biomass increase is not statistically significant, but still quantitative and relevant for crop improvement. Other parameters related to yield can be improved by adding microorganisms disclosed herein, such as but not limited to: plant vigor, NDVI score, photosynthetic capacity, nutrient utilization, stress tolerance. In some cases, microorganisms promote plant health, thereby further improving crop yield. When other parameters are improved, the yield parity under microbial and control conditions is a desired result.

[0441] Improvements in plant health or yield can be achieved under relatively stress-free conditions. In other cases, improvements can be achieved in plants grown under a variety of abiotic or biotic stresses, including drought stress, salt stress, high temperature stress, low temperature stress, low nutrient stress, nematode stress, insect herbivory stress, fungal pathogen stress, complex pathogen stress, or viral pathogen stress.

[0442] It is expected that plants grown from seeds treated with the agricultural composition will exhibit quantifiably higher yields and / or plants with improved health and / or plants with improved stress tolerance compared to control plants.

[0443] The yield from the plant of processing is high about 1% to 10%, high 10% to 20%, high 20% to 30%, high 30% to 40%, high 40% to 50%, high 50% to 60%, high 60% to 70%, high 70% to 80%, high 80% to 90% or more.The biomass from the plant of processing can be equal to about 1 bushel per acre compared with the control, or increase 2 bushels per acre, or increase 3 bushels per acre, or increase 4 bushels per acre, or increase 5 bushels per acre or more.In some cases, output improves under normal or typical conditions without biological or abiotic stress factors.In some cases, output is normal under the condition of biological and / or abiotic stress.In some cases, output increases under the condition of biological and / or abiotic stress.

[0444] The microorganisms described herein improve the health and / or yield of various crop plants, including those described below. The yield and / or plant health of a particular harvested material is improved under normal conditions as well as under conditions of abiotic stress (e.g., drought, herbicide application, pesticide application, reduction or elimination of applied nutrients such as nitrogen, phosphorus, potassium) or biotic stress (e.g., the presence of insects, larvae, nematodes, fungal diseases, bacterial diseases, viral diseases).

[0445] Caenorhabditis elegans ( C.elegans) MOA determination

[0446] To determine the possible mode of action of our nematicidal microbes, the model organism Caenorhabditis elegans, a free-living, soil-dwelling nematode, was used in several different assays. First, developmental tests were performed to determine activity. Young larvae were allowed to feed on the microbes. While doing so, the development and fecundity of the worms were tracked. Based on how quickly the C. elegans reached reproductive age and the resulting population of offspring, it was determined how these microbes affected the nematodes.

[0447] Secondly, the mortality of the adult worms was assessed. The adult worms were introduced to the microorganisms and the mortality was tracked. This demonstrated the toxicity of the microorganisms to the nematodes.

[0448] C. elegans is also tested in a chemotaxis assay. The worms are introduced to two different stimuli (e.g., microbial, chemical, metabolic, etc.). Based on the movement of the nematodes in response to these stimuli, a chemotaxis index, i.e., repulsion or attraction, is calculated.

[0449] Root-knot nematode (RKN) assay

[0450] Two weeks before treatment application, seeds of susceptible crops were planted in pots containing a 1:1 ratio mixture of soil and sand. An excess of seeds was sown per pot and then thinned to one seedling per pot.

[0451] Two weeks after planting, the microbial treatment solution was irrigated into the soil at the base of the plants at 1x10^7 CFU / mL per plant. The application amount was sufficient to cover the root zone in the pot.

[0452] Plants were not watered immediately before or after treatment application (no heavy watering that day - before or after treatment application). Regular watering was resumed the next day.

[0453] Forty-eight hours after treatment application, nematodes are administered at the desired concentration, for example, in some cases, the J2 stage is used instead of eggs to reduce variability.

[0454] On the day of nematode application, plants were sprayed with water in the morning prior to nematode infestation. Four weeks after nematode application, stem weights were harvested and roots washed for nematode assessment.

[0455] The roots were stained for egg masses and the egg masses, galls, eggs, etc. were counted. (Galls, eggs, egg masses, cysts, female insects, etc.)

[0456] Root dry weight was obtained, and the number of nematodes per gram of root dry weight was calculated.

[0457] Positive and negative controls include: untreated (no nematode inoculation, no microbial treatment, water only), inoculated control (nematode inoculation, no microbial treatment, water only), positive control (a known nematicide product against the test nematode - ideally both chemical nematicides and bionematicides are used as positive controls in the same experiment), negative microbial control - use E. coli DH5α or other available microorganisms that do not have nematicidal activity.

[0458] Strain 101136 reduced root-knot nematode J2 survival after hatching by 37.5% in an in vitro assay. In growth chamber trials, strain 101136 reduced RKN counts in cucumber by 84% and in tomato by 57%. In greenhouse trials, strain 101136 reduced RKN counts in tomato by 32%.

[0459] In growth chamber trials, line 101166 reduced RKN counts in tomatoes (72%) and cucumbers (61%). In greenhouse trials, line 101166 reduced RKN counts in tomatoes by 48%.

[0460] Row crop field trials

[0461] Corn seed treatment trials were conducted at 8 locations (6 replications) in which seeds were treated with each of the lines disclosed herein. Corn in-furrow trials were conducted at 2 locations (6 replications).

[0462] Corn plants grown from seeds treated with line 101136 showed an average yield increase of 7.9 bushels / acre (4.2% increase), a harvest rate of 87.5%, and outperformed the commercial chemical (increases ranged up to 23.6 bu / ac for line 101136). Nematodes were reduced by an average of 13%.

[0463] Corn plants grown from seeds treated with Line 101166 showed an average yield increase of 9.3 bu / acre (5.7%; 87.5% harvested). Corn plants treated in-furrow with Line 101166 showed an average yield increase of 5.0 bu / acre (2.1%; 100% harvested). Both the seed treatment and in-furrow application outperformed the commercial chemical control.

[0464] Wheat trials were conducted at 2 locations (6 replications) in which seeds were treated with each of the lines disclosed herein.

[0465] Wheat plants grown from seeds treated with line 101136 showed an average yield increase of 2.7 bu / acre (an 8.4% increase). Line 101136 performed better than the commercial chemical used as a control, which only increased yield by 6%.

[0466] Wheat plants grown from seeds treated with line 101166 showed an average yield increase of 7.5% (2.3 bu / acre). Line 101166 outperformed the commercial chemical, which only increased by 6%.

[0467] Soybean plants grown from seeds treated with Priesteria gigantea strain 101136 showed an average yield of 62.14 bu / ac in seven trials compared to untreated controls (60.82 bu / ac) and chemically treated controls (62.41 bu / ac).

[0468] Soybean plants grown from seeds treated with Paenibacillus cellulolyticus strain 101166 showed an average yield of 62.79 bu / ac (71% yield) in 7 trials compared to untreated controls (60.82 bu / ac) and chemically treated controls (62.41 bu / ac).

[0469] The soybean cyst nematode (SCN) results for lines 101136 and 101166 are shown in Table 3.

[0470] Table 3: Percent Change in SCN in Soybean Trials (Negative numbers indicate decreases)

[0471]

[0472] Vegetable field trials

[0473] A tomato trial was conducted at two locations (4 replications) with application via drench (8 qt / acre by drench / drench at 0, 7 and 14 days post planting (DPP)).

[0474] Tomato plants treated with line 101136 showed a 50% reduction in root-knot nematode counts per 100 cc of soil at 35 DPP and a 56% improvement in gall reduction compared to untreated controls.

[0475] Tomato plants treated with line 101166 showed a 52% reduction in root-knot nematode counts per 100 cc of soil at 35 DPP and a 49% improvement in gall reduction compared to untreated controls.

Claims

1. A synthetic composition comprising: a. a microbial cell, an exudate derived therefrom, or a culture broth derived therefrom, wherein the microbial cell is selected from the group consisting of: i. A microbial cell comprising a 16S sequence that shares at least 95% identity with any one of SEQ ID NOs: 1-14; ii. microbial cells obtained or derived from Priesteria gigantea strain 101136; deposit number NRRL B-68087; and iii. microbial cells obtained or derived from Bacillus cellulolyticus strain 101166, deposited as NRRL B-68088; and b. at least one heterologous composition selected from the group consisting of: a plant component, a formulation component, an agricultural composition, and any combination of the foregoing; wherein the microorganism is present at a concentration of at least about 10^2 CFU / mL in a liquid formulation or at least about 10^2 CFU / gram in a non-liquid formulation.

2. The synthetic composition according to claim 1, further comprising at least one additional microorganism.

3. The synthetic composition of claim 1, wherein the plant component is a seed.

4. The synthetic composition of claim 3, wherein the seed comprises a transgene.

5. The synthetic composition of claim 1, wherein the plant component is a leaf.

6. The synthetic composition of claim 1, wherein the plant component is a root.

7. The synthetic composition of claim 1, wherein the plant component is a whole plant.

8. The synthetic composition of claim 1, wherein the formulation components are selected from the group consisting of: compounds that improve the stability of the microorganism, preservatives, carriers, surfactants, anti-complexing agents, and any combination thereof.

9. The synthetic composition of claim 1, wherein the agricultural composition comprises a fungicide, a nematicide, a bactericide, an insecticide, a herbicide, or any combination thereof.

10. A plurality of synthetic compositions according to claim 1, wherein the synthetic composition is substantially enclosed within an article selected from the group consisting of: a tube, a bottle, a jar, an ampoule, a package, a container, a bag, a box, a storage box, an envelope, a carton, a container, a silo, a shipping container, a carriage, and a case.

11. A plurality of synthetic compositions according to claim 9, wherein the synthetic compositions are at a temperature below zero degrees Celsius.

12. The synthetic composition of claim 1, wherein the plant parts are obtained from a plant selected from the group consisting of: maize, soybean, wheat, cotton, cucumber, tomato, pepper, potato, strawberry, orange, lemon, lime, apple, snap bean, zucchini, pea, lettuce, broccoli, celery, cauliflower, sorghum and canola.

13. The synthetic composition of claim 1, wherein the agricultural composition comprises a growth medium.

14. The synthetic composition of claim 13, wherein the growth medium comprises soil.

15. A plurality of synthetic compositions according to claim 14, wherein the plurality of synthetic compositions are placed in the soil in a regular pattern with substantially equal spacing between each of the synthetic compositions.

16. A synthetic composition comprising: a. an exudate or culture broth of a plurality of cells, wherein the cells comprise at least one microbial cell selected from the group consisting of: i. A microbial cell comprising a 16S sequence that shares at least 95% identity with any one of SEQ ID NOs: 1-14; ii. microbial cells obtained or derived from Priesteria gigantea strain 101136; deposit number NRRL B-68087; and iii. microbial cells obtained or derived from Bacillus cellulolyticus strain 101166, deposited as NRRL B-68088; and b. At least one heterologous composition selected from the group consisting of a plant part, a formulation component, an agricultural composition, and any combination of the foregoing.

17. The synthetic composition of claim 16, further comprising at least one additional microorganism.

18. A method of modulating an agronomically important trait in a plant obtained or derived from a plant component, the method comprising treating the plant component with a formulation comprising a microbial cell, an exudate derived therefrom, or a culture broth derived therefrom, wherein the microbial cell is selected from the group consisting of: a. A microbial cell comprising a 16S sequence that shares at least 95% identity with any one of SEQ ID NOs: 1-14; b. microbial cells obtained or derived from Priesteria gigantea strain 101136; deposit number NRRL B-68087; and c. Microbial cells obtained or derived from Bacillus cellulolyticus strain 101166, deposited as NRRL B-68088.

19. The method of claim 18, further comprising at least one additional microorganism.

20. The method of claim 18, wherein the agronomically important trait is selected from the group consisting of disease resistance, drought tolerance, heat tolerance, cold tolerance, salt tolerance, metal tolerance, herbicide tolerance, chemical tolerance, improved water use efficiency, improved nitrogen use efficiency, improved nitrogen fixation, insect resistance, herbivore resistance, pathogen resistance, increased yield, increased yield under water limiting conditions, enhanced health, improved vigor, improved growth, increased photosynthetic capacity, enhanced nutrition, altered protein content, altered oil content, increased biomass, increased shoot length, increased root length, improved root architecture, increased seed weight, altered seed carbohydrate composition, altered seed oil composition, increased radicle length, number of pods, delayed senescence, stay-greenness, altered seed protein composition, increased dry weight of mature plant reproductive components, increased fresh weight of mature plant reproductive components, increased number of mature plant reproductive components per plant, increased chlorophyll content, increased number of pods per plant, increased pod length per plant, increased number of seeds per plant, increased seed weight per plant, decreased number of wilted leaves per plant, decreased number of severely wilted leaves per plant, increased number of non-wilted leaves per plant, or improved visual appearance of plants.

21. The method of claim 18, wherein the microbial cells, exudates therefrom, or culture broths therefrom are present in an amount capable of providing a benefit to a plant obtained from the plant parts compared to a plant obtained from the plant parts not treated with the microbial cells or exudates therefrom.

22. A method of cultivating a plant, the method comprising introducing into plant parts of the plant a microbial cell, an exudate derived therefrom, or a culture broth derived therefrom, wherein the microbial cell is selected from the group consisting of: a. A microbial cell comprising a 16S sequence that shares at least 95% identity with any one of SEQ ID NOs: 1-14; b. microbial cells obtained or derived from Priesteria gigantea strain 101136; deposit number NRRL B-68087; and c. microbial cells obtained or obtained from Bacillus cellulosus strain 101166, deposited as NRRL B-68088; wherein the microbial cell is heterologous to the plant component.

23. The method of claim 22, further comprising at least one additional microorganism.

24. The method of claim 22, wherein said introducing into said plant components is accomplished by an indirect method selected from the group consisting of: in-furrow application, soil drench application, and side application.

25. The method of claim 22, wherein said introducing into said plant component is accomplished by coating said plant component with a liquid formulation of said microorganism or an exudate therefrom.

26. The method of claim 22, wherein said introducing into said plant component is accomplished by coating said plant component with a substantially non-liquid formulation of said microorganism or an exudate therefrom.

27. The method of claim 22, wherein the plant component is a seed.

28. The method of claim 22, wherein the plant component is a leaf.

29. The method of claim 22, wherein the plant component is a root.

30. The method of claim 22, wherein the plant component is a whole plant.

31. A method of modulating an agronomically important trait in a harvested product, said method comprising introducing into said organism from which said harvested product is obtained a microbial cell, an exudate derived therefrom or a culture broth derived therefrom, wherein said microbial cell is selected from the group consisting of: a. A microbial cell comprising a 16S sequence that shares at least 95% identity with any one of SEQ ID NOs: 1-14; b. microbial cells obtained or derived from Priesteria gigantea strain 101136; deposit number NRRL B-68087; and c. Microbial cells obtained or derived from Bacillus cellulolyticus strain 101166, deposited as NRRL B-68088.

32. A method of modulating an agronomically important trait in a harvested product, the method comprising introducing into the harvested product a microbial cell, an exudate derived therefrom, or a culture broth derived therefrom, wherein the microbial cell is selected from the group consisting of: a. A microbial cell comprising a 16S sequence that shares at least 95% identity with any one of SEQ ID NOs: 1-14; b. microbial cells obtained or derived from Priesteria gigantea strain 101136; deposit number NRRL B-68087; and c. Microbial cells obtained or derived from Bacillus cellulolyticus strain 101166, deposited as NRRL B-68088.

33. The method of claim 31 or claim 32, further comprising at least one additional microorganism.

34. The method of claim 31 or claim 32, wherein the harvested product is a fruit.

35. The method of claim 31 or claim 32, wherein the harvested product is a vegetable.

36. The method of claim 31 or claim 32, wherein the harvested product is seed.

37. The method of claim 31 or claim 32, wherein the harvested product is fiber.

38. A substantially cell-free preparation obtained or derived from a culture of a microorganism, wherein the microorganism is selected from the group consisting of: a. A microbial cell comprising a 16S sequence that shares at least 95% identity with any one of SEQ ID NOs: 1-14; b. microbial cells obtained or derived from Priesteria gigantea strain 101136; deposit number NRRL B-68087; and c. Microbial cells obtained or derived from Bacillus cellulolyticus strain 101166, deposited as NRRL B-68088.

39. A purified composition prepared from the substantially cell-free preparation of claim 38.

40. Any of the methods of the compositions above, comprising Priesteria gigantea strain 101136 deposited as NRRL B-68087, Bacillus cellulolyticus strain 101166 deposited as NRRL B-68088, or an isolated bacterial strain having substantially similar morphological and physiological properties, substantially similar or substantially identical genetic properties, progeny, mutants, or genetically edited, altered or modified variants thereof.

41. An isolated bacterial strain comprising a polynucleotide sequence that shares at least 97% sequence identity with any one of SEQ ID NOs: 1-14.

42. An agricultural composition comprising: a) an isolated bacterial strain according to claim 40 or claim 41; and b) an agriculturally acceptable carrier; The bacterial strain is present in the agricultural composition in an amount effective to produce a modified phenotype in a plant with which it is associated.

43. The agricultural composition of claim 42, wherein the agricultural composition is formulated as a seed coating, a foliar spray, a soil drench, a drench treatment, an in-furrow treatment, a soil conditioner, a granule, a broadcast treatment, or a post-harvest disease control treatment.

44. A microbial cell and a plant component, the microbial cell comprising a nucleotide sequence that shares at least 90% identity with a sequence selected from SEQ ID NOs: 1-14; wherein the microbial cell is heterologously delivered to the plant component.

45. A composition comprising any of the preceding, wherein the composition produces a deleterious effect on a nematode, an insect or a pathogen.

46. ​​The composition of claim 45, wherein the nematode is a root-knot nematode.

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