Genome-edited microorganisms for improved fungicidal and bactericidal activity

Abstract

The present disclosure relates to methods and compositions for genetically engineering microorganisms for improved control of pathogens such as bacteria and / or fungi, which can be applied in various fields such as medicine and agriculture. The genetically engineered microorganisms may comprise deletions or knockout at one or more genomic loci that confer the ability to reduce mycelial growth of the fungi, reduce sporogenesis of the fungi, and / or other benefits.

Description

[0001] Cross-reference to related applications This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 484,449, filed February 10, 2023, pursuant to 35 USC 119(e), which is incorporated herein by reference in its entirety.

[0002] References to sequence lists submitted electronically An official copy of the sequence list is submitted electronically as a WIPO ST26 compliant XML sequence list and is submitted with this specification. The sequence list is named 23071-WO-PCT.xml, created on February 3, 2024, and is 24,530 bytes in size. The sequence list contained in that file is an integral part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure relates to methods and compositions of genetically modified microorganisms that produce metabolites, such as secondary metabolites, lipopeptides, and / or polyketides, which can be used in agriculture and other fields. This disclosure also relates to compositions comprising metabolites or metabolite-producing microorganisms, and methods for identifying them and using them in agriculture and other applications. Background Technology

[0004] According to the United Nations World Food Program, nearly 900 million people worldwide suffer from malnutrition. The malnutrition epidemic is particularly pronounced in developing countries, 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 to feed the growing world population, projected to reach 9 billion by 2037. The United Nations estimates that agricultural output must increase by 70% to 100% to feed the projected global population.

[0005] Many factors affect crop health and yield, including stress introduced by other living organisms (biotic stress) or by other factors (abiotic stress, such as insufficient or excessive water, poor nutrient availability, toxicity caused by salts, minerals or other compounds).

[0006] Biological stress in plants is caused by living organisms, particularly viruses, bacteria, fungi, nematodes, insects, spiders, and weeds. These factors cause biological stress, directly depriving their hosts of nutrients and potentially leading to plant death.

[0007] Despite progress made through technological innovations, such as genetically engineered crops and novel insecticides and herbicides, improved crop performance is still needed to meet the demands of the growing global population.

[0008] Therefore, novel compositions and methods are needed to enhance crops' resistance to biotic stress. Biocontrol of pests and diseases using live microorganisms or microbial products offers an attractive alternative to synthetic chemical insecticides and fungicides due to their reduced toxicity and higher biodegradability, thus mitigating environmental persistence concerns. The development of biocomposites is also typically faster and less expensive, making them commercially available within a certain timeframe, thereby benefiting growers and consumers. Summary of the Invention

[0009] This disclosure addresses the important question of how to improve crop performance, thereby narrowing the global yield gap, while providing ways to confer other beneficial traits onto plant species.

[0010] The solutions provided by this disclosure for improving crop performance and increasing yield are harmless to Earth's resources because they do not rely on increased water consumption or the input of synthetic chemicals into the system. Instead, this disclosure utilizes microorganisms or their metabolites to confer beneficial properties, including increased yield, onto desired plants.

[0011] Therefore, this disclosure provides an environmentally sustainable solution that enables farmers to increase the yield of important crops, especially those sensitive to one or more biotic stressors. Without limitation, biotic stressors can include pests such as nematodes and plant pathogens such as fungi.

[0012] In one aspect, this disclosure relates to a method for selecting microorganisms that produce one or more metabolites, said metabolites conferring one or more beneficial traits on plants, said method comprising: Obtain a first sample containing one or more metabolites from microorganisms; A first metabolite profile is obtained from a first sample; and when the first metabolite profile has one or more unique elements, microorganisms that produce metabolites that confer one or more beneficial traits to plants are selected, wherein at least one of the one or more unique elements corresponds to the one or more metabolites that confer the one or more beneficial traits to plants.

[0013] In some embodiments, the method of selecting microorganisms that produce one or more metabolites that confer one or more beneficial traits to plants includes comparing a first metabolite profile with a second metabolite profile, wherein the second metabolite profile is obtained from a second sample having one or more metabolites from a second microorganism that does not produce metabolites that confer one or more beneficial traits to plants.

[0014] In some implementations, the one or more beneficial traits conferred on the plant include protection against or biological control of plant pathogens. In some aspects, plant pathogens damage the plant before harvest. In other aspects, plant pathogens damage the harvested parts of the plant (e.g., fruit, seeds, lint, leaves).

[0015] In another aspect, this disclosure relates to compositions having a mixture of isolated metabolites, wherein the isolated metabolite mixture is derived from microorganisms selected via the methods disclosed herein. In some embodiments, the isolated metabolite mixture has one or more lipopeptides.

[0016] In some respects, lipopeptides are cyclic lipopeptides.

[0017] In some respects, cyclic lipopeptides are part of a lipopeptide assembly. In other respects, this assembly comprises a variety of different lipopeptides.

[0018] In some respects, this component contains multiple types of the same lipopeptides.

[0019] In some embodiments, this disclosure relates to compositions having a mixture of isolated metabolites, wherein the isolated metabolite mixture is derived from microorganisms that produce lipopeptides.

[0020] In another aspect, this disclosure relates to compositions having isolated microbial bodies, wherein the isolated microbial bodies are selected via the methods disclosed herein. In some embodiments, the isolated microorganisms produce a mixture of metabolites having one or more lipopeptides.

[0021] In another aspect, this disclosure relates to a method of conferring one or more beneficial traits on a plant, the method comprising applying a composition disclosed herein to the plant or the growing medium in which the plant is situated. In some embodiments, the one or more beneficial traits conferred on the plant are for the biological control of one or more pests and / or plant pathogens.

[0022] In another aspect, this disclosure relates to a method for producing one or more variant strains that produce an increased amount or relative ratio of controlling one or more biological stressors, such as one or more metabolites of fungi.

[0023] In another aspect, this disclosure relates to compositions having one or more isolated metabolites, wherein the one or more isolated metabolites are derived from microorganisms selected via the methods disclosed herein. In some embodiments, the isolated metabolites have one or more lipopeptides.

[0024] In some embodiments, this disclosure relates to a composition having one or more isolated metabolites, wherein: the one or more metabolites are one or more lipopeptides derived from genetically engineered strains.

[0025] In some embodiments, the composition is applied to the plant or plant parts before germination. In some embodiments, the composition is applied to the plant or plant parts before harvest. In some embodiments, the composition is applied to the plant or plant parts during the vegetative stage of the plant. In some embodiments, the composition is applied to the plant or plant parts during the reproductive stage of the plant. In some embodiments, the composition is applied to the plant or plant parts before harvest. In some embodiments, the composition is applied to the plant or plant parts after harvest. In some embodiments, the plant is cultivated in a field, harvested, stored in a warehouse, or distributed. In some embodiments, the plant or parts thereof are harvested. In some embodiments, the plant produces agriculturally important fruits, grains, cereals, fiber, food, feed, fuel, or seeds. In some embodiments, the parameters are selected from the group consisting of: the presence of pathogens, the distribution of pathogens, the quantification of pathogens, the type of pathogens, the biological state of pathogens, the activity of pathogens, the presence of pests, the distribution of pests, the quantification of pests, the type of pests, the biological state of pests, the activity of pests, visual assessment of the plant part, the biomass of the plant part, the biological state of the plant part, the activity of the plant part, and any combination of the foregoing parameters. In some embodiments, the plant part is a root, leaf, stem, flower, seed, bulb, or fruit. In some embodiments, the method further includes applying the multiple compositions of (b) to the plant. In some embodiments, the method includes applying multiple compositions in step (b). In some embodiments, the method includes applying multiple compositions in step (b), wherein each of the multiple compositions comprises a different feature of step (b), or any combination of the foregoing features. In some embodiments, the method further includes applying multiple compositions in step (b), wherein each of the multiple compositions comprises a different feature of step (b), or any combination of the foregoing features.

[0026] In some embodiments, the synthetic composition is substantially enclosed within an object selected from the group consisting of: bottles, wide-mouth bottles, ampoules, packaging, containers, bags, boxes, storage boxes, sleeves, cartons, containers, silos, shipping containers, wagons, and crates. In some embodiments, the synthetic composition of (b) is a cyclic lipopeptide. In some embodiments, the composition of (b) is derived from a genetically engineered strain. In some embodiments, the composition of (b) is substantially purified. In some embodiments, the synthetic composition further comprises one or more formulation components. In some embodiments, the one or more formulation components are selected from the group consisting of: salts, binders, surfactants, wetting agents, dispersants, emulsifiers, solubilizers, organic solvents, gelling agents, thickeners, antisettling agents, preservatives, stabilizers, antifreeze compounds, any of the foregoing substances, and any combination of the foregoing substances. In some embodiments, the synthetic composition further comprises one or more additional agents selected from the group consisting of: insecticides, herbicides, bactericides, fungicides, insecticides, viricides, acaricides, nematicides, scabies insecticides, plant growth regulators, rodenticides, algaecides, biocontrol agents, fertilizers, biopesticides, biostimulants, and any combination of the foregoing substances and / or multiple of the foregoing substances. In some embodiments, the synthetic composition further comprises fungi. In some embodiments, the plant or plant part of (a) produces agriculturally important fruits, grains, food, fuel, feed, cereals, fiber, or seeds. In some embodiments, the synthetic composition further comprises a growth medium. In some embodiments, the plant part is a root, leaf, stem, flower, seed, bulb, or fruit. In some embodiments, multiple synthetic compositions are provided. In some embodiments, each of the multiple compositions comprises a different feature of step (b), or any combination or multiple of the foregoing features.

[0027] In some respects, the synthetic composition may contain a variety of the same or different microorganisms and / or lipopeptides or combinations thereof.

[0028] In some aspects, a method is provided for producing a composition that enhances the tolerance of plants to biological stresses, the method comprising: obtaining a microorganism and culturing the microorganism under conditions suitable for growth and reproduction; and identifying the composition of the microorganism. Attached Figure Description

[0029] This disclosure will be more fully understood through the following detailed description and the accompanying drawings and sequence listings that form part of this application.

[0030] Figure 1AThe HPLC peak areas of cyclic lipopeptides (CLPs) in whole-cell culture broth from wild-type strain 7084 (7084-F) are shown compared to those of genetically modified strains 7084-G77, 7084-G93, and 7084-G105 (iturin, fengycin, surfactin, and total CLPs).

[0031] Figure 1B The HPLC peak areas of cyclic lipopeptides (CLPs) obtained from the wild-type strain 7084 (7084-F) are shown in repeated operations (Operation-1 and Operation-2, respectively) of whole-cell culture medium compared with the genetically modified strains 7084-G103, 7084-G77, 7084-G95 and 7084-G97.

[0032] Figure 2A The HPLC peak areas of polyketides (macrolides and difficidin) in the supernatant material obtained from wild-type strain 7084 (7084-F) are shown compared with those of genetically modified strains 7084-G77, 7084-G93 and 7084-G105.

[0033] Figure 2B The HPLC peak areas of polyketides (difenoxine 1, difenoxine 2, difenoxine 3) of supernatant materials obtained from wild-type strain 7084 (7084-F) are shown in repeated operations (operation-1 and operation-2, respectively) compared with those of genetically modified strains 7084-G103, 7084-G77, 7084-G95 and 7084-G97.

[0034] Figure 2C The HPLC peak areas of macrocyclic imides (macrocyclic imide 1, macrocyclic imide 2, macrocyclic imide 3, macrocyclic imide 4, macrocyclic imide 5) of the supernatant material obtained from wild-type strain 7084 (7084-F) are shown in repeated operations (operation-1 and operation-2, respectively) compared with the genetically modified strains 7084-G103, 7084-G77, 7084-G95 and 7084-G97.

[0035] Figure 2D The HPLC peak areas of baccilaene obtained from the supernatant material of wild-type strain 7084 (7084-F) are shown in repeated operations (Operation-1 and Operation-2, respectively) compared with those of genetically modified strains 7084-G103, 7084-G77, 7084-G95 and 7084-G97.

[0036] Figure 3A The HPLC chromatograms show two different production variants of the genetically modified strain 7084-G67 (magenta line = variant 1 / “high-yield”, royal blue line = variant 2 / “low-yield”) compared to the wild-type strain 7084 (black line). The cyan lines indicate the peak start / stop of the peak area integral.

[0037] Figure 3B A close-up of a portion of the HPLC chromatogram of variant 7084-G67 (royal blue line), showing (royal blue arrow) the retention time shift of one of the surfactants compared to wild-type strain 7084 (7084-F) (black line).

[0038] Figure 3C The relative yields of cyclic lipopeptides are shown for 7084 (WT, far right), 7084-G67 low-yield variant (variant 2, far left), and 7084-G67 high-yield variant (variant 1, middle).

[0039] Figure 3D The relative yields of polyketides are shown for 7084 (WT, far right), 7084-G67 low-yield variant (variant 2, far left), and 7084-G67 high-yield variant (variant 1, middle).

[0040] Figure 4 The image shows the HPLC chromatogram of the genetically modified strain 7084-G77 (magenta line) compared to the wild-type strain 7084 (black line). The cyan line indicates the peak start / stop at the peak area integral.

[0041] Figure 5 The image shows the HPLC chromatogram of the genetically modified strain 7084-G93 (brown line) compared to the wild-type strain 7084 (black line). The cyan line indicates the peak start / stop at the peak area integral.

[0042] Figure 6 The image shows the HPLC chromatogram of the genetically modified strain 7084-G105 (green line) compared to the wild-type strain 7084 (black line). The cyan line indicates the peak start / stop at the peak area integral.

[0043] Figure 7 This is the HPLC chromatogram of the genetically modified strain 7084-G68 (black line). The cyan line indicates the peak start / stop at the peak area integral.

[0044] Figure 8 This is the HPLC chromatogram of the genetically modified strain 7084-G75 (black line). The cyan line indicates the peak start / stop at the peak area integral.

[0045] Figure 9 This is the HPLC chromatogram of the genetically modified strain 7084-G76 (black line). The cyan line indicates the peak start / stop at the peak area integral.

[0046] Figure 10 This is an HPLC chromatogram of the genetically modified strain 7084-G95 (Royal Blue line). The cyan line indicates the peak start / stop at the peak area integral.

[0047] Figure 11 This is the HPLC chromatogram of the genetically modified strain 7084-G97 (magenta line). The cyan line indicates the peak start / stop at the peak area integral.

[0048] Figure 12 This is the HPLC chromatogram of the genetically modified strain 7084-G103 (brown line). The cyan line indicates the peak start / stop at the peak area integral.

[0049] Figure 13A The demonstration shows the effectiveness of whole-cell cultures (WCB) obtained from wild-type strain 7084 and genetically modified strain 7084-G77 against Fusarium graminearum at different dilutions. Fusarium gramminearum ( ) antifungal activity.

[0050] Figure 13B Photographs of cell culture plates of wild-type strain 7084 and genetically modified strain 7084-G77 at different dilutions against Fusarium graminearum are shown, compared with the untreated control inoculated with Fusarium graminearum.

[0051] Figure 14A This demonstrates the effectiveness of whole-cell cultures (WCB) obtained from wild-type strain 7084 and the genetically modified strain 7084-G77 against Penicillium expansum at different dilutions. Penicillium expansum ( ) antifungal activity.

[0052] Figure 14B Photographs of cell culture plates inoculated with Penicillium expansum at a 1:50 dilution are shown, comparing wild-type strain 7084 and genetically modified strain 7084-G77 with the untreated control inoculated with Penicillium expansum.

[0053] Figure 15A This demonstrates the effectiveness of whole-cell cultures (WCB) obtained from wild-type strain 7084 and the genetically modified strain 7084-G77 against Fusarium oxysporum tomato-specific strains at different dilutions. Fusarium oxysporum f.sp. lycopersici

[0054] Figure 15BPhotographs of cell culture plates of wild-type strain 7084 and genetically modified strain 7084-G77 at different dilutions against Fusarium oxysporum tomato-specific strain, compared to an untreated control inoculated with the untreated control.

[0055] Figure 16A This demonstrates the effectiveness of whole-cell cultures (WCB) obtained from wild-type strain 7084 and the genetically modified strain 7084-G77 against Fusarium oxysporum lettuce-specific strains at different dilutions. Fusarium oxysporum f.sp. lactucae

[0056] Figure 16B Photographs of cell culture plates of wild-type strain 7084 and genetically modified strain 7084-G77 (3 dpi) at different dilutions against Fusarium oxysporum lettuce-specific strain, compared to an untreated control inoculated with the lettuce-specific strain.

[0057] Figure 16C Photographs of cell culture plates of wild-type strain 7084 and genetically modified strain 7084-G77 (5 dpi) at different dilutions against Fusarium oxysporum lettuce-specific strain, compared to an untreated control inoculated with the lettuce-specific strain.

[0058] Figure 17A This demonstrates the effectiveness of whole-cell cultures (WCB) obtained from wild-type strain 7084 and the genetically modified strain 7084-G77 against Penicillium fingerling at different dilutions. Penicillium digitatum ( ) antifungal activity.

[0059] Figure 17B Photographs of cell culture plates of wild-type strain 7084 and genetically modified strain 7084-G77 (3 dpi) at different dilutions against Penicillium digitatum, compared to the untreated control inoculated with Penicillium digitatum.

[0060] Figure 18A This demonstrates the effectiveness of whole-cell cultures (WCB) obtained from wild-type strain 7084 and the genetically modified strain 7084-G77 against *Botrytis cinerea* at different dilutions. Botrytis cinerea ( ) antifungal activity.

[0061] Figure 18B Photographs of cell culture plates of wild-type strain 7084 and genetically modified strain 7084-G77 (3 dpi) at a 1:100 dilution against *Botrytis cinerea*, compared to an untreated control inoculated with *Botrytis cinerea*.

[0062] Figure 19This demonstrates the effectiveness of whole-cell cultures (WCBs) obtained from wild-type strain 7084 and genetically modified strains 7084-G77 and 7084-G95 against Agrobacterium tumefaciens at different dilutions. Agrobacterium tumefaciens ( ) antibacterial activity.

[0063] Figure 20A This is a photograph of a culture showing the main colony morphology of wild-type strain 7084, displaying smooth, round colonies. Most 7084 WT colonies exhibit this morphology.

[0064] Figure 20B Photographs of cultures showing alternative colony morphologies for wild-type strain 7084, displaying colonies with serrated edges. A small number of 7084 WT colonies exhibit this morphology. One edited strain, 7084-G67, shows only this alternative morphology and does not exhibit any smooth, round colonies.

[0065] Figure 21 The percentage of bioavailability of different secondary metabolites of the wild-type and edited strain 7084 is shown.

[0066] Figure 22A A photograph of colonies of wild-type strain 14416.

[0067] Figure 22B Photograph of a colony of 14416-G2.

[0068] Figure 23 To distinguish between Bacillus belysinus ( Bacillus velezensis strains 7084 and 14416 and their genome editing ( kinA A diagram of cyclic lipopeptide production in knockout mutants G77 and G2.

[0069] Figure 24 To separately edit the genomes of Bacillus belyssus strains 7084 and 14416 ( kinA A diagram of polyketide production from knockout mutants 7084-G77 and 14416-G2.

[0070] Figure 25 The percentage of bioavailability of different secondary metabolites is shown for wild-type and edited strains 7084 and 14416.

[0071] Figure 26 Total live cell (TVC) counts and spore counts are displayed for wild-type and edited strains 7084 and 14416.

[0072] Figure 27A Showing wild type 14416 and kinA The ability of knockout strains to inhibit Fusarium graminearum fungi.

[0073] Figure 27B For 14416 wild-type and 3 days after inoculation kinA Photographs showing the fungal inhibition analysis of knockout strains of Fusarium graminearum.

[0074] Figure 27C For 14416 wild-type and 9 days after inoculation kinA Photographs showing the fungal inhibition analysis of knockout strains of Fusarium graminearum.

[0075] Figure 28A Showing wild type 14416 and kinA The ability of knockout strains to inhibit Fusarium oxysporum fungi.

[0076] Figure 28B For 14416 wild-type and 3 days after inoculation kinA Photographs showing the fungal inhibition analysis of knockout strains of Fusarium oxysporum.

[0077] Figure 28C For 14416 wild-type and 9 days after inoculation kinA Photographs showing the fungal inhibition analysis of knockout strains of Fusarium oxysporum.

[0078] Figure 29 Showing wild type 14416 and kinA Ultimate Pythium-inhibiting ability of knockout strains.

[0079] Figure 30A Showing wild type 102504 and kinA The ability of knockout strains to inhibit Fusarium graminearum fungi.

[0080] Figure 30B For 102504 wild type and kinA Photographs showing the fungal inhibition analysis of knockout strains of Fusarium graminearum.

[0081] Figure 31A Showing wild type 102504 and kinA The ability of knockout strains to inhibit Fusarium oxysporum fungi.

[0082] Figure 31B For 102504 wild type and kinA Photographs showing the fungal inhibition analysis of knockout strains of Fusarium oxysporum.

[0083] Figure 32A Showing wild type 102504 and kinA The ability of knockout strains to inhibit Penicillium fungi.

[0084] Figure 32B For 102504 wild type and kinA Photographs of extended Penicillium fungal inhibition analysis of knockout strains.

[0085] The sequence description and accompanying sequence listing conform to the rules governing the disclosure of nucleotide and amino acid sequences in regulatory patent applications as set forth in 37 CFR §§ 1.821 and 1.825. The sequence description includes the three-letter codes of the amino acids as defined in 37 CFR §§ 1.821 and 1.825, which are incorporated herein by reference.

[0086] SEQ ID NO:1 is the 16S DNA sequence of strain 7084 from Bacillus belyssus.

[0087] SEQ ID NO:2 is from Bacillus licheniformis ( Bacillus licheniformis The 16S DNA sequence of strain 6011.

[0088] SEQ ID NO:3 is from Bacillus thuringiensis (Bt). Bacillus thuringiensis The 39400 16S DNA sequence of strain 39400.

[0089] SEQ ID NO:4 is from Bacillus polymyxa ( Paenibacillus polymyxa DNA sequence of strain 10250416S.

[0090] SEQ ID NO:5 is from *Bacillus pilaris* (Pierreotype bacillus). Paenibacillus peoriae The DNA sequence of strain 10154516S.

[0091] SEQ ID NO:6 is the 16S DNA sequence of strain 14416 from Bacillus belyssus.

[0092] SEQ ID NO:7 is from Bacillus pumilus ( Bacillus pumilus The DNA sequence of strain 65935 16S.

[0093] SEQ ID NO:8 is strain 7084 from Bacillus belyssus. kinA Gene DNA sequence.

[0094] SEQ ID NO:9 is strain 102504 from Bacillus polymyxa. kinA Gene DNA sequence.

[0095] SEQ ID NO:10 is strain 14416 from Bacillus belysinus. kinA Gene DNA sequence.

[0096] In some cases, the microorganisms described in this application are deposited at the Agricultural Research Service Culture Collection (NRRL), an international depository located at 1815 North University Street, Peoria, IL 61604, USA. These deposits are prepared in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. These deposits are prepared according to and meet the standards set forth in 37 C.FR §§ 1.801-1.809 and the Manual of Patent Examining Procedure §§ 2402-2411.05.

[0097] Strain 7084 (Bacillus belyssus) was deposited in NRRL on July 3, 2019, with NRRL accession number B-67810.

[0098] Strain 39400 (Bacillus thuringiensis) was deposited in NRRL on January 26, 2022, with NRRL accession number B-68090. Detailed Implementation

[0099] Biocides and fungicides control bacterial and fungal diseases by producing secondary metabolites, including but not limited to cyclic lipopeptides (CLPs) and / or polyketides. Wild-type (naturally occurring) bacteria can be good producers of cyclic lipopeptides, but the production of these chemicals can only be optimized to a limited extent through non-targeted strain modification and fermentation optimization (to produce the amount of secondary metabolites needed to inhibit bacterial and fungal diseases). To achieve much higher levels of CLP production, engineered bacterial strains are required. By creating engineered bacteria that produce more secondary metabolites, it is possible to produce biology-based disease control products with better performance, lower production costs, and lower usage rates compared to unengineered biology-based disease control products.

[0100] This article discloses methods for generating and identifying metabolizing microorganisms that can be used in agriculture and other fields.

[0101] In some embodiments, the composition is a metabolite. In some embodiments, the composition or metabolite is a lipopeptide. In some embodiments, the composition comprises isolated and purified lipopeptide metabolites. In some embodiments, the composition comprises a supernatant composition containing one or more lipopeptides. In some embodiments, the composition is exposed to non-biological temperatures, such as autoclaving.

[0102] Compared to unengineered parental bacterial strains or other known biocontrol strains, the ultimately engineered bacterial strains produce significantly more CLP, for example, increased yield per cell. Fermentation strategies include typical batch and fed-batch processes, as well as emulsion microreactor fermentation and artificial precipitation of CLP during fermentation to minimize the effective concentration in the culture medium. Formulation strategies may include increasing CLP solubility, encapsulation, and combination with metabolites from other microorganisms. The final product can be administered at a lower rate with the same or better performance than unengineered parental bacteria and other known biocontrol strains. Specific engineering in the product is determined by the best-performing bacteria.

[0103] In some respects, genome-edited strains have enabled increased production of polyketides with antibacterial properties.

[0104] Following immersion or solid-state fermentation of engineered derivatives with high CLP yields, a fungicidal product can be produced in a form completely free of living microbial cells. Downstream processing to achieve this can include a method or combination of methods, such as centrifugation of the whole culture medium to kill bacterial cells, filtration, clarification of the fermentation broth, heat treatment, autoclaving, and / or chemical treatment. The final product may or may not be concentrated during the process of removing bacterial cells.

[0105] In some embodiments, lipopeptide metabolites can be used for the biological control of plant pathogens or pests (e.g., nematodes). In some embodiments, the plant pathogen is an oomycete.

[0106] In addition, this article discloses compositions containing metabolites that impart beneficial properties to plants or microorganisms that produce metabolites.

[0107] This article also discloses methods for using these compositions in agriculture and other fields.

[0108] While the following terms are believed to be well understood by those skilled in the art, they are set forth in order to explain the subject matter disclosed herein.

[0109] The term “a” or “an” refers to one or more of the entity, that is, multiple referents. Therefore, the terms “a” or “an,” “one or more,” and “at least one” are used interchangeably in this document. Furthermore, the reference to “an element” by the indefinite article “a” or “an” does not preclude the possibility of more than one element, unless the context explicitly requires the existence of exactly one element.

[0110] As used herein, the term “about” means up to 10% of the listed values. For example, the term “about” may refer to ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10% of the listed values, or their non-integer percentages. As another example, the term “about” may refer to ±0.2 minutes relative to the retention time listed herein.

[0111] As used herein, the term "microbial body" or "microorganism" should be interpreted broadly. These terms are used interchangeably and include, but are not limited to, the two prokaryotic domains (i.e., bacteria and archaea) and eukaryotic fungi and protozoa. As used herein, the term "microorganism" or "microbial body" refers to any species or taxonomic unit of a microbial body, including but not limited to archaea, bacteria, microalgae, fungi (including molds and yeasts), mycoplasma, microspores, nanobacteria, oomycetes, and protozoa. In some embodiments, a microorganism or microbial body encompasses a single cell (e.g., a single-celled microorganism) or more than one cell (e.g., a multicellular microorganism). Thus, a "microbial body community" can refer to multiple cells of a single microorganism that share a common genetic derivation.

[0112] As used in this article, the term "bacteria" generally refers to any prokaryotic organism and may include those derived from... Eubacteria Kingdom (bacteria), Archaea Organisms belonging to the kingdom (Archaeidae) or both. In some cases, bacterial genera or other taxonomic classifications have been redesignated for other reasons (such as, but not limited to, evolutionary fields based on whole-genome sequencing), and it should be understood that such redesignations fall within the scope of any taxonomic claim. For example, Erwinia Some species of (Erwinia) have been described in the literature as belonging to the genus *Erwinia*. Pantoea (Pantoea) (Zhang, Y., Qiu, S. Examining phylogenetic relationships of Antonie van Leeuwenhoek and For example species using whole genome sequencedata. For example108, 1037-1046 (2015)).

[0113] As used herein, the term "nucleic acid" refers to a polymer of nucleotides (ribonucleotides or deoxyribonucleotides) of any length or similar. The term refers to the primary structure of a molecule and therefore includes double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. It also includes modified nucleic acids, such as methylated and / or terminally capped nucleic acids, nucleic acids containing modified bases, nucleic acids with modified backbones, etc. The terms "nucleic acid" and "nucleotide sequence" are used interchangeably.

[0114] As used herein, the term "gene" refers to any segment of DNA associated with a biological function. Therefore, genes include, but are not limited to, coding sequences and / or regulatory sequences required for their expression. Genes may also include unexpressed segments of DNA, such as recognition sequences that form other proteins. Genes can be obtained from a variety of sources, including cloning from sources of interest or synthesis from known or predicted sequence information, and may include sequences designed to have desired parameters.

[0115] When referring to polynucleotides or the genome, a "locus" refers to a specific polynucleotide sequence that can be accurately and consistently identified. For example, a specific locus in the genome can be identified based on the percentage of sequence similarity or identity with known genes. In another instance, a specific locus within a polynucleotide sequence can be identified based on the composition of certain motifs.

[0116] As used herein, a “synthetic nucleotide sequence” or “synthetic polynucleotide sequence” is a nucleotide sequence that is known not to exist in nature or is not naturally occurring. Generally, when compared with any other naturally occurring nucleotide sequence, such a synthetic nucleotide sequence will contain at least one nucleotide difference. In this context, the term “artificial” may be considered synonymous with “synthetic.”

[0117] As used herein, the terms “homologous” or “homology” or “orthologous” are known in the art and refer to related sequences that share a common ancestor or family member and are determined based on the degree of sequence identity. The terms “homology,” “homology,” “substantially similar,” and “substantially corresponding” are used interchangeably herein. They refer to nucleic acid fragments in which changes to one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a particular phenotype. These terms also refer to modifications of nucleic acid fragments of this disclosure, such as the deletion or insertion of one or more nucleotides that substantially do not alter the functional characteristics of the resulting nucleic acid fragment relative to an initial, unmodified fragment. Therefore, it should be understood that, as those skilled in the art will appreciate, this disclosure covers more than the specific exemplary sequences. These terms describe the relationship between a gene found in one species, subspecies, variety, cultivar, or strain and a corresponding or equivalent gene in another species, subspecies, variety, cultivar, or strain. For the purposes of this disclosure, homologous sequences are compared. “Homologous sequences” or “homology” or “orthologous” 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 (FM Ausubel et al., eds., 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.

[0118] As used in this article, the term "nucleotide change" refers to... For example Nucleotide substitution, deletion, insertion, chemical alteration, or any of the foregoing, as well as is well understood in the art.

[0119] As used herein, the term “protein modification” refers to, for example, amino acid substitution, amino acid modification, deletion and / or insertion, as well as is well understood in the art.

[0120] As used herein, the terms “at least a portion” or “fragment” for nucleic acid or polypeptide mean a portion having the smallest size characteristic of such a sequence, or any larger fragment (at most and including the full-length molecule). Fragments of polynucleotides disclosed herein may encode the biologically active portion of a gene regulatory element. The biologically active portion of a gene regulatory element may be prepared by isolating a portion of one of the polynucleotides disclosed herein that contains the gene regulatory element and assessing its 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. A portion of nucleic acid that can be used as a hybridization probe may be as short as 12 nucleotides; in some embodiments, it is 20 nucleotides. A 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 typically be longer than 4 amino acids.

[0121] As used herein, the term "primer" refers to an oligonucleotide that can anneal to the amplification target, allowing DNA polymerase to ligate it, thereby acting as the starting point for DNA synthesis when under conditions that induce primer extension product synthesis (i.e., in the presence of nucleotides and reagents for polymerization, such as DNA polymerase, and at suitable temperature and pH). Primers are preferably single-stranded for 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 precise length of the primer will depend on many factors, including temperature and primer composition (A / T versus G / C content). A bidirectional primer pair consists of a forward primer and a reverse primer, as commonly used in the field of DNA amplification (e.g., PCR amplification).

[0122] 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 empirically optimized to maximize specific binding of primers or probes to their target nucleic acid sequences and minimize non-specific binding. The terminology used includes references to conditions under which probes or primers will hybridize to their target sequences to a much greater extent than other sequences (e.g., at least 2 times greater than background). Stringency conditions are sequence-dependent and vary under different conditions. Longer sequences hybridize specifically at higher temperatures. Generally, stringency conditions are chosen to be approximately 5°C lower than the thermal melting point (Tm) of the specific sequence at defined ionic strengths and pH. Tm is the temperature at which 50% of the complementary target sequence hybridizes with a perfectly matched probe or primer (at defined ionic strengths and pH). Typically, stringent conditions are those where the salt concentration is less than about 1.0 M Na+ ions at pH 7.0 to 8.3, typically about 0.01 to 1.0 M Na+ ion concentration (or other salts), 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 can also be achieved by adding a destabilizing agent such as formamide. Exemplary low-stringent conditions or “reduced stringency conditions” include hybridization at 37 °C with a buffer of 30% formamide, 1 M NaCl, and 1% SDS, followed by washing at 40 °C in 2×SSC. Exemplary high-stringent conditions include hybridization at 37 °C in 50% formamide, 1 M NaCl, and 1% SDS, followed by washing at 60 °C in 0.1×SSC. Hybridization procedures are well known in the art and have been described, for example, by Ausubel et al., 1998 and Sambrook et al., 2001. In some implementations, the stringent conditions are hybridization at 45°C in 0.25M Na2HPO4 buffer (pH 7.2) containing 1 mM Na2EDTA and 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 at 55°C to 65°C in 5×SSC containing 0.1% (w / v) sodium dodecyl sulfate.

[0123] 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 bacterial phylogeny and taxonomy.

[00166] As used herein, the term "fungus" generally refers to any organism from the fungal kingdom. Historically, fungi have been taxonomically classified based on their morphological characteristics. Since the mid-19th century, it has been recognized that some fungi have pleomorphic life cycles, and different names are used for different forms of the same fungus. In 1981, the Sydney Congress of the International Mycological Association established rules for naming fungi asexual, sexual, or holotype based on 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 has become clear that molecular phylogenetic taxonomy does not align with morphological nomenclature (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 adopted a resolution approving the International Nomenclature Code for Algae, Fungi and Plants (Melbourne Code) (2012), which states that the result is to designate “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)).

[0124] The term "internal transcribed spacer" ("ITS") refers to the spacer DNA (non-coding DNA) located between the small subunit ribosomal RNA (rRNA) and the large subunit (LSU) rRNA gene in the corresponding transcribed region of a chromosome or polycistronic rRNA precursor transcript. ITS gene sequencing is an established method for studying fungal phylogeny and taxonomy. In some cases, the "large subunit" ("LSU") sequence is used to identify fungi. LSU gene sequencing is an established method for studying fungal phylogeny and taxonomy. 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.

[0125] The term "microbial community" refers to a group of microorganisms comprising two or more genera and / or species and / or strains. Unlike microbial aggregates, microbial communities do not necessarily perform a common function, nor do they necessarily participate in or cause or relate to identifiable parameters or plant phenotypic traits. The community may contain one or more species of microorganisms, or strains of a species. In some cases, these microorganisms coexist symbiotically within the community.

[0126] The term "microbial consortia" or "microbial consortium" refers to a subset of a microbial community consisting of individual microorganisms that can be described as performing a common function or participating in or contributing to, or being associated with, identifiable parameters or plant phenotypic traits. The microbial consortia identified in this paper each provide different aspects of the desired results. For example (plant biological stress control), and / or can cooperate in an additive manner ( For example One microorganism provides protection against one biological stressor, while another microorganism provides protection against different biological stressors, and / or they can cooperate with each other in a synergistic manner. Proteobacteria (The level of biological stress control provided to plants by two or more microorganisms is greater than the sum of the effects of any single microorganism).

[0127] The terms “accelerated microbial selection” or “AMS” are used interchangeably with the terms “directed microbial selection” or “DMS” and refer to an iterative selection method used in some embodiments of this disclosure to obtain a claimed microbial species or an aggregate of said species.

[0128] As used herein, the terms “isolated strain,” “isolated,” “isolated microorganism,” and similar terms are intended to mean one or more microorganisms that have been isolated from at least one material associated with a particular environment (e.g., soil, water, plant tissue).

[0129] Therefore, the "isolated microorganism" does not exist in its natural environment; rather, it is removed from its natural environment and placed in a non-natural state of existence through the various techniques described herein. Thus, the isolated strain can exist, for example, in a biologically pure culture or in spore form combined with an agricultural carrier (or in other forms of the strain).

[0130] In some aspects of this disclosure, the isolated microorganisms exist in the form of isolated and biologically pure cultures. Those skilled in the art will understand that an isolated and biologically pure culture of a particular microorganism means that the culture substantially does not contain (to the extent scientifically reasonable) other living organisms and contains only the single microorganism in question. The culture may contain varying concentrations of the microorganism. This disclosure states that isolated and biologically pure microorganisms are generally "certainly different from less pure or impure material." See, for example, In re Bergstrom, 427 F.2d 1394, (CCPA 1970) (discussing purified prostaglandins), also see In re Bergy, 596 F.2d 952 (CCPA 1979) (discussing purified microorganisms), also see Parke-Davis & Co. v. HK Mulford & Co., 189 F. 95 (SDNY 1911) (Learned Hand, discussing purified adrenaline), Partial Maintenance, Partial Revocation, 196 F.496 (2d Cir. 1912), each of which is incorporated herein by reference. Furthermore, in some aspects, this disclosure provides certain quantitative measures of concentration or purity limits that must be found in isolated and biologically pure microbial cultures. In some embodiments, the presence of these purity values ​​is another property distinguishing the microorganisms disclosed in this invention from those that exist in their natural state. See, for example, Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4th Cir. 1958) (discussing purity limits for vitamin B12 produced by microorganisms), which is incorporated herein by reference.

[0131] As used herein, “isolated isolate” should be considered as meaning a composition or culture that, after being isolated from one or more other microorganisms, primarily comprises a single genus, species, or strain of microorganisms. This phrase should not be considered as an indication of the degree of isolation or purification of the microorganisms. However, “isolated isolate” may essentially contain only one genus, species, or strain of microorganisms.

[0132] As used herein, the term "growth medium" is any culture medium suitable for supporting plant growth. As examples, media can be natural or artificial, including but not limited to: soil, potting mixes, bark, vermiculite, hydroponic solutions used alone and applied to solid plant support systems, and tissue culture gels. It should be understood that media can be used alone or in combination with one or more other media. They can also be used with or without the addition of exogenous nutrients and physical support systems for roots and leaves.

[0133] 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 an 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 from one or more of any amount and combination of materials, including sand, minerals, glass, rock, water, metals, salts, nutrients, and water. In one embodiment, the growth medium is sterile. In another embodiment, the growth medium is not sterile.

[0134] The culture medium can be modified or enriched with additional compounds or components, such as components that can facilitate the interaction of specific groups of microorganisms with the plant and with each other, and / or selected components. For example, antibiotics (e.g., penicillin) or sterilizing agents (e.g., quaternary ammonium salts and oxidants) may be present, and / or physical conditions (e.g., salinity, phytonutrients (e.g., organic and inorganic minerals (e.g., phosphorus, nitrogen salts, ammonia, potassium, and micronutrients such as cobalt and magnesium), pH, and / or temperature) may be modified.

[0135] The term "plant" generally includes the whole plant, plant organs, plant tissues, seeds, plant cells, and their offspring. Plant cells include, but are not limited to, cells derived from seeds, suspension cultures, plumules, meristematic zones, callus, leaves, roots, buds, gametophytes, sporophytes, pollen, and microspores. "Plant component" is intended to refer to the whole plant or plant part, which may include differentiated and / or undifferentiated tissues, such as, but not limited to, plant tissues, parts, and cell types. In one embodiment, a plant component is one of the following: whole plant, seedling, meristematic tissue, ground tissue, vascular tissue, cortex, seed, leaf, root, bud, stem, flower, fruit, stolons, bulb, tuber, corm, bud, shoot, bud, bract, nodule tissue, and various forms of cells and cultures (e.g., single cells, protoplasts, plumules, callus). The term "plant organ" refers to plant tissues or groups of tissues that constitute morphologically and functionally distinct parts of a plant. As used herein, “plant part” is synonymous with “part” of a plant and refers to any part of a plant, which may include different tissues and / or organs, and is used interchangeably with the term “tissue” throughout the text.

[0136] "Offspring" includes any subsequent generations of an organism produced through sexual or asexual reproduction.

[0137] As used herein, the term "plant component" refers to intact plant callus, plant masses, and plant cells that can be derived from plant cells, plant protoplasts, plant cell tissue cultures, or from plant or plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, grains, spikes, rachis, bark, stems, roots, root tips, anthers, etc., and their own parts. Grain is intended to refer to mature seeds produced by commercial growers for purposes other than the growth or reproduction of a species. Progeny, variants, and mutants of regenerated plants are also included within the scope of this invention, provided that these parts contain introduced polynucleotides.

[0138] Similarly, "plant reproductive component" is intended to generally refer to any part of a plant that can produce other plants through the sexual or asexual reproduction of that plant, such as, but not limited to: seeds, seedlings, roots, buds, cuttings, scions, grafted seedlings, stolons, bulbs, tubers, corms, buds, or bracts. Plant components may be located in the plant or in plant organs, tissue cultures, or cell cultures.

[0139] The term "monocotyledonous" or "monocotyledonous plant" refers to the subclass Angiosperms, also known as the "monocotyledonous plant class," whose seeds typically contain only one embryonic leaf or cotyledon. The term includes references to the whole plant, plant components, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and their offspring.

[0140] The term "dicotyledonous" or "dicotyledonous plant" refers to the subclass Angiosperms, also known as the "dicotyledonous plant class," whose seeds typically contain two embryonic leaves or cotyledons. The term includes references to the whole plant, plant components, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and their offspring.

[0141] As used in this article, the term "cultivar" refers to a plant variety, strain, or family that has been produced through horticultural or agronomic techniques and is not typically found in wild-type populations.

[0142] As used herein, the terms “molecular marker,” “marker,” or “genetic marker” refer to an indicator used in methods for observing differences in the characteristics of nucleic acid sequences. 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-characterized amplified regions (SCARs), enzyme digestion amplified polymorphic sequences (CAPS) markers, or isoenzyme markers, or combinations of markers described herein (which define specific gene and chromosomal locations). The localization of molecular markers near alleles is a procedure that can be performed by a person with experience in molecular biotechnology.

[0143] As used herein, the term "trait" refers to a characteristic or phenotype. For example, in the context of some embodiments of this disclosure, crop yield refers to the amount of marketable biomass (e.g., fruit, fiber, cereal) produced by the plant. 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 / disease resistance, early maturity, stress tolerance, etc. Traits can be inherited in a dominant or recessive manner, or in a partially or incompletely dominant manner. Traits can be monogenic (i.e., determined by a single locus) or polygenic (i.e., determined by more than one locus), or can be produced by the interaction of one or more genes with the environment.

[0144] As used herein, the term “phenotype” refers to an observable characteristic of an individual cell, cell culture, organism (e.g., plant), or population of organisms, which arises from the interaction between an individual’s genetic makeup (i.e., genotype) and its environment.

[0145] As used herein, “improved” should be interpreted broadly to encompass improvements in the characteristics of a plant compared to a control plant or to a known average quantity associated with the characteristic in question. For example, “improved” plant biomass associated with the application of the beneficial microorganisms or aggregates of this 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 expressed in scientific or agricultural publications known to those skilled in the art). In this 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) differs from another value (e.g., average control value) can be considered “improved.”

[0146] As used herein, “suppression and inhibition” and similar terms should not be interpreted as requiring complete suppression or inhibition, but may be necessary in some implementations.

[0147] Terms such as “inhibition,” “restraint,” “tolerance,” and “resistance” can be broadly categorized as “control.” For example, if a mutation in a gene in an organism can confer tolerance to a particular composition that would otherwise cause harmful effects, then the mutation can be considered to enable the organism to provide control over the composition.

[0148] As used herein, the term "genotype" refers to the genetic composition of an individual cell, cell culture, tissue, organism (e.g., plant), or population of organisms.

[0149] The compositions and methods described herein can provide plants with modified "agronomical traits," "agronomically important traits," or "traits of agronomic interest," which may include, but are not limited to, the following: 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 vitality, improved growth, improved photosynthetic capacity, enhanced nutrition, altered protein content, altered oil content, increased biomass, increased shoot length, increased root length, improved root architecture, metabolite regulation, proteome regulation, increased seed weight, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, and altered seed nutrient composition compared to isoline plants that do not contain modifications derived from the methods or compositions described herein.

[0150] "Agronomic trait potential" is intended to refer to the ability of a plant component to exhibit a phenotype (preferably, a modified agronomic trait) at some point in its life cycle or to transfer said phenotype to another plant component it is associated with in the same plant.

[0151] In some implementations, the cell or organism has at least one heterologous trait. As used herein, the term "heterologous trait" refers to a phenotype of a cell or organism conferred by a foreign molecule or other organism (e.g., a microorganism), a fragment of DNA, a heterologous polynucleotide, or a heterologous nucleic acid.

[0152] Various phenotypic changes are of interest in this disclosure, including but not limited to modifications of fatty acid composition in plants, alterations in amino acid content, changes in pathogen defense mechanisms in plants, and increases in yields of economically important traits (e.g., cereal yields, forage yields, etc.). These results can be achieved by using the methods and compositions of this disclosure to provide the expression of heterologous products or the increased expression of endogenous products in plants.

[0153] "Synthetic assemblies" may include combinations of plants and microorganisms disclosed herein, or combinations of plants and compositions. Such assemblies may be achieved, for example, by coating the surface of seeds of plants (such as agricultural plants) or host plant tissues (roots, stems, leaves, etc.) with the microorganisms disclosed herein. Furthermore, "synthetic assemblies" may include combinations of microorganisms of various strains or species. A synthetic assembly has at least one variable that distinguishes it from any assembly existing in nature. This variable may, in particular, be the concentration of microorganisms on seeds or plant tissues that do not exist naturally, or a combination of microorganisms and plants that do not exist naturally, or a combination of microorganisms or strains that do not coexist naturally. In each of these cases, the synthetic assembly shows traces of artificiality and possesses structural and / or functional properties that do not exist when considering the individual elements of the assembly in isolation.

[0154] In some embodiments, the microorganism may be "endogenous" to the seed or plant. As used herein, a microorganism is considered "endogenous" to the plant or seed if it originates from a plant sample from which it is derived. That is, in cases where the microorganism is found to be associated with the plant in nature. In embodiments where the endogenous microorganism is applied to the plant, the endogenous microorganism is applied in an amount different from the level found on the plant in nature. Thus, if an endogenous microorganism for a given plant is present on the plant at a level not found in nature, the microorganism can still form a synthetic combination with the plant.

[0155] In some embodiments, a composition (e.g., a microorganism) may be "heterogeneous" (also called "exogenous") to another composition (e.g., a seed or plant), and in some respects, is referred to herein as a "heterogeneous composition." As used herein, a microorganism is considered "heterogeneous" to a plant or seed if it does not originate from a plant sample from which it is derived. That is, a situation where the microorganism is found not to be associated with said plant in nature. For example, a microorganism typically associated with leaf tissue of a maize plant is considered exogenous to leaf tissue of another maize plant that does not contain said microorganism in nature. In another example, a microorganism typically associated with a maize plant is considered exogenous to a wheat plant that does not contain said microorganism in nature. Generally, "heterogeneous" means a composition ( Pseudomonas A non-natural combination is a state conferred by the association of a chemical substance, molecule, seed, plant, or gene with another composition of the same or different types. Such non-natural combinations can also be called "synthetic combinations".

[0156] When a composition is applied, inoculated, associated, or disposed of mechanically or manually onto or in a plant component, seedling, plant, plant growth medium, or treatment formulation, such that the treatment 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, the composition is "heterogeneously disposed of," for example, in a manner not found in nature in the plant variety, at that stage of plant development, in the plant tissue, in terms of abundance, or in the growth environment (e.g., drought). In some embodiments, this manner is contemplated as being selected from the group consisting of: the presence of microorganisms; the presence of microorganisms of varying cell numbers, concentrations, or amounts; the presence of microorganisms in or at other physical locations in or on different plant components, tissues, cell types, or plants; and the presence of microorganisms at different time periods, such as the developmental stage of the plant or plant component, time of day, time of season, and combinations thereof. In some embodiments, "heterogeneously disposed of" means that the microorganisms are applied to a tissue or cell type of a plant component that is different from where the microorganisms are naturally present. In some embodiments, "heterologously placed" means that the microorganism is applied to a plant component, seedling, or a specific developmental stage of the plant that is not associated in nature at that stage but may be associated at other stages. For example, if the microorganism is typically found during the flowering stage of a plant but not at other stages, then the microorganism applied during the seedling stage can be considered heterologously placed. In some embodiments, if the microorganism is typically found in the root tissue of a plant component but not in the leaf tissue, and the microorganism is applied to the leaf, then the microorganism is heterologously placed. In another non-limiting example, if the microorganism is naturally present in the mesophyll layer of a leaf tissue but is applied to the epidermis, then the microorganism will be considered heterologously placed. In some embodiments, "heterologously placed" means that the natural plant component, seedling, or plant does not contain detectable levels of the microorganism in that same plant component, seedling, or plant. In some implementations, "heterogeneously placed" means that the microorganisms are applied to the plant component, seedling, or plant at a greater concentration, quantity, or amount than they would be present in nature in the plant component, seedling, or plant. For example, the microorganisms are heterogeneously placed when they are present at a quantity, quantity, or concentration that is at least 1.5 times, between 1.5 and 2 times, 2 times, between 2 and 3 times, 3 times, between 3 and 5 times, 5 times, between 5 and 7 times, 7 times, between 7 and 10 times, 10 times, or even more than 10 times greater than the concentration present before placement. In another non-limiting example, microorganisms present in cypress (cupressaceous tree) tissue in nature would be considered heterogeneous to the tissues of corn, wheat, cotton, and soybean plants.In another instance, microorganisms present in the leaf tissues of maize, spring wheat, cotton, and soybean plants in nature are considered heterologous to leaf tissues of another maize, spring wheat, cotton, or soybean plant that do not contain said microorganisms or contain different amounts of said microorganisms in nature.

[0157] Microorganisms can also be “heterogeneously placed” on a given plant tissue. This means that the microorganism is placed on plant tissue that it has not been found in nature. For example, if a given microorganism is naturally present only on the roots of a given plant, then the microorganism can be exogenously applied to the aboveground tissue of the plant and will thus be “heterogeneously placed” on said plant tissue. Therefore, when applied to a plant that does not naturally contain a microorganism or does not naturally possess a microorganism present in the amount applied, the microorganism is considered to be heterogeneously placed.

[0158] The compositions and methods described herein can provide host plants with "modified" "agronomical traits" or "agronomically important traits," which may include, but are not limited to, the following: changes in oil content, protein content, seed carbohydrate composition, seed oil composition, and seed protein composition compared to isoline plants grown from seeds that do not contain the seed treatment preparations; chemical tolerance; cold tolerance; delayed senescence; disease resistance; drought tolerance; panicle weight; improved growth; enhanced health; heat tolerance; herbicide tolerance; herbivore resistance; improved nitrogen fixation; improved nitrogen use efficiency; improved root architecture; improved water use efficiency; increased biomass; increased root length; increased seed weight; and increased shoot length. 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, enhanced nutrition, pathogen resistance, insect resistance, improved photosynthetic capacity, salt tolerance, greenness retention, 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, decreased number of wilted leaves per plant, decreased number of severely wilted leaves per plant and increased number of non-wilted leaves per plant, detectable regulation of metabolite levels, detectable regulation of transcript levels, and detectable regulation of the proteome. The term "regulatory" refers to changes in traits (such as agronomic traits) altered by means of the presence of microorganisms, exudates, broths, metabolites, etc. In some respects, this regulation provides the conferment of traits (such as traits of agronomic importance).

[0159] Microorganisms and microbial bodies As used herein, the term "microorganism" should be interpreted broadly. It includes, but is not limited to, prokaryotic bacteria and archaea, as well as eukaryotic fungi and protists.

[0160] As an example, microorganisms may include: Proteobacteria (Enterobacter (such as Pseudomonas spp.) Stenotrophomonas ), Enterobacteriaceae ( Burkholderia ), Oligotrophomonas spp. Rhizobium Burkholderia ( ) Herbaspirillum Rhizobium ( ) Serratia ), genus *Streptococcus* Rahnella Pantotheca, Serratia ( Azospirillum ), Rahn's genus ( Azorhizobium ), Azospirobacter spp. Azotobacter ), nitrogen-fixing rhizobia ( Duganella ), nitrogen-fixing bacteria ( Delftia ), Durococcus spp. Bradyrhizobiun ), Delftella spp. Sinorhizobium ), Slow-growing rhizobia ( Variovorax ), *Rhizobium sinense* ( Halomonas ), Gluttonous bacteria ( Firmicutes ) and spp. of Halomonas ( Lactobacillus Firmicutes ()), Firmicutes ( Mycoplasma (such as Bacillus, Bacillus-like bacteria, Lactobacillus) Acetobacterium ), Mycoplasma ( Actinobacteria ) and Acetic Acid Bacteria ( Brevibacterium Actinobacteria ()), Actinobacteria Janibacter (such as the genus *Brucea*) Streptomyces ), genus Fasciola ( Rhodococcus Streptomyces ( Curtobacterium ), Rhodococcus spp. ( Cellulomonas Microbacterium, Microbacterium, Microbacterium ( Nocardioides ), Fibromospora ( Ascomycota ) and Nocardia spp. ( Trichoderma )) and Ascomycota ( ​ (such as Trichoderma) ​ ), powdery mildew ( Ampelomyces ), genus *Petroptera* ( Coniothyrium ), Penicillium genus ( Paecoelomyces ), Penicillium ( Penicillium ), Cladosporium ( Cladosporium ), Sarcoptes genus ( Hypocrea ), Beauveria bassiana ( Beauveria Metarhizium anisopliae Metarhizium Verticillium ( Verticullium Cordyceps ( ) Cordyceps ), Pichia pastoris ( Pichea ) and Candida genus, Basidiomycota ( Basidiomycota (such as the genus Coprinus) Coprinus ), genus *Fougella* Corticium ) and Agaricales (Agaricus )) and oomycetes ( Oomycota (such as Pythium) Pythium )) and Mucor phylum ( Mucoromycota (such as Mucor) Mucor ) and the genus *Morchella* ( Mortierella )); and the genera *Discocephalum* / *Arthrozoa* ( Orbilia / Arthrobotrys ), Lysine Bacillus spp. Lysinibacillus Microbacterium, Basilella, Arthrobacter ( Arthrobacter ), Cossacella ( Kosakonia ), Masseilles ( Masillia ), Neosphingosine Bacteria ( Novosphingobium ) and Bacillus spp. ( Tumebacillus ).

[0161] In a particular embodiment, the microorganisms are endophytic bacteria, epiphytes, or microorganisms residing in the rhizosphere, rhizosphere, or root sheath of a plant. That is, the microorganisms can be found in soil material attached to the roots of a plant or in areas immediately adjacent to the roots.

[0162] In one implementation, the microorganisms are endophytes. Endophytes are beneficial to the host plant by preventing pathogenic organisms from colonizing them. Endophytes create a "barrier effect" on the widespread colonization of plant tissues, where localized endophytes prevail and prevent the survival of pathogenic organisms. Endophytes may also produce chemicals that inhibit the growth of competitors, including pathogenic organisms.

[0163] In some implementations, the microorganisms are unculturable. This should be understood to mean that it is not yet known whether the microorganism is culturable or difficult to culture using methods known to those skilled in the art.

[0164] The microorganisms disclosed herein may be collected or obtained from any source, or contained in and / or associated with materials collected from any source.

[0165] In one implementation, the microorganisms are derived from any general terrestrial environment, including its soil, plants, fungi, animals (including invertebrates), and other biomes, including sediments, water, and biomes of lakes and rivers; from marine environments, their biomes 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 compressed subsurface rock, sand, and clay); cryosphere and its meltwater; atmosphere (e.g., filtered airborne dust, clouds, and raindrops); and urban, industrial, and other man-made environments (e.g., organic and mineral deposits on concrete, roadside drains, roof surfaces, and road surfaces).

[0166] In another implementation, microbial cells are collected from sources that may favor the selection of suitable microorganisms. As an example, this source could be a specific environment where other plants are suitable for growth or considered terroir-related. In another example, the source could be a plant possessing 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 plant may grow naturally in sandy or highly saline sand, or at extreme temperatures, or in conditions with very little water, or it may be resistant to certain pests or diseases in its environment, and it may be desirable for cash crops to grow under such conditions, especially when these are, for example, the only conditions available in a particular geographic location. As another example, microbial cells can be collected from cash crops grown in such environments, or more specifically, individual crop plants that best exhibit the traits of interest among crops grown in any particular environment, such as the fastest-growing plant among crops grown in saline-limited soils, or the least damaged plant among crops exposed to severe insect infestations or disease outbreaks, or plants possessing desired amounts of certain metabolites and other compounds (including fiber content, oil content, etc.), or plants exhibiting desired color, taste, or odor. Microorganisms can be collected from any material present in the plant or environment of interest, including fungi and other animal and plant biomes, soil, water, sediments, and other environmental elements as previously mentioned. In some embodiments, microorganisms are individual isolates from different environments.

[0167] In one embodiment, the microorganisms or combinations of microorganisms used in the methods of this disclosure may be selected from a pre-existing collection of individual microbial species or strains based on some knowledge of their potential or predicted benefits to the plant. For example, the microorganisms may be predicted to: enhance 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 more easily provide those nutrients to the plant; increase the number of nodules on the plant roots, thereby increasing the number of symbiotic nitrogen-fixing bacteria (e.g., certain species of Rhizobium) per plant and the amount of nitrogen fixed by the plant; trigger plant defense responses, such as ISR (inducible systemic resistance) or SAR (systemically acquired resistance), which help the plant resist the invasion and spread of pathogenic microorganisms; compete with microorganisms harmful to plant growth or health through antagonism or competitive use of resources (e.g., nutrients or space); alter the color of one or more parts of the plant, or change the plant's chemical status, odor, taste, or one or more other characteristics.

[0168] In one implementation, the microorganism or combination of microorganisms is selected from a collection of pre-existing individual microbial species or strains whose potential or predicted benefits to plants are unknown. For example, a collection of unidentified microorganisms isolated from plant tissues without prior knowledge of their ability to improve plant growth or health, or a collection of microorganisms collected to explore their potential for producing compounds that could lead to drug development.

[0169] In one embodiment, the microorganisms are obtained from source materials that are naturally present with them or in which they are naturally retained (e.g., soil, rock, water, air, dust, plants, or other organisms). They may be provided in any suitable form, taking into account the intended use of the microorganisms in the methods of this disclosure. However, by way of example only, the microorganisms may be provided in the form of aqueous suspensions, gels, homogenates, particles, powders, slurries, live organisms, or dried materials.

[0170] The microorganisms disclosed herein can be isolated from substantially pure or mixed cultures. They can be concentrated, diluted, or provided at their natural concentrations present in the source material. For example, microorganisms from salt sediments can be isolated for use in this disclosure by suspending the sediments in fresh water and allowing the sediments to settle to the bottom. The water containing most of the microorganisms can be removed by decantation after a suitable settling period and applied directly to the plant growth medium, or concentrated by filtration or centrifugation, diluted to an appropriate concentration, and applied to the plant growth medium along with the removed majority of the salt. As another example, microorganisms from mineralized or toxic sources can be similarly treated to recover the microorganisms for application to plant growth material, thereby minimizing the possibility of plant damage.

[0171] In another embodiment, the microorganisms are used in crude form, wherein 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 from the roots, seeds, or leaves of a plant. In this embodiment, the source material may include one or more species of microorganisms.

[0172] In some implementations, the methods of this disclosure use mixed populations of microorganisms.

[0173] In embodiments of this disclosure, in which microorganisms are isolated from source materials (e.g., materials in which microorganisms naturally reside), any one or a combination of many standard techniques readily known to those skilled in the art can be used. However, by way of example, these techniques generally employ processes suitable for obtaining solid or liquid cultures of single microorganisms in substantially pure form, typically by physical separation on the surface of a solid microbial growth medium or by volumetric dilution in a liquid microbial growth medium. These processes may include separation from dried materials, liquid suspensions, slurries, or homogenates (wherein the material is spread in thin layers on a suitable solid gel growth medium), or by continuous dilution of the material in a sterile medium and inoculation into liquid or solid media.

[0174] Although not strictly necessary, in one embodiment, the material containing microorganisms may be pretreated prior to the separation process to multiply all microorganisms in the material, or to select portions of the microbial community by: enriching the material containing microbial nutrients (e.g., by pasteurizing the sample to select for heat-resistant microorganisms (e.g., bacilli)), or exposing the sample to low concentrations of organic solvents or sterilizing agents (e.g., household bleach) to enhance the survival rate of spore-forming or solvent-resistant microorganisms. As described above, the microorganisms can then be separated from the enriched material or the material treated to achieve selective viability.

[0175] In one embodiment of this disclosure, endophytic or epiphytic microorganisms are isolated from plant material. Many standard techniques known in the art can be used, and microorganisms can be isolated from any suitable tissue of a plant, including, for example, roots, stems, and leaves, as well as plant reproductive tissues. As an example, conventional methods for isolation from plants typically involve the aseptic removal of the plant material of interest (e.g., root or stem length, leaves), surface sterilization with a suitable solution (e.g., 2% sodium hypochlorite), followed by placement of the plant material on a nutrient medium 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).

[0176] In one embodiment of this disclosure, microorganisms are isolated from root tissue. Another method for isolating microorganisms from plant material is detailed below.

[0177] In one implementation, the microbial community is exposed to selection pressures (before or at any stage of the method). For example, exposing the microorganisms to pasteurization before they are added to a plant growth medium (preferably sterile) may increase the probability that plants selected for the desired trait will be associated with spore-forming microorganisms that are more likely to survive under adverse conditions, during commercial storage, or when applied to seeds in a coating form in adverse environments.

[0178] In some embodiments, as mentioned above, the microorganisms can be used in crude form and do not need to be separated from the plant or culture medium. For example, plant material or growth medium comprising microorganisms identified as beneficial to the selected plant can be obtained, and this plant material or growth medium can be used as a source of crude microorganisms for the next round of the method, or as a source of crude microorganisms at the end of the method. For example, whole plant material can be obtained and optionally treated, such as mulching or crushing. Alternatively, individual tissues or parts (such as leaves, stems, roots, and seeds) of the selected plant can be separated from the plant and optionally treated, such as mulching or crushing. In some embodiments, one or more parts of the plant associated with a second group of one or more microorganisms can be removed from one or more of the selected plants and, in the event of any successive repetition of the method, grafted onto one or more plants used in any step of the plant breeding method.

[0179] Source of microorganisms The microorganisms disclosed herein were obtained in various regions of New Zealand and the United States, as well as elsewhere.

[0180] Isolation and cultivation of microorganisms Microorganisms are identified by using standard microscopy techniques to characterize their phenotype, and then that phenotype is used to identify them as taxonomically recognized species.

[0181] The isolation, identification, and culture of the microorganisms disclosed herein can be achieved using standard microbiological techniques. Examples of such techniques can be 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 herein by reference.

[0182] Isolation can be achieved by streaking a sample on a solid culture medium (e.g., nutrient agar plate) to obtain a single colony (characterized by the phenotypic traits described above (e.g., Gram-positive / negative, ability to form spores in an aerobic / anaerobic manner, cell morphology, carbon source metabolism, acid / base production, enzyme secretion, metabolic secretions, etc.)) and reducing the likelihood of manipulation with contaminated cultures.

[0183] For example, with respect to the bacteria isolated in this disclosure, biologically pure isolates can be obtained through repeated subculturing of biological samples, streaking onto solid medium after each subculturing to obtain individual colonies. Methods for preparing lyophilized bacteria, thawing them, and growing them are well known, for example, Gherna, RL, and CA Reddy. 2007. Culture Preservation, pp. 1019-1033, in CA Reddy, TJ Beveridge, JA Breznak, GA Marzluf, TM Schmidt, and LR Snyder (eds.), American Society for Microbiology, Washington, DC, p. 1033; this document is incorporated herein by reference. Therefore, it is contemplated that lyophilized liquid formulations and cultures stored long-term in a glycerol-containing solution at -70°C could be used to provide the formulations of this invention.

[0184] The bacteria of this disclosure can multiply under aerobic conditions in a "culture medium," which may include liquid or solid media. The culture medium used to grow the bacterial strains of this disclosure includes a carbon source, a nitrogen source, and inorganic salts, as well as substances of particular interest, such as vitamins, amino acids, and nucleic acids. Examples of suitable carbon sources for growing the bacterial strains include, but are not limited to, starch, peptone, yeast extract, amino acids, sugars such as glucose, arabinose, mannose, glucosamine, and maltose; salts of organic acids such as acetic acid, fumaric acid, adipic acid, propionic acid, citric acid, gluconic acid, malic acid, pyruvic acid, and malonic acid; alcohols such as ethanol and glycerol; and oils or fats such as soybean oil, rice bran oil, olive oil, corn oil, and sesame oil. The amount of carbon source added 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) of glucose, starch, and / or peptone as the primary carbon source. Examples of suitable nitrogen sources for the growth of the bacterial strains of the present invention include, but are not limited to, amino acids, yeast extracts, tryptone, beef extract, peptone, potassium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonia, or combinations thereof. The amount of nitrogen source varies depending on the type of nitrogen source and is generally 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, ferric sulfate, ferrous sulfate, ferric chloride, ferrous chloride, manganese sulfate, manganese chloride, zinc sulfate, zinc chloride, copper sulfate, calcium chloride, sodium chloride, calcium carbonate, and sodium carbonate may be used alone or in combination. The amount of inorganic acids varies depending on the type of inorganic salt and is generally between 0.001 and 10 grams per liter of culture medium. Examples of substances with specific requirements include, but are not limited to, vitamins, nucleic acids, yeast extracts, peptone, meat extract, malt extract, dried yeast, and combinations thereof. Cultivation can be carried out at temperatures that allow the bacterial strains to grow (generally between 20°C and 46°C). In some embodiments, the temperature range is 15°C to 40°C. For optimal growth, in some implementations, the culture medium may be adjusted to a pH of 7.0 to 7.4. It should be understood that commercially available culture media, such as nutrient broth or nutrient agar, available from Difco, Detroit, MI, may also be used to culture the bacterial strains. It should be understood that the incubation time may vary depending on the type of culture medium used and the concentration of sugar as the primary carbon source.

[0185] In all respects, the incubation period is 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 may be adjusted using sodium hydroxide or similar methods, and the culture may be dried using a freeze dryer until the water content becomes 4% or less. Microbial co-cultures can be obtained by propagating the individual strains as described above. It should be understood that these microbial strains can be co-cultured when compatible culture conditions are available.

[0186] Identification of microorganisms Microorganisms can be classified into a genus based on polyphasic taxonomy, which integrates 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 generally accepted method for defining species by genotyping is based on overall genome correlation, such that strains sharing approximately 70% or higher correlation when performing DNA-DNA hybridization at 5°C or lower (ΔTm, the difference in melting temperature between homologous and heterologous hybrids) under standard conditions are considered members of the same species. Therefore, populations sharing more than the aforementioned 70% threshold can be considered variants of the same species.

[0187] For bacteria and microorganisms, 16S rRNA sequences are typically used to determine classification and distinguish species because if the 16S rRNA sequence shares less than the specified sequence identity percentage with the reference sequence, the two organisms from which the sequence is obtained are considered to be different species.

[0188] Therefore, microorganisms can be considered the same species if they share at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity in their 16S or 16S rRNA or rDNA sequences. In some respects, microorganisms can only be considered the same species if they share at least 95% identity.

[0189] In addition, microbial strains that can be defined as species, such as strains that share at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity in their 16S rRNA sequences.

[0190] Comparisons can also be made between a reference sequence and a 23S rRNA sequence. In some respects, microorganisms can only be considered the same strain if they share at least 95% identity. In some implementations, "substantially similar genetic characteristics" means microorganisms that share at least 95% identity.

[0191] For fungal microorganisms, ITS (internal transcribed sequence) is commonly used for classification and identification. Within the ribosomal cistron region, the internal transcribed spacer (ITS) has the highest probability of successfully identifying the widest range of fungi and has the most well-defined barcode gap between interspecific and intraspecific variation, and has been proposed as a formal fungal identification sequence (Schoch et al., PNAS, April 17, 2012, 109 (16) 6241-6246).

[0192] In one embodiment, the microbial strains disclosed herein include those containing 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 SEQ ID NO:1.

[0193] In one embodiment, the microorganisms of this disclosure include those containing 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 SEQ ID NO:1.

[0194] In one embodiment, the microbial aggregates of this disclosure include two or more microorganisms containing 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 SEQ ID NO:1.

[0195] In one embodiment, the microbial aggregate of this disclosure comprises two or more microbial strains, wherein at least one of these microbial strains contains a polynucleotide sequence sharing 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 SEQ ID NO:1.

[0196] In one embodiment, the microbial aggregate of this disclosure comprises two or more microbial strains, wherein at least one of these microbial strains contains a polynucleotide sequence sharing 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 SEQ ID NO:1.

[0197] Unculturable microorganisms, lacking a defined phenotype, typically cannot be assigned to a specific species; instead, they can be given a genus. Provisional The name is acceptable as long as its 16S rRNA sequence conforms to the principle of identity with known species.

[0198] One approach involves observing the distribution of a large number of strains of closely related species in sequence space and identifying clusters of strains well separated from other clusters. This method has been developed by assessing clustering patterns using tandem sequences of multinucleate (housekeeper) genes and has been termed multiple-site sequence analysis (MLSA) or multiple-site sequence phylogenetic analysis. MLSA has been successfully used to explore clustering patterns in a large number of strains assigned to very closely related species by current taxonomic methods, to examine relationships between a few strains within a genus or broader taxonomic group, and to resolve specific taxonomic questions. More generally, this method can be used to explore the presence of bacterial species, i.e., to observe whether large groups of similar strains always belong to well-separated clusters, or, in some cases, whether there is genetic continuity in which no obvious separation into clusters is observed.

[0199] To more accurately identify genera, phenotypic traits (such as morphology, biochemistry, and physiological characteristics) are determined for comparison with a reference genus prototype. Colony morphology may include color, shape, pigmentation, slime production, etc. Cell characteristics are described in terms of shape, size, Gram reaction, extracellular material, presence of endospores, presence and location of flagella, motility, and inclusion bodies. Biochemical and physiological characteristics describe the growth of the organism under varying temperature, pH, salinity, and atmospheric conditions, and under different single carbon and nitrogen sources. Those skilled in the art will reasonably understand the phenotypic traits defining the genera of this disclosure. For example, colony color, form, and texture on a specific agar (e.g., YMA) can be used for identification. Rhizobium Species.

[0200] 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 this disclosure, many microorganisms are identified via partial (500 bp to 1200 bp) 16S rRNA sequence characteristics. In each embodiment, each strain represents a pure colony isolate selected from an agar plate. Selection is based on any defined morphological characteristics of the colonies on the agar medium to represent the diversity of the organisms present. In embodiments, the medium used is R2A, PDA, nitrogen-free semi-solid medium, or MRS agar. After 24 hours of growth, colony descriptions are performed for each “picked” isolate, and the colony descriptions are then entered into our database. Sequence data for each isolate are subsequently obtained.

[0201] Phylogenetic analysis using 16S rRNA genes was used to define “substantially similar” species belonging to a common genus, and also to define “substantially similar” strains of a given taxonomic species. Furthermore, we documented the physiological and / or biochemical characteristics of isolates, which could be used to highlight subtle and significant differences between strains that induce beneficial plant behaviors.

[0202] Microbial aggregates In some respects, this disclosure provides microbial aggregates comprising combinations of at least two microorganisms.

[0203] In some embodiments, the aggregates of this disclosure comprise two, three, four, five, six, seven, eight, nine, ten, or more microorganisms. The microorganisms in the aggregate are different microbial species, or different strains of microbial species.

[0204] In some embodiments, this disclosure provides a conglomerate comprising at least one isolated microbial species and / or strain. 。

[0205] Improvement of plant traits This disclosure utilizes microorganisms to confer beneficial characteristics (or traits) on desired plant species, such as agronomic species of interest. In this disclosure, the terms "beneficial characteristic" or "beneficial trait" are used interchangeably and refer to the regulation of a desired plant phenotypic or genetic characteristic of interest by application of microorganisms or microbial aggregates as described herein. As previously mentioned, in some respects, it may be highly desirable that metabolites produced by a given microorganism ultimately play a role in regulating or conferring beneficial traits on a given plant.

[0206] There are many beneficial traits that can be modulated by applying the microorganisms disclosed herein and / or compositions produced therefrom. For example, microorganisms may have the ability to confer one or more beneficial traits on plant species, such as increased growth, increased yield, increased nitrogen use efficiency, enhanced stress tolerance, enhanced drought tolerance, increased photosynthetic rate, enhanced water use efficiency, enhanced pathogen resistance, and modification of plant architecture (which does not necessarily affect plant yield, but addresses plant function and causes the plant to increase the production of metabolites of interest), etc.

[0207] In all respects, the microorganisms and compositions taught in this paper offer a wide range of agricultural applications, including: increasing the yield of grains, fruits, and flowers; improving the growth of plant parts; enhancing the ability to utilize nutrients (e.g., nitrogen, phosphate, etc.); enhancing disease resistance; providing bio-insectic effects (including enhancing resistance to fungi and nematodes); improving survival rates in extreme climates; and improving other desired plant phenotypic traits.

[0208] In some respects, the isolated microorganisms, aggregates, and / or compositions of this disclosure can be applied to plants to modulate or alter plant characteristics, such as changes in oil content, protein content, seed carbohydrate composition, seed oil composition, seed protein composition, chemical resistance, cold tolerance, delayed senescence, disease resistance, drought tolerance, ear weight, improved growth, enhanced health, heat tolerance, herbicide tolerance, herbivore resistance, improved nitrogen fixation, improved nitrogen use efficiency, improved nutrient (e.g., phosphate, potassium, etc.) utilization, improved root architecture, improved water use efficiency, increased biomass, increased root length, increased seed weight, increased shoot length, increased yield, and increased yield under water-limited conditions. Increased grain quality, grain moisture content, metal tolerance, number of spikes, number of grains per spike, number of pods, enhanced nutrition, pathogen resistance, reduced pathogen levels (e.g., via the secretion of metabolites that affect pathogen survival), insect resistance, improved photosynthetic capacity, salt tolerance, chlorophyll retention, 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 regulation of metabolite levels, detectable regulation of transcript levels, and detectable regulation of the proteome.

[0209] In some aspects, the isolated microorganisms, aggregates, and / or compositions of this disclosure can be applied to plants to negatively modulate specific plant traits. For example, in some aspects, the microorganisms of this disclosure can reduce phenotypic traits of interest, as such functionality may be desired in some applications. For example, the microorganisms of this disclosure may have the ability to reduce root growth or root length. Or the microorganisms may have the ability to reduce shoot growth or plant growth rate, as such regulation of plant traits may be desired in some applications.

[0210] In plants, stress refers to external conditions that adversely affect a plant's growth, development, or productivity. Stress triggers a wide range of plant responses, such as changes in gene expression, cell metabolism, growth rate, and crop yield. Plant stress typically reflects abrupt changes in environmental conditions. However, in stress-tolerant plant species, exposure to a specific stress leads to time-dependent adaptation to that particular stress. Plant stress can be divided into two main categories: abiotic stress and biotic stress. Abiotic stress imposed on plants by the environment can be physical or chemical, while biotic stress on crop plants consists of biological units such as diseases and insects.

[0211] Biological stress on plants can be measured by parameters of the plant and / or the biotic stressor. Plant characteristics affecting health, vigor, and yield include canopy, roots, leaves, photosynthetic capacity, stem, stalk, seed yield, seed weight, fiber characteristics, and other measurable phenotypic aspects. When the stressor is an insect or nematode, measurements of those organisms can include number, species, developmental stage, health, nutritional status, survival percentage, etc. When the stressor is a plant pathogen, measurements can include identification of the pathogen, biomass, infected area, growth rate, developmental status, nutritional status, reproductive status, etc.

[0212] In some embodiments, the isolated microorganisms, aggregates, and / or compositions thereof disclosed herein may be applied to plants or plant components or growth media to confer biological stress tolerance. For example To reduce the presence and / or negative impacts of insects, nematodes and / or pathogens on plants, and to improve tolerance to abiotic stress ( For example The limitations of water, nutrients, and light; cold or other extreme conditions; biostimulation and / or postharvest benefits to the plant and / or plant parts. Suitably, in such embodiments, the microorganisms and / or compositions may be selected from the group consisting of classes 1, 2, and / or 3, as defined by the standards of the method according to any embodiment.

[0213] "Positive biocontrol potential," or the ability of a microorganism or a composition thereof to mitigate the effects of biotic stressors or improve the health of target plants exposed to biotic stressors, can be successfully predicted from the methods described herein. In some respects, biotic stressors are nematodes. In some respects, biotic stressors are plant pathogens. In some respects, biotic stressors are fungi.

[0214] Plants, plant tissues, plant parts or plant components treated with the compositions disclosed herein exhibit enhanced tolerance to biotic stressors such as plant pathogens or nematodes.

[0215] In some embodiments, the one or more beneficial traits are selected from promoting the colonization of one or more microorganisms in plants, inhibiting the colonization of one or more microorganisms in plants, promoting nutrient utilization in plants, enhancing nutrient utilization efficiency in plants, controlling plant pathogens in plants, and biocontrolling plant pathogens in plants. In some embodiments, the one or more beneficial traits include promoting the colonization of one or more microorganisms in plants. In some embodiments, the one or more beneficial traits include inhibiting the colonization of one or more microorganisms in plants. In some embodiments, the one or more beneficial traits include promoting nutrient utilization efficiency in plants. In some embodiments, the one or more beneficial traits include enhancing nutrient utilization efficiency in plants. In some embodiments, the one or more beneficial traits include controlling plant pathogens in plants. In some embodiments, the one or more beneficial traits include biocontrolling plant pathogens in plants. In some embodiments, the one or more beneficial traits include biocontrolling plant pathogens in plants, wherein the plant pathogens include one or more microorganisms selected from the genera selected from the group consisting of: *Pythium* (… Pythium ), Penicillium ( Penicillium ), Phytophthora spp. Phoma ), Botrytis ( Botrytis Fusarium ( ) Fusarium Mucor ( ) Mucor Anthrax genus ( Colletotrichum ) and Geotrichum ( GeotrichumIn some embodiments, the one or more beneficial traits include biocontrol of plant pathogens in plants, wherein the plant pathogens include one or more microorganisms selected from the group consisting of: *Pythium*, *Penicillium*, *Stemona*, *Botrytis*, and *Fusarium*. In some embodiments, the one or more beneficial traits include biocontrol of plant pathogens in plants, wherein the plant pathogens include one or more microorganisms selected from the group consisting of: *Pythium*, *Penicillium*, *Stemona*, and *Fusarium*. In some embodiments, the plant pathogen belongs to the genus *Pythium*. In some embodiments, the plant pathogen belongs to the genus *Penicillium*. In some embodiments, the plant pathogen belongs to the genus *Stemona*. In some embodiments, the plant pathogen belongs to the genus *Botrytis*. In some embodiments, the plant pathogen belongs to the genus *Fusarium*. In some embodiments, the plant pathogen belongs to the genus *Mucor*. In some embodiments, the plant pathogen belongs to the genus *Anthracis*. In some embodiments, the plant pathogen belongs to the genus *Geotrichum*. In some embodiments, the one or more beneficial traits include biocontrol of plant pathogens in plants, wherein the plant pathogens include one or more microorganisms selected from the group consisting of: *Pythium terrestris*, *Penicillium expansum*, etc. Penicillium expansum ), Penicillium finger ( Penicillium digitatum ), Staphylococcus aureus ( Botrytis cinerea Fusarium oxysporum ( Fusarium oxysporum Fusarium graminearum ( ), Fusarium graminarum Mucor ( ) Mucor circinelloides ), Colloidal anthrax bacteria ( Colletotrichum gloeosporoides ) and white globulus ( Geotrichum candidum In some embodiments, the one or more beneficial traits include biocontrol of plant pathogens in plants, wherein the plant pathogens include one or more microorganisms selected from the group consisting of: *Pythium terrestris*, *Penicillium expansum*, *Penicillium digitatum*, *Botrytis cinerea*, and *Fusarium oxysporum*. In some embodiments, the plant pathogen is *Pythium terrestris*. In some embodiments, the plant pathogen is *Penicillium expansum*. In some embodiments, the plant pathogen is *Penicillium digitatum*. In some embodiments, the plant pathogen is *Penicillium digitatum*. In some embodiments, the plant pathogen is... Botrytis cinerea. In some embodiments, the plant pathogen is Fusarium oxysporum. In some embodiments, the plant pathogen is Fusarium graminearum. In some embodiments, the plant pathogen is Mucor. In some embodiments, the plant pathogen is Colletotrichum gloeosporioides. In some embodiments, the plant pathogen is Geotrichum candida. In some embodiments, the microorganisms that produce metabolites that confer one or more beneficial traits on the plant belong to genera selected from the group consisting of Bacillus, Pseudomonas, and Bacillus-like organisms. In some embodiments, the microorganisms belong to the Bacillus genus. In some embodiments, the microorganisms belong to the Pseudomonas genus. In some embodiments, the microorganisms belong to the Bacillus-like organisms genus.

[0216] In some embodiments, the microorganisms that produce metabolites that confer one or more beneficial traits to the plant are derived from or originate from species of the genus *Bacillus*. In some aspects, the microorganisms are selected from *Bacillus licheniformis*, *Bacillus thuringiensis*, and *Bacillus tekira*. Bacillus tequilensis ), Bacillus pumilus, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), Methyltrophic Bacillus ( Bacillus methylotrophicus ) and Bacillus belesii.

[0217] In some embodiments, the microorganisms that produce metabolites that confer one or more beneficial traits to the plant are obtained from or derived from species of the genus *Bacillus*. In some aspects, the microorganisms are selected from *Bacillus polymyxa* and *Bacillus pilaris*.

[0218] Methods for identifying microorganisms that produce compositions conferring beneficial traits In one aspect, methods for identifying microorganisms that produce metabolites useful for a variety of applications in agriculture or other fields are disclosed. For example, in some embodiments, methods for identifying microorganisms that produce metabolites conferring one or more beneficial traits to plants are disclosed. In some embodiments, these methods identify microorganisms that produce metabolites that can be used to promote the colonization of one or more microorganisms in plants. In some embodiments, these methods identify microorganisms that produce metabolites that can be used to inhibit the colonization of one or more microorganisms in plants. In some embodiments, these methods identify microorganisms that produce metabolites that can be used to promote nutrient use efficiency in plants. In some embodiments, these methods identify microorganisms that produce metabolites that can be used to enhance nutrient use efficiency in plants. In some embodiments, these methods identify microorganisms that produce metabolites that can be used for the biocontrol of plant pathogens in plants. In some embodiments, a method for identifying microorganisms that produce metabolites conferring one or more beneficial traits to plants includes: obtaining a first sample having one or more metabolites from the microorganism; obtaining a first metabolite profile from the first sample; and selecting microorganisms that produce one or more beneficial traits to plants when the first metabolite profile has one or more unique elements, wherein at least one of the one or more unique elements corresponds to the one or more metabolites conferring the one or more beneficial traits to the plant.

[0219] As used herein, the term "unique" refers to a feature present in one article but not in another. As an example, this disclosure relates to a metabolite spectrum having "unique elements." In this case, the described metabolite spectrum possesses elements, markers, or features not present in a reference metabolite spectrum compared to a first metabolite spectrum. For example, if the metabolite spectrum is a chromatogram, a "unique element" might be a peak within the chromatogram that is not present in a reference chromatogram compared to a first chromatogram.

[0220] In some embodiments, the microorganisms may be cultured in a liquid medium for 1 to 14 days before obtaining a sample containing one or more metabolites from the microorganisms. In some embodiments, the microorganisms may be cultured in a solid medium (…) before obtaining a sample containing one or more metabolites from the microorganisms. For exampleThe microorganisms are cultured on agar (or similar agar) for 1 to 14 days. In some embodiments, the microorganisms may be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days prior to obtaining a sample containing one or more metabolites. In some embodiments, the microorganisms may be cultured for approximately 3 to approximately 5 days prior to obtaining a sample containing one or more metabolites. In some embodiments, the microorganisms may be cultured for 4 days prior to obtaining a sample containing one or more metabolites.

[0221] In some embodiments, a first sample containing one or more metabolites from a microorganism that produces metabolites that confer one or more beneficial traits to a plant is selected from a supernatant sample of a culture containing the microorganism, a whole broth sample of a culture containing the microorganism, and an extract of a culture containing the microorganism. In some embodiments, the first sample containing one or more metabolites from a microorganism that produces metabolites that confer one or more beneficial traits to a plant is derived from a supernatant sample of a culture containing the microorganism. The supernatant sample can be prepared by centrifuging the culture containing the microorganism and separating the supernatant from the precipitated cells and other solid components of the culture. Alternatively, the supernatant sample can be prepared by filtering the culture to separate the supernatant from the cells and other solid components of the culture. Those skilled in the art will understand that many other methods for separating supernatants from cultures are available and will therefore be compatible with the methods disclosed herein. In some embodiments, the first sample containing one or more metabolites from a microorganism that produces metabolites that confer one or more beneficial traits to a plant is derived from a whole broth sample of a culture containing the microorganism. In some embodiments, a first sample containing one or more metabolites from a microorganism that produces metabolites that confer one or more beneficial traits to a plant is derived from an extract of a culture containing that microorganism. This extract can be prepared by lysing cultured cells using techniques known to those skilled in the art, followed by separation of the soluble extract from insoluble cell debris and other components of the culture (e.g., by centrifugation).

[0222] In some implementations, samples containing one or more metabolites from microorganisms include one or more lipopeptides.

[0223] In some embodiments, obtaining a first metabolite profile includes subjecting a first sample having one or more metabolites to analytical techniques to identify the elements constituting the first sample. The analytical techniques may be any techniques known to those skilled in the art capable of identifying component metabolites within a first sample having one or more metabolites.

[0224] For example, the analytical technique can be, but is not limited to, chemical separation, chromatographic separation, nuclear magnetic resonance spectroscopy, mass spectrometry, etc.

[0225] In some implementations, obtaining a first metabolite profile includes subjecting a first sample having one or more metabolites to chromatographic separation.

[0226] In some embodiments, obtaining a first metabolite profile includes subjecting a first sample having one or more metabolites to chromatographic separation, wherein subjecting the first sample having one or more metabolites to chromatographic separation includes subjecting the first sample to high-performance liquid chromatography (HPLC).

[0227] In some implementations, high-performance liquid chromatography includes: The sample is subjected to a column; and one or more metabolites are eluted with a gradient of first and second mobile phase solvents. "Gradient of first and second mobile phase solvents" refers to the change in the composition of the mixture of the first and second mobile phase solvents over time. In some embodiments, the concentration of the first mobile phase solvent increases over time relative to the concentration of the second mobile phase solvent. In some embodiments, the concentration of the second mobile phase solvent increases over time relative to the concentration of the first mobile phase solvent.

[0228] Those skilled in the art will recognize that the gradient used to elute the one or more metabolites can include any compatible mobile phase solvent that can be used to separate a sample having one or more metabolites into its component metabolites. For example, compatible solvents may include, but are not limited to, water, acetonitrile, methanol, ethanol, ethyl acetate, hexane, etc., and these solvents may optionally also include one or more additives. Compatible additives may include acids or bases, wherein the acid or base may be selected from, but is not limited to, formic acid, acetic acid, trifluoroacetic acid, ammonium acetate, etc. Similarly, those skilled in the art will recognize that the gradient can be run at any suitable flow rate sufficient to separate a sample having one or more metabolites into its component metabolites for any suitable time period.

[0229] In some embodiments, the gradient includes water as a mobile phase solvent. In some embodiments, the gradient includes water as a mobile phase solvent, wherein the mobile phase solvent further includes trifluoroacetic acid as an additive. In some embodiments, the gradient includes water supplemented with 0.01% trifluoroacetic acid as a mobile phase solvent. In some embodiments, the gradient includes acetonitrile as a mobile phase solvent. In some embodiments, the gradient includes acetonitrile as a mobile phase solvent, wherein the mobile phase solvent further includes trifluoroacetic acid as an additive.

[0230] In some implementations, the gradient includes acetonitrile supplemented with 0.01% trifluoroacetic acid as the mobile phase solvent.

[0231] In some embodiments, the gradient has an initial concentration of approximately 40% of the second mobile phase solvent and a final concentration of approximately 100% of the second mobile phase solvent. In some embodiments, the gradient has a run time of approximately 15 minutes to approximately 45 minutes. In some embodiments, the gradient has a run time of approximately 30 minutes. In some embodiments, the gradient has a flow rate of approximately 0.5 mL / min to approximately 1.5 mL / min. In some embodiments, the gradient has a flow rate of approximately 0.8 mL / min.

[0232] In some implementations, the first metabolite spectrum is a high-performance liquid chromatogram.

[0233] In some embodiments, the high-performance liquid chromatography (HPLC) method includes: subjecting a sample to a C18 column having a diameter of 4.6 mm and a length of 100 mm at a temperature of about 20°C to about 40°C; and eluting the one or more metabolites with a gradient having first and second mobile phase solvents, wherein: the first mobile phase solvent comprises water; the second mobile phase solvent comprises acetonitrile; the gradient has an initial concentration of about 40% of the second mobile phase solvent and a final concentration of about 100% of the second mobile phase solvent; and the gradient has a run time of about 30 minutes and a flow rate of about 0.8 mL / min.

[0234] In some embodiments, the one or more unique elements have one or more retention times selected from the group consisting of: 6.8 minutes, about 8.3 minutes, about 8.6 minutes, about 8.7 minutes, about 9.0 minutes, about 10.5 minutes, and about 12.1 minutes, wherein the retention time is determined by the aforementioned HPLC method. In some embodiments, the one or more unique elements have one or more retention times selected from the group consisting of: about 8.7 minutes, about 9.0 minutes, and about 12.1 minutes, wherein the retention time is determined by the aforementioned HPLC method. In some embodiments, the one or more unique elements have one or more retention times selected from the group consisting of: about 6.8 minutes, about 8.3 minutes, about 8.6 minutes, and about 10.5 minutes, wherein the retention time is determined by the aforementioned HPLC method. Those skilled in the art will understand that retention times may vary slightly, for example, due to changes in column or instrument performance leading to slight differences between replicates. Therefore, the aforementioned retention times should be understood to cover retention times within ±0.2 minutes of the listed values. As an example, the listed retention time of 8.7 minutes is equivalent to a retention time in the range of 8.5 minutes to 8.9 minutes.

[0235] In some embodiments, the method for identifying microorganisms that produce one or more metabolites conferring one or more beneficial traits to plants further includes comparing a first metabolite profile with a second metabolite profile. In some embodiments, the second metabolite profile is obtained from a second sample having one or more metabolites from a second microorganism. In some embodiments, the second metabolite profile is obtained from a second sample having one or more metabolites from a second microorganism, wherein the second microorganism does not produce metabolites conferring one or more beneficial traits to plants. The second sample having one or more metabolites can be prepared from a supernatant sample of a culture containing the second microorganism, a whole broth sample of a culture containing the second microorganism, or an extract of a culture containing the second microorganism. In some embodiments, the second sample having one or more metabolites is prepared from a supernatant sample of a culture containing the second microorganism. The supernatant sample can be prepared by centrifuging a culture containing the microorganism and separating the supernatant from the precipitated cells and other solid components of the culture. In some embodiments, the second sample having one or more metabolites from a microorganism that produces metabolites conferring one or more beneficial traits to plants is from a whole broth sample of a culture containing the microorganism. In some embodiments, a second sample containing one or more metabolites from a microorganism that produces metabolites that confer one or more beneficial traits to a plant is derived from an extract of a culture containing that microorganism. This extract can be prepared by lysing cultured cells using techniques known to those skilled in the art, followed by separation of the soluble extract from insoluble cell debris and other components of the culture (e.g., by centrifugation).

[0236] In some embodiments, the second metabolite profile is obtained by subjecting a first sample containing one or more metabolites to an analytical technique to identify the elements constituting the second sample. This analytical technique can be any technique known to those skilled in the art capable of identifying component metabolites within the first sample containing one or more metabolites. For example, the analytical technique can be, but is not limited to, chemical separation, chromatographic separation, nuclear magnetic resonance spectroscopy, mass spectrometry, etc. In some embodiments, the second metabolite profile is obtained by subjecting a first sample containing one or more metabolites to chromatographic separation. In some embodiments, obtaining the second metabolite profile includes subjecting a second sample containing one or more metabolites to chromatographic separation, wherein subjecting the second sample containing one or more metabolites to chromatographic separation includes subjecting the second sample to high-performance liquid chromatography (HPLC). In some embodiments, HPLC includes: subjecting the sample to a column; and eluting the one or more metabolites with a gradient of first and second mobile phase solvents. "Gradient of first and second mobile phase solvents" refers to a change in the composition of the mixture of first and second mobile phase solvents over time. In some embodiments, the concentration of the first mobile phase solvent increases over time relative to the concentration of the second mobile phase solvent. In some embodiments, the concentration of the second mobile phase solvent increases over time relative to the concentration of the first mobile phase solvent.

[0237] Those skilled in the art will recognize that the gradient used to elute the one or more metabolites can include any compatible mobile phase solvent that can be used to separate a sample having one or more metabolites into its component metabolites. For example, compatible solvents may include, but are not limited to, water, acetonitrile, methanol, ethanol, ethyl acetate, hexane, etc., and these solvents may optionally also include one or more additives. Compatible additives may include acids or bases, wherein the acid or base may be selected from, but is not limited to, formic acid, acetic acid, trifluoroacetic acid, ammonium acetate, etc. Similarly, those skilled in the art will recognize that the gradient can be run at any suitable flow rate sufficient to separate a sample having one or more metabolites into its component metabolites for any suitable time period.

[0238] In some embodiments, the gradient includes water as a mobile phase solvent. In some embodiments, the gradient includes water as a mobile phase solvent, wherein the mobile phase solvent further includes trifluoroacetic acid as an additive. In some embodiments, the gradient includes water supplemented with 0.01% trifluoroacetic acid as a mobile phase solvent. In some embodiments, the gradient includes acetonitrile as a mobile phase solvent. In some embodiments, the gradient includes acetonitrile as a mobile phase solvent, wherein the mobile phase solvent further includes trifluoroacetic acid as an additive.

[0239] In some implementations, the gradient includes acetonitrile supplemented with 0.01% trifluoroacetic acid as the mobile phase solvent.

[0240] In some embodiments, the gradient comprises approximately 40% initial concentration of a second mobile phase solvent and approximately 100% final concentration of a second mobile phase solvent. In some embodiments, the gradient has a run time of approximately 15 minutes to approximately 45 minutes. In some embodiments, the gradient has a run time of approximately 30 minutes. In some embodiments, the gradient has a flow rate of approximately 0.5 mL / min to approximately 1.5 mL / min. In some embodiments, the gradient has a flow rate of approximately 0.8 mL / min.

[0241] In some embodiments, the second metabolite spectrum is a high-performance liquid chromatography (HPLC) chromatogram. In some embodiments, the second metabolite spectrum is an HPLC chromatogram obtained by subjecting a second sample having one or more metabolites to the same HPLC method used to obtain the first metabolite spectrum.

[0242] In some embodiments, a second microorganism from which a second sample containing one or more metabolites is prepared can be cultured in a liquid culture medium for 1 to 14 days before obtaining a sample containing one or more metabolites. In some embodiments, the microorganisms can be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days before obtaining a sample containing one or more metabolites. In some embodiments, the microorganisms can be cultured for about 3 to about 5 days before obtaining a sample containing one or more metabolites. In some embodiments, the microorganisms can be cultured for 4 days before obtaining a sample containing one or more metabolites.

[0243] In some embodiments, the second microorganism from which a second sample having one or more metabolites is prepared belongs to a genus selected from the group consisting of Bacillus, Pseudomonas, and Bacillus-like organisms. In some embodiments, the first and second microorganisms from which a first and second sample having one or more metabolites are prepared respectively belong to a genus selected from Bacillus, Pseudomonas, and Bacillus-like organisms. In some embodiments, the first and second microorganisms from which a first and second sample having one or more metabolites are prepared respectively belong to the genus Bacillus.

[0244] In some embodiments, the method for identifying microorganisms that produce one or more metabolites conferring one or more beneficial traits to plants further includes comparing a first metabolite profile with a second metabolite profile, and selecting microorganisms that produce one or more metabolites conferring one or more beneficial traits to plants when the first metabolite profile has one or more unique elements, wherein the one or more unique elements identified in the first metabolite profile are not present in the second metabolite profile, and at least one of the one or more unique elements corresponds to the one or more unique elements. As an example, the first metabolite profile may be a high-performance liquid chromatogram (HPLC) including multiple peaks corresponding to one or more elements of a first sample having one or more metabolites. The chromatogram of the first metabolite profile may be compared with a second metabolite profile, which is an HPLC chromatogram including multiple peaks corresponding to one or more elements of a second sample having one or more metabolites. The chromatogram of the first metabolite profile may include unique peaks not present in the chromatogram of the second metabolite profile, wherein the unique peaks correspond to one or more metabolites conferring beneficial traits to plants.

[0245] On the other hand, this disclosure relates to a method for selecting a microbial strain or species that produces one or more metabolites that combat one or more biotic stressors on or in a plant, the method comprising: obtaining a sample containing one or more metabolites; obtaining a metabolite profile from a first sample; and selecting the microorganism as a species that produces metabolites that combat one or more biotic stressors on or in a plant when the metabolite profile contains lipopeptides having one or more retention times selected from the group consisting of 6.8 min, 8.3 min, 8.6 min, 8.7 min, 9.0 min, 10.5 min, and 12.1 min, wherein the retention times are determined by high-performance liquid chromatography (HPLC) comprising: subjecting the sample to a C18 column having a diameter of 4.6 mm and a length of 100 mm. mm and the temperature is 25°C; and the one or more metabolites are eluted with a gradient comprising first and second mobile phase solvents, wherein: the first mobile phase solvent comprises water; the second mobile phase solvent comprises acetonitrile; the gradient comprises 40% initial concentration of the second mobile phase solvent and approximately 100% final concentration of the second mobile phase solvent; and the gradient comprises a run time of 30 minutes and a flow rate of 0.8 mL / min.

[0246] In some embodiments, the one or more lipopeptides have one or more retention times selected from the group consisting of 8.7 minutes, 9.0 minutes, and 12.1 minutes. In some embodiments, the one or more lipopeptides have a retention time of 8.7 minutes. In some embodiments, the one or more lipopeptides have a retention time of 9.0 minutes. In some embodiments, the one or more lipopeptides have a retention time of 12.1 minutes. In some embodiments, the one or more lipopeptides have one or more retention times selected from the group consisting of 6.8 minutes, 8.3 minutes, 8.6 minutes, and 10.5 minutes. In some embodiments, the one or more lipopeptides have a retention time of 6.8 minutes. In some embodiments, the one or more lipopeptides have a retention time of 8.3 minutes. In some embodiments, the one or more lipopeptides have a retention time of 8.6 minutes. In some embodiments, the one or more lipopeptides have a retention time of 10.5 minutes. Those skilled in the art will understand that retention times may vary slightly, for example, due to variations in column or instrument performance, resulting in slight differences between replicates. Therefore, the aforementioned retention times should be understood to cover retention times within ±0.2 minutes of the listed values. As an example, the listed retention time of 8.7 minutes is equivalent to a retention time in the range of 8.5 minutes to 8.9 minutes.

[0247] In some embodiments, the methods disclosed herein can be used to identify Bacillus species that produce a dual metabolite profile, as described in Example 1 herein. In some embodiments, the Bacillus species that produce a dual metabolite profile include Bacillus amyloliquefaciens, Bacillus methyltrophicus, Bacillus tekirae, and Bacillus belesii.

[0248] In some embodiments, the methods disclosed herein can be used to identify Bacillus species that produce a three-class metabolite profile, as described in Example 2 herein. In some embodiments, the Bacillus species that produce a three-class metabolite profile include Bacillus amyloliquefaciens, Bacillus methyltrophicus, and Bacillus belesii.

[0249] Microbial-produced compositions In some cases, the microorganisms disclosed herein may produce one or more compounds and / or have one or more activities, such as producing one or more of the following: producing metabolites, producing cyclic lipopeptides, producing plant hormones (such as auxins), producing acetoin, producing antimicrobial compounds, producing siderophores, producing polyketides, producing phenazines, producing cellulase, producing pectinase, producing chitinase, producing glucanase, producing xylanase, fixing nitrogen, or dissolving mineral phosphates.

[0250] For example, the microorganisms disclosed herein can produce plant hormones selected from the group consisting of: auxins, cytokinins, gibberellins, ethylene, brassinolide, and abscisic acid.

[0251] Therefore, "metabolites produced by the microorganisms of this disclosure" is intended to encompass any molecule (small molecules, vitamins, minerals, proteins, nucleic acids, lipids, fats, carbohydrates, etc.) produced by microorganisms. Typically, the precise mechanism by which the microorganisms of this disclosure confer beneficial traits on a given plant species is unknown. It is presumed that, in some cases, microorganisms produce metabolites beneficial to plants. Therefore, in some respects, cell-free or inactivated preparations of microorganisms are beneficial to plants, because the microorganisms need not be alive to confer beneficial traits on a given plant species, as long as the preparation includes metabolites produced by said microorganisms that are beneficial to plants.

[0252] In one embodiment, the microorganisms of this disclosure can produce auxin (e.g., indole-3-acetic acid (IAA)). Auxin production 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 a culture. Auxins play a crucial role in altering plant physiology, including the extent of root growth.

[0253] Therefore, in one embodiment, the microorganisms of this disclosure exist in the form of a community disposed on the surface or within the tissues of a given plant species. The microorganisms can effectively produce a detectably increased amount of the composition, such as metabolites, found on or within the plant when compared to a reference plant not treated with the microorganisms of this disclosure or with a cell-free or inactive preparation. The composition produced by the microbial community can be beneficial to the plant species.

[0254] Compositions produced by such microorganisms may be present in cell culture broths or media in which the microorganisms grow, or may encompass exudates produced by the microorganisms. As used herein, “exudate” refers to one or more compositions secreted by or extracted from one or more microbial cells. As used herein, “culture medium” refers to the common composition of cell culture media after the microbial cells have been placed in the culture medium. The composition of the culture medium may change over time, during different stages of microbial growth and / or development. The culture medium and / or exudate may improve the traits of the plant to which it becomes associated.

[0255] cyclic lipopeptide (CLP) CLPs produced by bacteria are known to have fungicidal and / or bactericidal activity (see, for example, Malfanova et al., "Cyclic lipopeptide profile of the plant-beneficial endophyticbacterium"). Bacillus subtilis HC8”, Archives of Microbiology Volume 194, pp. 893-899 (2012). CLPs are amphiphilic molecules containing a fatty acid tail linked to a short oligopeptide, forming a macrocyclic structure. These CLPs include several major types of compounds, such as ituronin, fentanyl, and surfactants.

[0256] Ikumin is derived from Bacillus subtilis ( Bacillus subtilis A family of lipopeptides extracted from the culture media of various strains of ) . These amphiphilic compounds are characterized by a peptide ring of seven amino acid residues, including the invariant D-Tyr2, in which the constant chiral sequence LDDLLDL is blocked by C14-C17 aliphatic β-amino acids. (Maget-Dana and Peypoux, “Iturins, a special class of pore-forming lipopeptides: biological and physicochemical properties”, Toxicology 87(1-3):151-74, 1994).

[0257] Fengyuan lipopeptide is partially cyclic, and the lactone bond formation occurs between the third amino acid l-Tyr and the last amino acid l-Ile (Samel et al., 2006). It consists of anionic cyclic decapeptides linked to an N-terminus of a β-hydroxy fatty acid.

[0258] Surfactin is an anionic cyclic lipopeptide composed of heptapeptides linked to β-hydroxy fatty acids. Due to its amphiphilic nature, surfactant is incorporated into the phospholipid bilayer and induces permeability and perturbation of target cells. (Seydlova and Svobodova, “Review of Surfactin chemical properties and the potential biomedical applications”, Central European Journal of Medicine, Vol. 3, pp. 123-133, 2008) polyketides Polyketides are structurally diverse and biologically active secondary metabolites produced by bacteria and other organisms. Polyketides are a large family of compounds formed through the condensation of acyl-thioester units, such as malonyl-CoA and methylmalonyl-CoA, to produce metabolites with diverse structures and biological activities. Examples of polyketides include, but are not limited to: macrolides (e.g., defisatin, macrolides), ansamycin, polyenes (e.g., dithiol-like compounds), polyethers, tetracyclines, and acetogenin.

[0259] Three types of polyketide synthases (PKS) have been identified in bacteria. Multimodal PKS consist of one or more large, multidomain polypeptides in which the growing polyketide chain is sequentially passed from one active site to the next. These macrosyntheses generate chemical diversity and complexity in a stepwise manner, depending on the nature of their constituent catalytic domains. Iterative PKS contain a set of catalysts that assemble polyketides with controlled chain lengths through repeated use of active sites. In both cases, the nascent polyketide product is frequently acted upon by other custom enzymes to produce antibiotics. The third type of PKS (called type III PKS) is distinct in that the growing polyketide chain is never directly linked to a protein. (Ridley et al., “Evolution of polyketide synthases in bacteria,” PNAS, Vol. 105, No. 12, pp. 4595-4600, 2008).

[0260] Agricultural Composition In some embodiments, the microorganisms of this disclosure are combined with agricultural compositions. Agricultural compositions generally refer to organic and inorganic compounds that may include compositions that promote the cultivation of microorganisms and / or plant components; compositions involved in the formulation of microorganisms to be applied to plant components (e.g., but not limited to: wetting agents, compatibilizers (also called "compatibilizers"), defoamers, detergents, chelating agents, drift reducers, neutralizers and buffers, corrosion inhibitors, dyes, odorants, spreaders (also called "dispersants"), penetration aids (also called "penetrators"), adhesives (also called "binders" or "binding agents"), dispersants, thickeners (also called "thickeners"), stabilizers, emulsifiers, freezing point inhibitors, antimicrobial agents, etc.); compositions involved in conferring protection to plant components or plants (e.g., but not limited to: insecticides, nematicides, fungicides, bactericides, herbicides, etc.); and other compositions that may be of interest for a particular application.

[0261] In some embodiments, the compositions of this disclosure are solids. When using solid compositions, it may be desirable to include one or more carrier materials with isolated active microorganisms or aggregates. In some embodiments, this disclosure teaches the use of carriers, including but not limited to: mineral soils such as silica, silica gel, silicates, talc, kaolin, activated clay, limestone, chalk, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, milled synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, thiourea and urea, plant-derived products such as grain flour, bark powder, wood flour and nutmeg powder, cellulose powder, palygorskite, montmorillonite, mica, vermiculite, synthetic silica and synthetic calcium silicate, or combinations of these substances.

[0262] growth composition In some embodiments, compositions that promote growth and development are provided to microorganisms and / or plant components. Exemplary compositions include liquids (e.g., broths, culture media) and / or solids (e.g., soil, nutrients). Various organic or inorganic compounds, alone or in combination with plant components, may be added to the growth composition to benefit the health of the microorganisms, such as, but not limited to, amino acids, vitamins, minerals, carbohydrates, monosaccharides, and lipids.

[0263] formulation composition One or more compositions other than microorganisms or microbial-derived compositions may be combined for various applications, stability, activity and / or storage reasons. Additional compositions may be referred to as "formulation components".

[0264] In some embodiments, the compositions disclosed herein are liquids. Therefore, in some embodiments, this disclosure teaches that the compositions disclosed herein may comprise compounds or salts such as monoethanolamine salts, sodium sulfate, potassium sulfate, sodium chloride, potassium chloride, sodium acetate, ammonium bisulfate, ammonium chloride, ammonium acetate, ammonium formate, ammonium oxalate, ammonium carbonate, ammonium bicarbonate, ammonium thiosulfate, ammonium diphosphate, ammonium monophosphate, sodium ammonium hydrogen phosphate, ammonium thiocyanate, ammonium aminosulfonate, or ammonium carbamate.

[0265] In some embodiments, this disclosure teaches that the composition may comprise a binder such as: polyvinylpyrrolidone, polyvinyl alcohol, partially hydrolyzed polyvinyl acetate, carboxymethyl cellulose, starch, ethylene pyrrolidone / vinyl acetate copolymer, and polyvinyl acetate, or a combination of these substances; a lubricant such as magnesium stearate, sodium stearate, talc, or polyethylene glycol, or a combination of these substances; a defoamer such as silicone emulsion, long-chain alcohol, phosphate ester, ethynyl glycol, fatty acid, or organofluorine compound; and a complexing agent such as: a salt of ethylenediaminetetraacetic acid (EDTA), a salt of triazinotriacetic acid, or a salt of polyphosphate, or a combination of these substances.

[0266] In some embodiments, the composition comprises a surfactant. In some embodiments, the surfactant is added to the liquid agricultural composition. In other embodiments, the surfactant is added to solid formulations, particularly those designed to be diluted with a carrier prior to application. Therefore, in some embodiments, the composition comprises a surfactant. Surfactants are sometimes used alone or in combination with other additives (such as minerals or vegetable oils) as adjuvants to spray can mixtures to improve the biocompatibility of microorganisms with a target. The type of surfactant used in bioenhancers typically depends on the nature and mode of action of the microorganisms. Surfactants can be anionic, cationic, or nonionic and can be used as emulsifiers, wetting agents, suspending agents, or for other purposes. In some embodiments, the surfactant is a nonionic surfactant, such as alkyl ethoxylates, linear fatty alcohol ethoxylates, and fatty amine ethoxylates. Surfactants commonly used in the field of pharmaceutical formulations and also applicable to the formulations of this invention are described in McCutcheon's Detergents and Emulsifiers Annual, MC Publishing Corp., Ridgewood, NJ, 1998 and Encyclopedia of Surfactants, Volumes I-III, Chemical Publishing Co., New York, 1980-81. In some embodiments, this disclosure teaches the use of surfactants comprising alkali metal, alkaline earth metal, or ammonium salts of fatty acids, including aromatic sulfonic acids (e.g., lignin sulfonic acid, phenol sulfonic acid, naphthalene sulfonic acid, and dibutylnaphthalene sulfonic acid), 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 naphthalene sulfonic acid with phenol and formaldehyde; condensates of phenol or phenol sulfonic acid with formaldehyde; condensates of phenol with formaldehyde and sodium sulfite; polyoxyethylene octylphenyl ether; ethoxylated isooctylphenol, ethoxylated octylphenol, or ethoxylated nonylphenol; tributylphenyl polyethylene glycol ether; alkyl aryl polyether alcohol; isotetrazol; ethoxylated castor oil; ethoxylated triarylphenol; salts of phosphorylated triarylphenol ethoxylates; dodecyl alcohol polyethylene glycol ether acetate; sorbitol esters; lignin-sulfite waste or methylcellulose; or combinations thereof.

[0267] In some embodiments, this disclosure teaches other suitable surfactants, including salts of alkyl sulfates, such as diethanolammonium dodecyl sulfate; alkyl aryl sulfonates, such as calcium dodecylbenzenesulfonate; alkylphenol-epoxide addition products, such as nonylphenol-C18 ethoxylate; ethanol-epoxide addition products, such as tridecyl alcohol-C16 ethoxylate; soaps, such as sodium stearate; alkyl naphthalene-sulfonates, such as sodium dibutylnaphthalenesulfonate; and dialkyl esters of sulfosuccinates, such as di(2-ethylhexyl)sulfonate. Sodium succinate; sorbitol esters, such as sorbitol oleate; quaternary ammonium, 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.

[0268] In some embodiments, the composition comprises a wetting agent. A wetting agent is a substance that, when added to a liquid, improves the spreading or penetrating ability of the liquid by reducing the interfacial tension between the liquid and the surface over which it spreads. Wetting agents are used in agrochemical formulations for two main functions: improving the wetting rate of powders in water during processing and manufacturing to prepare soluble liquid concentrates or suspension concentrates; and shortening the wetting time of wettable powders and improving the penetration rate of water into water-dispersible particles during mixing of the product with water in a spray can or other container. Examples of wetting agents used in the compositions of this disclosure (including wettable powders, suspension concentrates, and water-dispersible particle formulations) in some embodiments are: sodium dodecyl sulfate; sodium dioctyl sulfosuccinate; alkylphenol ethoxylates; and fatty alcohol ethoxylates.

[0269] In some embodiments, the compositions of this disclosure include a dispersant. A dispersant is a substance that adsorbs onto the surface of particles and helps maintain the dispersed state of the particles and prevents them from re-aggregating. In some embodiments, a dispersant is added to the compositions of this disclosure to promote dispersion and suspension during manufacturing and to ensure that the particles are redispersed in water in a spray can. In some embodiments, the dispersant is used in wettable powders, suspension concentrates, and water-dispersible particles. Surfactants used as dispersants have the ability to strongly adsorb onto the particle surface and provide charged or steric barriers that prevent particle re-aggregation. In some embodiments, the most commonly used surfactants are anionic, nonionic, or a mixture of both.

[0270] In some embodiments, sodium lignosulfonate is the most commonly used dispersant for wettable powder formulations. In some embodiments, polyelectrolytes (e.g., sodium naphthalenesulfonate formaldehyde condensates) are used in suspension concentrates to provide excellent adsorption and stabilization. In some embodiments, styrene-phenol ethoxylate phosphate esters are also used. In some embodiments, alkylaryl ethylene oxide condensates and EO-PO block copolymers are sometimes combined with anionic surfactants as dispersants for suspension concentrates.

[0271] In some embodiments, the compositions of this disclosure comprise a polymeric surfactant. In some embodiments, the polymeric surfactant has an extremely long hydrophobic "backbone" and a large number of ethylene oxide chains that form the "teeth" of a "comb-like" surfactant. In some embodiments, these high molecular weight polymers can give the suspension concentrate excellent long-term stability because the hydrophobic backbone has many points anchored to the particle surface. Examples of dispersants used in the compositions of this disclosure in some embodiments are: sodium lignin sulfonate; sodium naphthalene sulfonate formaldehyde condensate; tristyrene-phenol ethoxylate phosphate; fatty alcohol ethoxylates; alkyl ethoxylates; EO-PO block copolymers; and graft copolymers.

[0272] In some embodiments, the compositions of this disclosure include an emulsifier. An emulsifier is a substance that stabilizes a suspension of droplets in one liquid phase in another liquid phase. Without an emulsifier, the two liquids would separate into two immiscible liquid phases. In some embodiments, the most commonly used emulsifier blends comprise alkylphenols or fatty alcohols having 12 or more ethylene oxide units and oil-soluble calcium salts of dodecylbenzenesulfonic acid. A hydrophilic-lipophilic balance (“HLB”) value in the range of 8 to 18 will generally provide a well-stabilized emulsion. In some embodiments, emulsion stability can sometimes be improved by adding a small amount of EO-PO block copolymer surfactant.

[0273] In some embodiments, the compositions of this disclosure include a solubilizer. The solubilizer is a surfactant that forms micelles in water at concentrations exceeding the critical micelle concentration. The micelles then enable the dissolution or solubilization of water-insoluble materials within the hydrophobic portion of the micelles. Commonly used surfactants for dissolution are nonionic surfactants: sorbitan monooleate; sorbitan monooleate ethoxylate; and methyl oleate.

[0274] In some embodiments, the compositions of this disclosure comprise organic solvents. Organic solvents are primarily used in formulations of emulsifiable concentrates, ULV formulations, and to a lesser extent in granule formulations. Sometimes mixtures of solvents are used. In some embodiments, this disclosure teaches the use of solvents including aliphatic paraffin oils, such as kerosene or refined paraffin. In other embodiments, this disclosure teaches the use of aromatic solvents, such as xylene and higher molecular weight fractions of C9 and C10 aromatic solvents. In some embodiments, chlorinated hydrocarbons may be used as co-solvents to prevent pesticide crystallization when the formulation is emulsified in water. Sometimes alcohols are used as co-solvents to improve solubility.

[0275] In some embodiments, the composition comprises a gelling agent. Thickeners or gelling agents are primarily used in the formulation of suspension concentrates, emulsions, and suspension emulsions to alter the rheology or flowability of the liquid and prevent the separation and sedimentation of dispersed particles or droplets. Thickeners, gelling agents, and anti-settling agents generally fall into two categories: water-insoluble particles and water-soluble polymers. It is possible to use clay and silica to produce suspension concentrate formulations. In some embodiments, the composition comprises one or more thickeners, including but not limited to: montmorillonite, such as bentonite; magnesium aluminum silicate; and palygorskite. In some embodiments, this disclosure teaches the use of polysaccharides as thickeners. 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, this disclosure teaches the use of thickeners, including but not limited to: guar gum; locust bean gum; carrageenan; alginate; methylcellulose; sodium carboxymethyl cellulose (SCMC); and hydroxyethyl cellulose (HEC). In some embodiments, this disclosure teaches the use of other types of antisettling agents, such as modified starch, polyacrylates, polyvinyl alcohol, and polyethylene oxide. Another good antisettling agent is xanthan gum.

[0276] In some embodiments, the presence of surfactants (which reduce interfacial tension) can cause foaming in water-based formulations during preparation and mixing operations during application via a spray can. Therefore, in some embodiments, to reduce the tendency to foam, defoamers are typically added during the preparation stage or before filling into the bottle / spray can. Generally, there are two types of defoamers: silicone and non-silicone. Silicone defoamers are typically aqueous emulsions of dimethylpolysiloxane, while non-silicone defoamers are water-insoluble oils, such as octanol and nonanol, or silica. In both cases, the function of the defoamer is to transfer the surfactant from the air-water interface.

[0277] In some embodiments, the composition contains a preservative.

[0278] In some embodiments, the composition may be formulated as: soil irrigation agent, foliar spray, impregnation treatment agent, furrow treatment agent, soil conditioner, granules, broadcast treatment agent, post-harvest disease control treatment agent, or seed treatment agent. In some embodiments, the composition may be applied alone or in combination with other agricultural products according to a rotary spraying procedure.

[0279] In some embodiments, the composition is tank-compatible. In some embodiments, the composition is tank-compatible with other agricultural products. In some embodiments, the composition is compatible with equipment used for ground, air, and irrigation application.

[0280] In some embodiments, the composition may be applied to genetically modified seeds or plants.

[0281] protective composition Furthermore, individual microorganisms, microbial aggregates, or microbial communities developed according to the disclosed methods can be combined with known active agents available in the agricultural field, such as insecticides, herbicides, fungicides, insecticides, viricides, acaricides, nematicides, scabicides, plant growth regulators, rodenticides, anti-algae agents, biocontrol agents, or beneficial reagents. Additionally, microorganisms, microbial aggregates, or microbial communities developed according to the disclosed methods can be combined with known fertilizers. Such combinations can exhibit synergistic properties. Furthermore, individual microorganisms, microbial aggregates, or microbial communities developed according to the disclosed methods can be combined with inert ingredients. Additionally, in some aspects, the disclosed microorganisms are combined with bioactive agents.

[0282] In some embodiments, individual microorganisms, microbial aggregates, or microbial communities developed according to the disclosed methods can be combined with biopesticides that act as herbicides, fungicides, insecticides, viricides, acaricides, nematicides, scabies mites, rodenticides, and / or antialgae agents. Such biopesticides can be, but are not limited to, macroorganisms (e.g., beneficial nematodes, etc.) and microbial organisms (e.g., Serenade®, Bacillus thuringiensis…). Bacillus thuringiensis Plant extracts (e.g., Timorex Gold), biochemicals (e.g., insect pheromones), and / or minerals and oils (e.g., canola oil).

[0283] pesticides and biopesticides In some embodiments, the compositions of this disclosure comprise an insecticide used in combination with the taught microorganisms. In some embodiments, the compositions of this disclosure comprise a bio-insecticide used in combination with the taught microorganisms.

[0284] In some implementations, individual microorganisms or microbial aggregates or communities developed according to the disclosed methods can be combined with known pesticides in the agricultural field, such as pesticides that act as herbicides, fungicides, insecticides, viricides, acaricides, nematicides, scabicides, rodenticides and / or antialgae agents.

[0285] In some implementations, individual microorganisms or microbial aggregates or communities developed according to the disclosed methods can be combined with known biopesticides in the agricultural field, such as biopesticides that act as herbicides, fungicides, insecticides, viricides, acaricides, nematicides, scabicides, rodenticides and / or antialgae agents.

[0286] As used herein, the term "control" refers to the regulation or management of species that are considered to have a negative impact on agricultural processes or products, such as plants. Control of a species can be achieved through the use of chemical or biological agents. Control of a species may involve eradicating the species from agricultural processes or products, reducing the species population to a level where the species no longer has a negative impact on agricultural processes or products, or protecting agricultural processes or products from the effects of the species.

[0287] As used herein, "control of one or more plant pathogens on or in a plant" refers to the control of plant pathogen species that have already infected or otherwise established on a plant. For example, chemical or biological agents may be applied to the plant or the culture medium in which it grows to eradicate or reduce populations of plant pathogens on, in, or around the plant. Population reduction can be sufficient to prevent the negative effects of plant pathogen infection or colonization.

[0288] Alternatively, plants can be protected from infection or colonization by plant pathogens. For example, the application of biological or chemical agents can prevent plants from being infected or colonized by plant pathogens, such as by killing or otherwise inactivating the pathogens before they establish infection or colonization on, in, or around the plant.

[0289] As used herein, the term “biological control” is equivalent to the term “biological control” and refers to the use of biological organisms or their products to control species that are considered to have a negative impact on agricultural processes or products such as plants. For example, biological control organisms may involve eradicating the species from plants, reducing the population of the species to a level where the species no longer has a negative impact on agricultural processes or products, or protecting agricultural processes or products from the effects of the species.

[0290] As used herein, "biocontrol of one or more plant pathogens on or in a plant" refers to the use of a biological organism or its products to biocontrol a species of plant pathogen that has infected or otherwise colonized a plant. For example, a biological organism may be applied to a plant or the culture medium in which it grows to eradicate or reduce a population of plant pathogens on, in, or around the plant. Population reduction may be sufficient to prevent the negative effects of infection or colonization by plant pathogens. Alternatively, plants may be protected from infection or colonization by plant pathogens. As an example, the applied biological organism or its products may prevent infection or colonization by plant pathogens, for example, by killing or otherwise inactivating the plant pathogens before they are established on, in, or around the plant.

[0291] For example, in some embodiments, this disclosure teaches compositions comprising one or more of the following active ingredients: macroorganisms (e.g., beneficial nematodes, etc.), microorganisms (e.g., Serenade, Bt, etc.), plant extracts (e.g., Timorex Gold, etc.), biochemical substances (e.g., insect pheromones, etc.), and / or minerals and oils (e.g., canola oil).

[0292] In some embodiments, the individual microorganisms, microbial aggregates, or microbial communities developed according to the disclosed methods may be combined with herbicides selected from the group consisting of: acetamides selected from the group consisting of: acetochlor, metolachlor, butachlor, succinyl-methyl, fenfluroxychlor, fluthiamethoxam, bensulfuron-methyl, metolachlor, pyrazosulfuron, diltiazem, naproxen, clethodim, pretilachlor, thiamethoxam, and thifenoxysulfuron; amino acid derivatives selected from the group consisting of: bisphosphonium, glufosinate, and glyphosate; and aryloxyphenoxypropionates selected from the group consisting of: clodinafop-propionate, cyhalofop-butyl, quizalofop-p-ethyl, haloxyfop-P-ethyl, oxadiazon, oxychlorpyrifos, quizalofop-P-ethyl, and quizalofop-P-ethyl. The following are listed as examples of herbicides: diquat and paraquat; thiocarbamates selected from the following groups: chlorpyrifos, butachlor, carbaryl, betaine, piperazine, EPTC, quizalofop-p-ethyl, chlorpyrifos, berberine, betaine, berberine, barnyardgrass, quizalofop-p-ethyl, and oxadiazon; cyclohexanediones selected from the following groups: butylbenzyl, clethodim, thiamethoxam, cyclobenzyl, haloxyfop-methyl, pyrazosulfuron, and oxadiazon; dinitroanilines selected from the following groups: flurbichlor, ethylbutadiene, azoxystrobin, pendimethalin, ambroxol, and trifluralin; diphenyl ethers selected from the following groups: trifluralin, bensulfuron-methyl, chlorpyrifos, chlorfluazon, flufenoxuron, quizalofop-p-ethyl, and ethoxyfluorfen. Ethers; hydroxybenzonitrile selected from the group consisting of: bromobenzonitrile, chlorpyrifos, and iodobenzonitrile; imidazolinone selected from the group consisting of: imazalil, methoxymethyl imazalil, methyl imazalil, metribuzin, metribuzin, and imazalil; phenoxyacetic acid selected from the group consisting of: chlorfenapyr, 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4-DB, dt-propionic acid, MCPA, MCPA-thioethyl ester, MCPB, and 2,4-methylchloropropionic acid; pyrazine selected from the group consisting of: chlorpyrifos, flupyridaben, cyhalofop-butyl, flupyridaben, and pyrazosulfuron; pyridine selected from the group consisting of: chlorpyrifos, dichlorpyrifos, pyrfluthrin, flusulfanilamide, flupyridaben, and chlorpyrifos. Atrazine, flupyrazole and thiamethoxam; sulfonylureas selected from the group consisting of: sulfadiazine, tetrazolium sulfadiazine, bensulfadiazine, chlorpyrifos, chlorsulfuron, ethersulfuron, cypromethazine, ethoxysulfuron, pyrimisulfuron, flupyrsulfuron, flupyrsulfuron, formamidesulfuron, chlorpyrifos, azoxysulfuron, iodosulfuron, mesosulfuron, mesosulfuron, nicosulfuron, epoxysulfuron, flupyrsulfuron, flusulfuron, pyrimisulfuron, sulfadiazine, mesosulfuron, sulfonylsulfuron, thifensulfuron, etherbensulfuron, benzylsulfuron, trifluridinesulfuron, flumethanil, trifluridinesulfuron and 14(2-chloro-6-propyl-imidazol[1,2]pyridazin-3-yl)sulfonyl)-3-(4,6-dimethoxy-pyrimidin-2-yl)urea;Triazine compounds selected from the group consisting of the following: atrazine, atrazine, cyprodinil, isoamyl, etaziclomefone, cyclomethonium, benzoate, cyprodinil, promethazine, simazine, terbufenozide, decazine, and triazine fluroxypyr; urea compounds selected from the group consisting of the following: chlormequat, chlorfluazuron, diuron, fenfluroxypyr, isoproturon, linuron, methylbenzylthiazoline, and butyrazoline; acetolactate synthase inhibitors selected from the group consisting of the following: bispyribac-sodium, chlorpyrifos-sulfuron-methyl, dichlorvos-sulfuron-methyl, fluroxypyr, pyrazopyr-sulfuron-methyl, sulfadiazine-sulfuron-methyl, penoxsulam, propanil-sulfuron-methyl, propyltriazine. Glufosinate, pyrimisulfuron, cyclopyrimisulfuron, pyrimisulfuron, pyrimisulfuron, sulfonylpyrazosulfuron, and pyrazosulfuron; and compounds selected from the group consisting of: azoxystrobin, aminotriazole, sparphos, flubutyrazole, glyphosate, bencarbazone, benfluresate, pyrazosulfuron, bentazon, dicyclosulfuron, chlorpyrifos, brobutyrazole, flupropyrazosulfuron, phosmet, pyrazosulfuron, pyrazosulfuron, indole-methyl, diflubenzuron, cyclohexane, isoxaflutole, bensulfuron, propargite, dicamba, fenvalerate, flupyrazole, and *Drechslera*. Monoceras), Herbicides, Ethoxybenzamide, Tetracycline, Fluroxypyr, Propyleneflunomide, Flumetsulam, Fluroxypyr, Fluroxypyr, Furazolidone, Indoxime, Isoxazolidone, Isoxazolidone, Cyclopyralid, Propanil, Pendimethalin, Quinolinic acid, Chlormethalin, Nitrosulfuron, Methylarsic acid, Herbicides, Propyleneoxadiazon, Oxychlor, Oxychlor, Cyclopyralid, Cyclopyralid, Bisoxazolidin, Pyrazosulfuron ... 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)-ethyl acetate, methyl 6-amino-5-chloro-2-cyclopropyl-pyrimidin-4-carboxylate, 6-chloro-3-(2-cyclopropyl-6-methyl-phenoxy)-pyridazin-4-ol, 4-amino-3-chloro-6-(4-chloro-phenyl)-5-fluoro-pyridin-2-carboxylic acid, methyl 4-amino-3-chloro-6-(4-chloro-2-fluoro-3-methoxy-phenyl)-pyridin-2-carboxylate, and methyl 4-amino-3-chloro-6-(4-chloro-3-dimethylamino-2-fluoro-phenyl)-pyridin-2-carboxylate.

[0293] In some embodiments, the individual microorganisms, microbial aggregates, or microbial communities developed according to the disclosed methods may be combined with insecticides selected from the group consisting of: organo(thio)phosphates selected from the group consisting of: acephate, methyl pyrazophos, phosmet, chlorpyrifos, methyl chlorpyrifos, chlorpyrifos, diazinon, dichlorvos, chlorpyrifos, dimethoate, phorate, ethion, fenitrothion, fenthion, isoxazophos, malathion, methamidophos, chlorpyrifos, methyl parathion, phosmet, phosmet, phosmet, parathion, parathion, parathion, phosmet, phosmet, phosmet, phorate, phorate, phorate, phosmet, phorate, phorate, phosmet, phorate, phosmet, phorate, phosmet, phorate, phosmet, terbufos, phosmet Phosphorus, triazophos, and trichlorfon; carbamates selected from the group consisting of: carbaryl, aldicarb, cypermethrin, carbofuran, carbofuran, thiocarb, fenoxycarb, furazolidone, methiocarb, methomyl, chlorpyrifos, propoxur, thiamethoxam, and pymetrozine; pyrethroids selected from the group consisting of: allethrin, bifenthrin, cypermethrin, deltamethrin, cypermethrin, α-cypermethrin, β-cypermethrin, ζ-cypermethrin, deltamethrin, fenvalerate, fenvalerate, cypermethrin, cypermethrin, cypermethrin, lambda-cyhalothrin, permethrin, pyrethrin I and II, benzalkonium chloride, flusilazole, and deltamethrin. Heptafluthrin, methamidophos, tetrabromopyrethrin, tetrafluorobenzyl, propofol, and tetrafluoromethrin; insect growth regulators selected from the group consisting of: a) chitin synthesis inhibitors, wherein the chitin synthesis inhibitors are benzoylureas selected from the group consisting of: flufenoxuron, cyramazin, diflubenzuron, flufenoxuron, flufenoxuron, flufenoxuron, lufenuron, flufenoxuron, flufenoxuron, flufenoxuron, chlorfenapyr; thiamethoxam, bensulfuron, thiamethoxam, etoxazole, and tetradifon; b) ecdysone antagonists selected from the group consisting of: chlorfenapyr, methoxyfenozide, tebufenozide, and azadirachtin; c) juvenile hormone analogs selected from the group consisting of: pyriproxyfen, tebufenozide, and phenoxyfenozide. d) Lipid biosynthesis inhibitors selected from the group consisting of: spirodiclofen, spirodiclofen, and spirotetramat; Nicotinic receptor agonists / antagonists selected from the group consisting of: thiamethoxam, fipronil, imidacloprid, thiamethoxam, acetamiprid, acetamiprid, thiamethoxam, and 1-(2-chloro-thiazo-5-ylmethyl)-2-nitromimino-3,5-dimethyl-[1,3,5]triazine; GABA antagonists selected from the group consisting of: endosulfan, acetamiprid, fipronil, flupyradifurone, pyrazinoflurane, pyrazolium, and 5-amino-1-(2,6-dichloro-4-methyl-phenyl)-4-sulfinylamino-1H-pyrazole-3-thiocarbamate;Macrolide insecticides selected from the following groups: abamectin, emamectin, mibamectin, rapamycin, spinosad, and ethyl spinosad; mitochondrial electron transport inhibitor (METI) I scabies insecticides selected from the following groups: quinfenoxam, pyridaben, pymetrozine, azoxystrobin, and pyrimethanil; METI insecticides selected from the following groups... Compounds II and III: acaricide, fluacyprim, and flufenoxuron; brofenoxuron; oxidative phosphorylation inhibitors selected from the group consisting of: tricyclic tin, bufenozide, fenbutatin, and chlorfenapyr; cryomazine; synergistic ethers; sodium channel blockers selected from the group consisting of: indoxacarb and cyfluthrin; and compounds selected from the group consisting of: benclothiazide, bifenazate, batan, flonicamid, acetamiprid, pymetrozine, sulfur, chlorfenapyr, flufenoxuron, chlorantraniliprole, brofenoxuron (HGW86), pyridaben, pyrimethanil, dicofol, sulfadiazine, imicyafos, diflubenzuron, and pyrifluquinazon.

[0294] In some embodiments, the present invention teaches the synergistic use of the microorganisms or microbial aggregates disclosed herein with known pesticides in the agricultural field, such as pesticides that act as herbicides, fungicides, insecticides, viricides, acaricides, nematicides, scabicides, rodenticides, and / or antialgae agents.

[0295] In some embodiments, the present invention teaches the synergistic use of the microorganisms or microbial aggregates disclosed herein with known biopesticides in the agricultural field, such as biopesticides that act as herbicides, fungicides, insecticides, viricides, acaricides, nematicides, scabicides, rodenticides, and / or antialgae agents.

[0296] In some embodiments, when microorganisms or microbial aggregates identified according to the taught methods are combined with insecticides, an additive effect on the plant phenotypic trait of interest is observed. In other embodiments, when microorganisms or microbial aggregates identified according to the taught methods are combined with insecticides, a synergistic effect on the plant phenotypic trait of interest is observed.

[0297] In some embodiments, when microorganisms or microbial aggregates identified according to the taught methods are combined with biopesticides, an additive effect on the plant phenotypic trait of interest is observed. In other embodiments, when microorganisms or microbial aggregates identified according to the taught methods are combined with biopesticides, a synergistic effect on the plant phenotypic trait of interest is observed.

[0298] The synergistic effect obtained through the taught method 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, the full text of which is incorporated herein by reference. Therefore, the term “synergistic” refers to the component that, in its presence, increases the expected effect beyond the cumulative amount.

[0299] The isolated microorganisms and aggregates disclosed herein can synergistically enhance the efficacy of agricultural active pesticide compounds and agricultural auxiliary pesticide compounds.

[0300] The isolated microorganisms and aggregates disclosed herein can synergistically enhance the efficacy of agricultural active biological pesticide compounds and agricultural auxiliary biological pesticide compounds.

[0301] plant growth regulators and biostimulants In some embodiments, the compositions disclosed herein comprise plant growth regulators and / or biostimulants used in combination with the taught microorganisms.

[0302] In some implementations, individual microorganisms or microbial aggregates or 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-producing agents, growth inhibitors, and growth retardants.

[0303] For example, in some embodiments, this disclosure teaches compositions comprising one or more of the following active ingredients, including: cyclopropionylpyridinol, sec-butylamine, alcohol, chlormequat chloride, cytokinin, butyrylhydrazine, ethephon, furazolidone, gibberellic acid, gibberellin mixture, indole-3-butyric acid (IBA), maleic hydrazine, mefludide, mepiquat pentaborate, naphthaleneacetic acid (NAA), 1-naphthylacetamide (NAD), n-decyl alcohol, placlobutrazol, calcium cyclohexane, anti-bacterial ester, uniconazole, salicylic acid, abscisic acid, ethylene, brassinolide, jasmonic acid, polyamine, nitric oxide, strigolactone, or karrikins, etc.

[0304] In some embodiments, individual microorganisms, microbial aggregates, or microbial communities developed according to the disclosed methods can be combined with seed inoculants known in the agricultural field, such as QUICKROOTS.® VAULT ® RHIZO-STICK ® NODULATOR ® DORMAL ® SABREX ® Etc. In some implementations, slow-growing rhizobia (… Bradyrhizobium The inoculum can be used in combination with any single microorganism or microbial aggregate disclosed herein. In certain respects, when one of the aforementioned inoculums (e.g., QUICKROOTS) is used... ® Synergistic effects were observed when *Or slow-growing rhizobia* were combined with microorganisms or microbial aggregates as taught herein.

[0305] In some embodiments, the compositions disclosed herein comprise plant growth regulators comprising: kinetin, gibberellic acid, and indolebutyric acid, as well as copper, manganese, and zinc.

[0306] In some embodiments, this disclosure teaches compositions comprising one or more commercially available plant growth regulators, including but not limited to: Abide®, A-Rest®, Butralin®, Fair®, Royaltac M®, Sucker-Plucker®, Off-Shoot®, Contact-85®, Citadel®, Cycocel®, E-Pro®, Conklin®, Culbac®, Cytoplex®, Early Harvest®, Foli-Zyme®, Goldengro®, Happygro®, Incite®, Megagro®, Ascend®, Radiate®, Stimulate®, Suppress®, Validate®, X-Cyte®, B-Nine®, Compress®, Dazide®, Bol Buster®, BolD®, Cerone®, Cotton Quik®, Ethrel®, Finish®, Flash®, Florel®, Mature®, MFX®, Prep®, Proxy®, Quali-Pro®, SA-50®, Setup®, Super Boll®, Whiteout®, Cutless®, Legacy®, Mastiff®, Topflor®, Ascend®, Cytoplex®, Ascend®, EarlyHarvest®, Falgro®, Florgib®, Foli-Zyme®, GA3®, GibGro®, Green Sol®, Incite®, N-Large®, PGR IV®, Pro-Gibb®, Release®, Rouse®, Ryzup®, Stimulate®, BVB®, Chrysal®, Fascination®, Procone®, Fair®, Rite-Hite®, Royal®, Sucker Stuff®, Embark®, Sta-Lo®, Pix®, Pentia®, DipN Grow®, Goldengro®, Hi-Yield®, Rootone®, Antac®, FST-7®, Royaltac®, Bonzi®, Cambistat®, Cutdown®, Downsize®, Florazol®, Paclo®, Paczol®, Piccolo®, Profile®, Shortstop®, Trimmit®, Turf Enhancer®, Apogee®, ArmorTech®, Goldwing®, Governor®, Groom®, Legacy®, Primeraone®, Primo®, Provair®, Solace®, T-Nex®, T-Pac®, Concise® and Sumagic®.

[0307] In some embodiments, the present invention teaches the synergistic use of the microorganisms or microbial aggregates disclosed herein with plant growth regulators and / or stimulants (such as plant hormones or chemicals that affect the production or destruction of plant growth regulators).

[0308] 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 their production, such as being regulated by ACC synthase and destroyed by ACC deaminase).

[0309] In some implementations, individual microorganisms, microbial aggregates, or microbial communities developed according to the disclosed methods may be combined with biostimulants. Such biostimulants may be, but are not limited to, microbial organisms, plant extracts, algae, acids, biochar, etc.

[0310] In some embodiments, individual microorganisms, microbial aggregates, 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.), phosphates, and potash fertilizers. Such fertilizers may also contain micronutrients, including but not limited to sulfur, iron, and zinc.

[0311] In some embodiments, the present invention teaches additional plant growth-promoting chemicals that can work synergistically with the microorganisms and microbial aggregates disclosed herein, such as humic acid, fulvic acid, amino acids, polyphenols, and protein hydrolysates.

[0312] Therefore, in some embodiments, this disclosure provides for the application of the taught microorganism in combination with Ascend® to any crop. Furthermore, this disclosure provides for the application of the taught microorganism in combination with Ascend® to any crop and for any method or application rate.

[0313] In some embodiments, this disclosure teaches compositions having a biostimulant.

[0314] 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.

[0315] The level of microorganisms in soil or growth media 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 nourish and grow plants, in some embodiments, biostimulants provide biodegradable carbon (e.g., molasses, carbohydrates (e.g., sugars)) to nourish and grow microorganisms. Unless otherwise explicitly stated, biostimulants may contain a single ingredient or a combination of several different ingredients, which, due to the effects of one or more of these ingredients (acting alone or in combination), can enhance microbial activity or plant growth and development.

[0316] In some embodiments, biostimulants are compounds that produce non-nutritive plant growth responses. In some embodiments, the many important benefits of biostimulants are based on their ability to influence hormone activity. Hormones in plants (plant hormones) 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 plant hormones. In some embodiments, compounds in biostimulants can alter the hormonal state of plants and have a significant impact on their growth and health. Therefore, in some embodiments, this disclosure teaches kelp, humic acid, fulvic acid, and vitamin B as common components of biostimulants. In some embodiments, the biostimulants of this disclosure enhance antioxidant activity, thereby enhancing the plant's defense system. In some embodiments, vitamin C, vitamin E, and amino acids (such as glycine) are antioxidants contained in the biostimulant.

[0317] 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 containing specific organic seed extracts (e.g., soybean) are used in combination with microbial inoculants, the biostimulants can stimulate the growth of microorganisms included in the microbial inoculum. Therefore, in some embodiments, this disclosure teaches one or more biostimulants that, when used with microbial inoculants, can 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.

[0318] Combinations of plant components, microorganisms and / or compositions In some embodiments, this disclosure teaches compositions or combinations thereof, consisting of a single microorganism or a microbial aggregate or community or any combination thereof, that can be applied to plant components, optionally in combination with any agricultural composition, to improve plant phenotype.

[0319] Isolated microorganisms, communities, or aggregates (often interchangeably referred to as "microbes" or "microbes") can be applied to heterologous plant components to form a synthetic assemblage. Microorganisms are considered heterologous to plant components if they are not normally associated with plant components in nature or are applied in quantities different from those found in nature. In some embodiments, microorganisms may be present in one part of a plant but not in another, and the introduction of microorganisms into another part of the plant is considered a heterologous association.

[0320] It is further envisioned that isolated microorganisms, or those in combination with plants or plant components, could be further associated with one or more agricultural compositions, such as those described above.

[0321] The concept of microbial and plant components, microbial and agricultural compositions, and synthetic combinations of microbial and plant components and compositions (often referred to as "synthetic compositions," i.e., compositions containing components that are not typically found in nature) is envisioned.

[0322] plant component treatment In some embodiments, this disclosure also relates to the finding that treating plant components with one or more of the microorganisms or compositions of this disclosure prior to sowing or planting can enhance desired plant traits, such as plant growth, plant health, and / or plant insect resistance.

[0323] Therefore, in some embodiments, this disclosure teaches the use of one or more microorganisms or microbial aggregates as plant component treatment agents. A plant component treatment agent can be a plant component coating applied directly to untreated and "naked" plant components. However, a plant component treatment agent can be a plant component coating applied to plant components that have already been coated with one or more previous plant component coatings or plant component treatment agents. Previous plant component treatment agents may include one or more active compounds (chemical or biological) and one or more inert components.

[0324] The term "plant component treatment agent" generally refers to the application of material to plant components before or during planting in soil. Plant component treatment agents having the microorganisms and other compositions disclosed herein have the advantage of delivering the treatment agent to areas where plant components are planted shortly before germination and emergence.

[0325] In other embodiments, this disclosure also teaches that the use of plant component treatment agents can minimize the amount of microorganisms or agricultural compositions required for successful plant treatment and further limit worker exposure to microorganisms and compositions compared to application techniques such as spraying over soil or over germinating plant components.

[0326] Furthermore, in some embodiments, this disclosure teaches that the microorganisms disclosed herein are important for enhancing plant life in the early stages (e.g., within the first thirty days after the emergence of plant components). Therefore, in some embodiments, delivering the microorganisms and / or compositions of this disclosure in the form of a plant component treatment agent allows them to be placed in the area of ​​action at a time when microbial activity is critical.

[0327] In some embodiments, the microbial compositions of this disclosure are formulated as plant component treatment agents. In some embodiments, it is envisioned that one or more layers of the microorganisms and / or compositions disclosed herein can be substantially uniformly coated onto plant components using treatment agent application equipment specially designed and manufactured to apply the plant component treatment product precisely, safely, and effectively, using conventional mixing, spraying methods, or combinations thereof. Such equipment utilizes various types of coating technologies, such as rotary coaters, drum coaters, fluidized bed technology, fountain beds, rotary sprays, or combinations thereof. Liquid plant component treatment agents (such as the liquid plant component treatment agents of this disclosure) can be applied via a spinning "atomizer" disc or nozzle, which distributes the plant component treatment agent uniformly onto the plant component as it moves in a spray mode. In various embodiments, the plant component is subsequently mixed or tumbled for a period of time to achieve further treatment agent distribution and drying.

[0328] Prior to coating with the microbial composition, the plant components may be induced or uninitiated to improve the uniformity of germination and emergence. In an alternative embodiment, the dry powder formulation may be metered onto the mobile plant components and allowed to mix until fully distributed.

[0329] In some embodiments, at least a portion of the surface area of ​​the plant component is coated with the microbial composition according to the present disclosure. In some embodiments, the plant component coating containing the microbial composition is applied directly to the bare plant component. In some embodiments, an outer coating of the plant component containing the microbial composition is applied to a plant component that has already been coated with a plant component coating. In some aspects, the plant component may have a plant component coating containing, for example, thiamethoxam and / or Bacillus thuringiensis (Bt). Bacillus firmusThe composition of the invention is applied to a plant component coated with PONCHO™ VOTiVO™ in the form of a plant component coating, such as metalaxyl and / or thiamethoxam and / or Bacillus thuringiensis-I-1582. In some aspects, the taught microbial composition is applied to a plant component treated with PONCHO™ VOTiVO™ in the form of a plant component coating. In some aspects, the taught microbial composition is applied to a plant component treated with ACCELERON™ in the form of a plant component coating.

[0330] In some embodiments, the plant components treated with microorganisms have the following microbial spore concentrations or microbial cell concentrations: approximately 10^2 to 10^12, 10^2 to 10^11, 10^2 to 10^10, 10^2 to 10^9, 1^02 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.

[0331] In some embodiments, the plant components treated with microorganisms have the following microbial spore concentrations or microbial cell concentrations: approximately 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.

[0332] In some embodiments, the plant components treated with microorganisms have the following microbial spore concentrations or microbial cell concentrations: approximately 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.

[0333] In some embodiments, the plant components treated with microorganisms have the following microbial spore concentrations or microbial cell concentrations: 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.

[0334] In some implementations, the plant components treated with microorganisms have a microbial spore concentration or microbial cell concentration of approximately 10⁵ to 10⁹ per plant component.

[0335] In some embodiments, the plant components treated with microbial bodies have the following microbial spore concentrations or microbial cell concentrations: 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.

[0336] In some embodiments, the amount of one or more of the microorganisms and / or composition applied to the plant component depends on the final formulation and the size or type of the plant or plant component utilized. In some embodiments, one or more of the microorganisms are present at about 2% w / w to about 80% w / w of the total formulation. In some embodiments, by weight, one or more of the microorganisms used in the composition are about 5% w / w to about 65% w / w, or 10% w / w to about 60% w / w of the total formulation.

[0337] In some embodiments, the plant component 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.

[0338] In some embodiments, the plant component coatings of the present disclosure can be up to 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, 1120μm, 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, 1310μm, 1320 μm, 1330 μm, 1340 μm, 1350 μm, 1360μm, 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, 1550 μm, 1560 μm, 1570 μm, 1580 μm, 1590 μm, 1600 μm, 1610 μm, 1620 μm, 1630 μm, 1640 μm, 1650 μm, 1660 μm, 1670 μm, 1680 μm, 1690 μm, 1700 μm, 1710 μm, 1720 μm, 1730 μm, 1740 μm, 1750 μm, 1760 μm, 1770 μm, 1780 μm, 1790 μm, 1800 μm, 1810 μm, 1820 μm, 1830 μm, 1840 μm, 1850 μm, 1860 μm, 1870 μm, 1880 μm, 1890 μm, 1900 μm, 1910 μm, 1920 μm, 1930 μm, 1940 μm, 1950 μm, 1960 μm, 1970 μm, 1980 μm, 1990 μm, 2000 μm, 2010 μm, 2020 μm, 2030 μm, 2040 μm, 2050 μm, 2060 μm, 2070μm, 2080 μm, 2090 μm, 2100 μm, 2110 μm, 2120 μm, 2130 μm, 2140 μm, 2150 μm, 2160 μm, 2170 μm, 2180 μm, 2190 μm, 2200 μm, 2210 μm, 2220 μm, 2230 μm, 2240 μm, 2250 μm, 2260 μm, 2270 μm, 2280 μm, 2290 μm, 2300 μm, 2310 μm, 2320 μm, 2330 μm, 2340 μm, 2350 μm, 2360 μm, 2370 μm, 2380 μm, 2390 μm, 2400 μm, 2410 μm、2420 μm、2430 μm、2440 μm、2450 μm、2460 μm、2470 μm、2480 μm、2490 μm、2500 μm、2510 μm、2520 μm、2530 μm、2540 μm、2550 μm、2560 μm、2570 μm、2580 μm、2590 μm、2600 μm、2610μm, 2620 μm, 2630 μm, 2640 μm, 2650 μm, 2660 μm, 2670 μm, 2680 μm, 2690 μm, 2700 μm, 2710 μm, 2720 μm, 2730 μm, 2740 μm, 2750 μm, 2760 μm, 2770 μm, 2780 μm, 2790 μm, 2800 μm, 2810 μm, 2820 μm, 2830 μm, 2840 μm, 2850 μm, 2860 μm, 2870 μm, 2880 μm, 2890 μm, 2900 μm, 2910 μm, 2920 μm, 2930 μm, 2940 μm, 2950 μm, 2960 µm, 2970 µm, 2980 µm, 2990 µm or 3000 µm thick.

[0339] In some embodiments, the thickness of the plant component coating disclosed herein may 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.

[0340] In some embodiments, the plant component coating of this disclosure may 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%, or 24% of the 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%.

[0341] In some embodiments, the microorganisms and / or the composition may be freely coated onto the plant component or may be formulated in a liquid or solid composition prior to coating onto the plant component. For example, a solid composition containing microorganisms may be prepared by mixing a solid carrier with a suspension of spores until the solid carrier is impregnated with a suspension of spores or cells. The mixture may then be dried to obtain the desired particles.

[0342] In some other embodiments, the solid or liquid microbial compositions of this disclosure are envisioned to also contain functional agents, such as activated carbon, nutrients (fertilizers), and other agents capable of improving the germination and quality of the product or combinations thereof.

[0343] Plant component coating methods and compositions known in the art can be particularly useful when modified by adding one of the embodiments of this disclosure. Such coating methods and their application devices are disclosed, for example, in U.S. Patent 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.

[0344] Plant component coating compositions are disclosed in, for example, U.S. Patent 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.

[0345] In some embodiments, a variety of additives may be added to the plant component treatment formulation comprising the composition of the present invention. A binder may be added, and said binder comprises a natural or synthetic adhesive polymer that has no phytotoxic effect on the coated plant component. The binder may be selected from polyvinyl acetate; polyvinyl acetate copolymers; ethylene-vinyl acetate (EVA) copolymers; polyvinyl alcohol; polyvinyl alcohol copolymers; cellulose, including ethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, and carboxymethyl cellulose; polyvinylpyrrolidone; polysaccharides, including starch, modified starch, dextrin, maltodextrin, alginate, and deacetylated chitosan; fats; oils; proteins, including gelatin and corn gluten; gum arabic; shellac; vinylidene chloride and vinylidene chloride copolymers; calcium lignosulfonate; acrylic acid copolymers; polyvinyl acrylate; polyethylene oxide; acrylamide polymers and copolymers; hydroxyethyl polyacrylate, methacrylamide monomers; and polychloroprene.

[0346] A variety of colorants can be used, including organic chromophores classified as nitroso; nitro; azo, including monoazo, diazo, and polyazo; acridine, anthraquinone, azazine, diphenylmethane, indoleamine, indophenol, methine, oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, and xanthracene. Other additives may include micronutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum, and zinc.

[0347] Polymers or other dust control agents can be applied to leave the treatment on the surface of the plant components.

[0348] In some specific embodiments, in addition to microbial cells or spores, the coating may also comprise 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; polyvinylpyrrolidone; polysaccharides; proteins; fats; oils; gum arabic; gelatin; syrups; and starch. Further examples can be found, for example, in U.S. Patent No. 7,213,367, which is incorporated herein by reference.

[0349] Plant component treatment formulations may also contain various additives, such as adhesives, dispersants, surfactants, nutrients, and buffering agents. Other common 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, solid, or dry powder, 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 various phosphorus compounds.

[0350] In some embodiments, the plant component coating composition may comprise at least one filler, which is an organic or inorganic, natural or synthetic component, wherein the active components are combined to facilitate their application to the plant component. In various respects, the filler is an inert solid, such as clay, natural or synthetic silicate, silica, resin, wax, solid fertilizer (e.g., ammonium salt), natural soil minerals (e.g., kaolin, clay, talc, lime, quartz, palygorskite, montmorillonite, bentonite, or diatomaceous earth) or synthetic minerals (e.g., silica, alumina, or silicates, especially aluminum silicate or magnesium silicate).

[0351] In some embodiments, the plant component treatment formulation may also include one or more of the following ingredients: other insecticides, including compounds that act only below ground level; fungicides, such as captan, thiram, metalaxyl, fludioxonil, oxadixyl, and isomers of each of these materials; herbicides, including compounds selected from glyphosate, carbamates, thiocarbamates, acetamides, triazines, dinitroaniline, glyceryl ethers, pyridazinones, uracil, phenoxy compounds, urea, and benzoic acid; herbicidal safeners, such as benzoxazine, diphenylmethyl derivatives, N,N-diallyl dichloroacetamide, various dihaloacetyl, oxazolidinyl and thiazolyl compounds, acetone, naphthalenecarboxylic anhydride compounds, and oxime derivatives; chemical fertilizers; biofertilizers; and biocontrol agents, such as those derived from rhizobia. Bacillus Other naturally occurring or recombinant bacteria and fungi, including *Pseudomonas*, *Serratia*, *Trichoderma*, *Glomus*, *Gliocladium*, and mycorrhizal fungi. These components may be added as a separate layer on the plant component or alternatively as part of the plant component coating composition disclosed herein.

[0352] In some embodiments, the formulations used in this disclosure for treating plant components may be in the following forms: suspensions; emulsions; slurries of particles in an aqueous medium (e.g., water); wettable powders; wettable particles (dry and flowable); and dried particles. If formulated as a suspension or slurry, the concentration of the active ingredient in the formulation may be from about 0.5% by weight to about 99% by weight (w / w), or from 5% by weight to 40% by weight, or otherwise formulated by those skilled in the art.

[0353] 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 binders; dispersants, such as methylcellulose, which acts as a combined dispersant / binder for plant component treatments; polyvinyl alcohol; lecithin; polymeric dispersants (e.g., polyvinylpyrrolidone / vinyl acetate); thickeners (e.g., clay thickeners used to increase viscosity and reduce sedimentation of particulate suspensions); emulsion stabilizers; surfactants; antifreeze compounds (e.g., urea); dyes; colorants, etc. Further inert ingredients that may be used in this disclosure can be found in McCutcheon's, Volume 1, “Emulsifiers and Detergents,” MC Publishing Company, GlenRock, NJ, USA, 1996, which is incorporated herein by reference.

[0354] The plant component coating formulations of this disclosure can be applied to plant components by a variety of methods, including but not limited to: mixing in a container (e.g., bottle or bag), mechanical application, tumbling, spraying, and immersion. A variety of active or inert materials can be used to contact the plant components with the microbial composition according to this disclosure.

[0355] In some embodiments, the amount of microorganisms 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 the treatment will involve contacting the plant component with an agriculturally effective amount of the composition of the present invention.

[0356] As discussed above, an effective amount means an amount of the composition of the present invention sufficient to affect beneficial or desired results. An effective amount may be applied in one or more applications.

[0357] In some implementations, in addition to the coating layer, the plant components may be treated with one or more of the following: other insecticides, including fungicides and herbicides; herbicide-safe agents; fertilizers and / or biocontrol agents. These components may be added as a separate layer or alternatively added to the coating layer.

[0358] In some embodiments, the plant component coating formulations of this disclosure can be applied to the plant components using a variety of techniques and machines, such as fluidized bed technology, roller mill methods, roller electrostatic plant component processors, and drum coating machines. Other methods, such as fountain beds, can also be useful. The plant components can be pre-sized before coating. After coating, the plant components are typically dried and then transferred to a sizing machine for further sizing. Such procedures are known in the art.

[0359] In some embodiments, the plant components treated with microorganisms may also be coated with an external film to protect the coating. Such coatings are known in the art and can be applied using fluidized bed and cylindrical film coating techniques.

[0360] In other embodiments of this disclosure, the composition according to this disclosure can be introduced onto a plant component by using a solid matrix. For example, a certain amount of the composition of the present invention can be mixed with a solid matrix material, and then the plant component can be placed in contact with the solid matrix material for a period of time to allow the composition to be introduced into the plant component. The plant component can then optionally be separated from the solid matrix material and stored or used, or the mixture of the solid matrix material and the plant component can be stored or grown directly. Solid matrix materials that can be used in this disclosure include polyacrylamide, starch, clay, silica, alumina, soil, sand, polyurea, polyacrylate, or any other material capable of absorbing or adsorbing the composition of the present invention for a period of time and releasing the composition into or onto the plant component. It is useful to ensure that the composition of the present invention and the solid matrix material are compatible with each other. For example, the solid matrix material should be selected such that it can release the composition at a reasonable rate (e.g., over a period of minutes, hours, or days).

[0361] In some embodiments, this disclosure teaches that individual microorganisms or microbial aggregates or microbial communities developed according to the disclosed methods can be combined with any plant biostimulant.

[0362] In some embodiments, this disclosure teaches compositions comprising one or more commercially available biostimulants, including but not limited to: Vitazyme®, Diehard™ Biorush®, Diehard™ Biorush® Fe, Diehard™ Soluble Kelp, Diehard™ Humate SP, Phocon®, Foliar Plus™, PlantPlus™, Accomplish LM®, Titan®, Soil Builder™, Nutri Life, Soil Solution™, SeedCoat™, PercPlus™, Plant Power®, CropKarb®, Thrust™, Fast2Grow®, Baccarat®, and Potente®.

[0363] In some embodiments, when microorganisms or microbial aggregates identified according to the taught methods are combined with active chemical agents, an additive effect on the plant phenotypic trait of interest is observed. In other embodiments, when microorganisms or microbial aggregates identified according to the taught methods are combined with active chemical agents, a synergistic effect on the plant phenotypic trait of interest is observed.

[0364] In some embodiments, when microorganisms or microbial aggregates identified according to the taught methods are combined with fertilizers, an additive effect on the plant phenotypic trait of interest is observed. In other embodiments, when microorganisms or microbial aggregates identified according to the taught methods are combined with fertilizers, a synergistic effect on the plant phenotypic trait of interest is observed.

[0365] In some embodiments, when microorganisms or microbial aggregates identified according to the taught methods are combined with plant growth regulators, an additive effect on the plant phenotypic trait of interest is observed. In some embodiments, a synergistic effect is observed when microorganisms or microbial aggregates identified according to the taught methods are combined with plant growth regulators. In some aspects, the microorganisms of this disclosure are combined with Ascend... ® The study combined and observed synergistic effects on one or more phenotypic traits of interest.

[0366] In some embodiments, an additive effect on the plant phenotypic trait of interest is observed when microorganisms or microbial aggregates identified according to the taught methods are combined with biostimulants. In some embodiments, a synergistic effect is observed when microorganisms or microbial aggregates identified according to the taught methods are combined with biostimulants.

[0367] The synergistic effect obtained through the taught method 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, the full text of which is incorporated herein by reference. Therefore, the term “synergistic” refers to the component that, in its presence, increases the expected effect beyond the cumulative amount.

[0368] The isolated microorganisms and aggregates disclosed herein can synergistically enhance the efficacy of agricultural active compounds and agricultural auxiliary compounds.

[0369] In other implementations, synergistic effects were observed when microorganisms or microbial aggregates identified according to the taught methods were combined with fertilizers.

[0370] Furthermore, in some embodiments, this disclosure utilizes synergistic interactions to define microbial aggregates. That is, in some aspects, this disclosure combines certain isolated microbial species that exert synergistic effects into aggregates that confer beneficial traits to plants, or are associated with enhancing beneficial plant traits.

[0371] The compositions developed according to this disclosure can be formulated with certain adjuvants to enhance the activity of known active agricultural compounds. This has the advantage of reducing the amount of active ingredient in the formulation while maintaining the efficacy of the active compound, thus keeping costs as low as possible and complying with any official regulations. In standalone cases, it is also possible to broaden the scope of action of the active compound, since plants (where treatment with a specific active ingredient without addition is not sufficiently successful) can actually be successfully treated by adding certain adjuvants as well as the disclosed microbial isolates and aggregates. Furthermore, when environmental conditions are unfavorable, the performance of the active substance can be enhanced in standalone cases through suitable formulations.

[0372] Such adjuvants, which can be used in agricultural compositions, are typically adjuvants. Adjuvants are usually in the form of surfactants or salt compounds. Based on their mode of action, they can be broadly classified as regulators, activators, fertilizers, pH buffers, etc. Regulators affect the wetting, adhesion, and spreading properties of formulations. Activators disrupt the waxy surface of plants and increase the penetration of active ingredients into the surface (short-term (minutes) and long-term (hours)). Fertilizers (e.g., ammonium sulfate, ammonium nitrate, or urea) increase the absorption and solubility of active ingredients and can reduce antagonistic behavior of active ingredients. pH buffers are routinely used to adjust formulations to the optimal pH.

[0373] Further embodiments of the compositions disclosed herein See "Chemistry and Technology of Agrochemical Formulations", edited by DA Knowles, copyright 1998, Kluwer Academic Publishers, which is incorporated herein by reference.

[0374] Plant and agricultural benefits A wide variety of plants (including those cultivated in agriculture) can benefit from the application of microorganisms (such as those described herein, including single microorganisms, aggregates, and / or compositions produced therefrom, or containing any of the foregoing). Many different plants, including mosses, lichens, and algae, can be used in the methods of this disclosure. In embodiments, the plants have economic, social, or environmental value. For example, plants may include those used as food crops, fiber crops, oil crops, for forestry, for the pulp and paper industry, as feedstock for biofuel production, and as ornamental plants.

[0375] In other embodiments, the plant may be economically, socially, or environmentally undesirable, such as weeds. The following is a list of non-limiting examples of plant types to which the methods of this disclosure can be applied, including the following plant parts or plant types.

[0376] food crops cereals, for example Corn, rice, wheat, barley, sorghum, millet, oats, rye, black wheat, and buckwheat; leafy vegetables, for example Cruciferous vegetables, such as cabbage, broccoli, bok choy, and arugula; salad greens, such as spinach, watercress, and lettuce; fruit and flowering vegetables, for example Avocados, sweet corn, artichokes; melons, such as zucchini, cucumbers, cantaloupes, squash, and pumpkins; nightshade vegetables / fruits, such as tomatoes, eggplants, and peppers; podded vegetables, for example Peanuts, peanuts, peas, soybeans, kidney beans, lentils, chickpeas, okra; bulb and stem vegetables, For example, asparagus, celery, and alliums ( Allium Crops, such as garlic, onions, and leeks; roots and tubers Vegetables, such as carrots, beets, bamboo shoots, cassava, yams, ginger, Jerusalem artichokes, parsnip, radishes, potatoes, sweet potatoes, taro, turnips, and wasabi; Sugar crops, including sugar beets ( Beta vulgaris ),sugar cane( Saccharum officinarum ); Crop cultivation for the production of non-alcoholic beverages and stimulants, such as coffee, black tea, herbal tea and green tea, cocoa, and tobacco; fruit crops Fruits such as true berries (e.g., kiwi, grape, currant, gooseberry, guava, feijoa, pomegranate), citrus fruits (e.g., orange, lemon, lime, grapefruit), superior fruits (e.g., banana, cranberry, blueberry), aggregate fruits (blackberry, raspberry, boysonberry), compound fruits (e.g., pineapple, fig), stone fruits (e.g., apricot, peach, cherry, plum), pome fruits (e.g., apple, pear), and other fruits such as strawberries and sunflower seeds; culinary and medicinal herbs, for example Rosemary, basil, bay leaf, coriander, mint, dill, St. John's wort ( Hypericum ), foxglove, aloe vera, and rosehip; crop plants producing aromas, for example Black pepper, cumin, cinnamon, nutmeg, ginger, cloves, saffron, cardamom, nutmeg skin, red chili pepper, masala, star anise; Planting crops for the production of nuts, for example Almonds and walnuts, Brazil nuts, cashews, coconuts, chestnuts, macadamia nuts, pistachios, peanuts, pecans; Cultivate crops used to make beer, wine and other alcoholic beverages, such as grapes and hops; Oilseed crops, for exampleSoybeans, peanuts, cotton, olives, sunflowers, sesame, lupins, and brassica crops (e.g., canola / rapeseed); and Edible fungi, for example White mushrooms, shiitake mushrooms, and oyster mushrooms; Plants for agroforestry Leguminosae: Clover ( Trifolium ) species, genus Alfalfa ( Medicago ) species and the genus Lobelia ( Lotus Species; white clover (T. repens); Red clover (T. pratense); Caucasian clover （T. ambigum); Earth Trifoliate (T. subterraneum); Alfalfa (Medicago sativum); Annual alfalfa; Tribulus terrestris; alfalfa zebrina; red clover (Onobrychis viciifolia); Hundred Veins Root (Lotus corniculatus); Great Hundred Veins Root (Lotus pedunculatus); Seed legumes / dried beans, including peas ( Pisum sativum ),kidney bean( Phaseolus vulgaris ),broad bean( Vicia faba ),green beans( Vigna radiata ),cowpea( Vigna unguiculata ), chickpeas ( Cicer arietum Lupinus (certain species of the genus Lupinus) Lupinus species Cereals, including corn / maize ( Zea mays ), sorghum ( Sorghum spp .),Millet( Panicum miliaceum, P. sumatrense ), rice ( Oryza sativa indica, Oryza sativa japonica ),wheat( Triticum aestivum ),barley( Hordeum vulgare ),rye( Secale cereale Black wheat () Triticum X Secale ),oat( Avena sativa ); Forage and woodland grasses: temperate grasses, such as ryegrass ( Lolium ) species; Festuca ( ) Festuca ) species; genus *Gnaphalium* ( Agrostis ) species, perennial ryegrass ( Lolium perenne ); hybrid ryegrass ( Lolium hybridum ); annual ryegrass ( Lolium multiflorum ), tall fescue ( Festuca arundinacea ); cowtail grass ( Festuca pratensis ); Festuca rubra fescue ( ) Festuca ovina ); Festuloliums(Festuca ryegrass hybrid ( Lolium X Festuca crosses )); duckgrass ( Dactylis glomerata Kentucky bluegrass ( ); Kentucky bluegrass ( Poa pratensis Kentucky bluegrass (); Poa palustris Kentucky bluegrass in woodland (); Poa nemoralis ); Common Kentucky bluegrass ( Poa trivialis Kentucky bluegrass ( ); Kentucky bluegrass ( Poa compresa ); Broccoli ( Bromus ) species; genus *Gnaphalium* ( Phalaris (Tigris genus) Phleum ) species); Limnipotentifolia ( Arrhenatherum elatius ); genus *Agropyron* ( Agropyron ) species; rough-haired oats ( Avena strigosa Millet (); Setaria italic ); Tropical grasses, such as: *Gnaphalium* ( ) Phalaris ) species; genus *Armillaria* ( Brachiaria ) species; genus *Lysimachia* ( Eragrostis ) species; millet ( ) Panicum Species; Bahia grass (Paspalum notatum); Brachypodium (Brachypodium ) species; and grasses used for biofuel production, such as switchgrass. (Panicum virgatum) and Miscanthus ( Miscanthus ) species; Fiber crops Cotton, kapok, jute, coconut, sisal, flax (Flame genus) Linum ) species), New Zealand flax (New Zealand hemp genus ( Phormium (Species); planted and natural forest species harvested for paper and engineered wood fiber products, such as coniferous and broadleaf forest species.

[0377] Trees and shrub species planted in forestry and biofuel crops Pine (Pinus) Pinus ) species); fir (Dalbergia genus ( Pseudotsuga ) species); spruce (genus spruce ( Picea ) species); cypress (Cypress genus ( Cupressus ) species); Acacia tree (Acacia genus ( Acacia ) species); Alder (Alder genus ( Alnus ) species); oak species (Quercus genus ( Quercus ) species); Sequoia (Giant Sequoia) Sequoiadendron ) species); willow (willow genus ( Salix ) species); birch (Betula genus ( Betula ) species); Cedar (Cedar genus ( Cedurus ) species); Ash (Fraxinus genus ( Fraxinus ) species); larch (Larch genus (Larix ) species); Eucalyptus genus ( Eucalyptus ) species; bamboo (Bambusae tribe ( Bambuseae ) species) and poplar (poplar genus ( Populus (Species).

[0378] Plants grown for conversion into energy, biofuel or industrial products by extraction, biological, physical or biochemical processing Hevea brasiliensis Oil-producing plants, such as oil palm, jatropha, soybean, cotton, and flaxseed; latex-producing plants, such as rubber tree (…). Castilla elastica ) and Panama rubber trees ( Crops producing natural products useful in the pharmaceutical, agricultural, nutritional food and cosmeceutical industries Plants used as direct or indirect feedstocks for the production of biofuels (i.e., following chemical, physical (e.g., thermal or catalytic), or biochemical (e.g., enzymatic pretreatment), or biological (e.g., microbial fermentation) conversions 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, sugarcane), starch-producing crops (e.g., C3 and C4 cereals and tuber crops), cellulosic crops such as trees (e.g., pine, eucalyptus), and grasses and gramineous plants such as bamboo, switchgrass, and miscanthus; crops used in energy, biofuel, or industrial chemical production by gasification and / or microbial or catalytic conversion into biofuels or other industrial feedstocks (e.g., solvents or plastics, producing or not producing biochar), such as biomass crops such as coniferous, eucalyptus, tropical or broadleaf trees, grasses and gramineous plants such as bamboo, switchgrass, miscanthus, sugarcane, or cork such as poplar and willow; and biomass crops used for the production of biochar.

[0379] Flowering, ornamental and beautifying plants grown for their aesthetic or environmental properties Crops that produce pharmaceutical precursors or compounds or nutritional food and cosmeceutical compounds and materials, such as star anise (shikimic acid), Japanese knotweed (resveratrol), and kiwifruit (soluble fiber, proteolytic enzymes).

[0380] Plants grown for bioremediation Flowers, such as roses, tulips, and chrysanthemums.

[0381] Ornamental shrubs, such as boxwood, privet, rose, azalea, and ivy.

[0382] Beautifying plants, such as sycamore, Mexican orange, mouse thorn, euphorbia, and mosses.

[0383] Mosses, such as peat moss.

[0384] Hybrid plants and genetically modified plants (GM) improvement In some respects, the microorganisms of this disclosure are applied to hybrid plants to enhance the beneficial traits of said hybrids. In other respects, the microorganisms of this disclosure are applied to genetically modified plants to enhance the beneficial traits of said GM plants. The microorganisms taught herein can be applied to hybrids and GM plants, and thus can maximize the superior genetic and trait techniques of these plants.

[0385] It should be understood that plants may be provided as seeds, seedlings, cuttings, propagules, or any other plant material or tissue form capable of growth. In one embodiment, seeds may be surface-sterilized with materials such as sodium hypochlorite or mercuric chloride to remove microorganisms from contaminated surfaces. In one embodiment, propagules are grown in pure cultures, such as as sterile seedlings in tissue cultures, before being placed in a plant growth medium.

[0386] Bacillus firmus In some respects, the microorganisms of this disclosure are applied to hybrid plants to enhance the beneficial traits of said hybrids. In other respects, the microorganisms of this disclosure are applied to genetically modified plants to enhance the beneficial traits of said GM plants. The microorganisms taught herein can be applied to hybrids and GM plants, and thus can maximize the superior genetic and trait techniques of these plants.

[0387] It should be understood that plants may be provided as seeds, seedlings, cuttings, propagules, or any other plant material or tissue form capable of growth. In one embodiment, seeds may be surface-sterilized with materials such as sodium hypochlorite or mercuric chloride to remove microorganisms from contaminated surfaces. In one embodiment, propagules are grown in pure cultures, such as as sterile seedlings in tissue cultures, before being placed in a plant growth medium.

[0388] Application method Microorganisms can be applied to plants, seedlings, cuttings, propagules, etc., and / or growth media containing said plants, using any suitable technique known in the art.

[0389] However, as an example, isolated microorganisms, aggregates, or compositions containing them and / or compositions derived therefrom can be applied to plants, seedlings, cuttings, propagules, etc. by spraying, coating, sprinkling, or any other method known in the art.

[0390] In another embodiment, the isolated microorganisms, aggregates, or compositions containing them can be applied directly to plant seeds prior to sowing.

[0391] In another embodiment, isolated microorganisms, aggregates, compositions derived therefrom, or compositions containing them can be applied directly to plant seeds in the form of seed coating.

[0392] In one embodiment of this disclosure, the isolated microorganisms, aggregates, or compositions containing them are supplied in the form of granules or fillers applied to plant growth media or soil irrigation.

[0393] In other embodiments, the isolated microorganisms, aggregates, or compositions containing them are supplied in the form of foliar application, such as foliar sprays or liquid compositions. Foliar sprays or liquid applications may be applied to growing plants or growth media, such as soil.

[0394] In some embodiments, the isolated microorganisms, aggregates, or compositions comprising them are supplied in forms selected from: soil irrigation agents, foliar sprays, impregnation treatments, furrow treatments, soil conditioners, granules, broadcast treatments, post-harvest disease control treatments, or seed treatments. In some embodiments, the composition may be applied alone or in a rotary spraying procedure.

[0395] In some embodiments, the isolated microorganisms, aggregates, or compositions containing them are compatible with the tank. In some embodiments, the composition is compatible with tanks containing other agricultural products. In some embodiments, the composition is compatible with equipment used for ground, air, and irrigation applications.

[0396] In another embodiment, the isolated microorganisms, aggregates, or compositions containing them may be formulated into granules and applied next to the seeds during planting. Alternatively, the granules may be applied after planting. Or, the granules may be applied before planting.

[0397] In some embodiments, isolated microorganisms, aggregates, or compositions containing them are applied to plants or growth media in the form of topical application and / or irrigation to improve crop growth, yield, and quality. Topical application can be carried out using dry mixtures, powders, or dusting compositions, or it can be a liquid-based formulation.

[0398] In embodiments, the isolated microorganisms, aggregates, or compositions containing them may be formulated as: (1) solutions; (2) wettable powders; (3) spreadable 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 immediate-release); (10) microencapsulated granules or suspensions; (11) as irrigation components; and (12) components of fertilizers, pesticides, and other compatibility modifiers, etc. In some respects, the compositions may be diluted in an aqueous medium prior to conventional spray application. The compositions of this disclosure may be applied to soil, plants, seeds, rhizosphere, root sheath, rhizosphere, or other areas where application of the microbial composition will be beneficial. Furthermore, shock treatment may be used as a means of introducing endophytic microorganisms.

[0399] The composition is applied to the leaves of plants in various ways. It can be applied to the leaves as an emulsion or suspension concentrate, a liquid solution, or a foliar spray. Application can be carried out in a laboratory, growth chamber, greenhouse, or field.

[0400] In another embodiment, microorganisms can be inoculated into plants by pruning roots or stems and exposing the plant surface to microorganisms by spraying, dipping, or otherwise applying a liquid microbial suspension, gel, or powder.

[0401] In another embodiment, the microorganisms can be injected directly into the leaf or root tissue, or otherwise inoculated directly into or onto the leaf or root pruning site, or into detached plumules, radicles, or coleoptiles. These inoculated plants can then be further exposed to a growth medium containing additional microorganisms; however, this is not necessary.

[0402] In other embodiments, particularly where the microorganisms are unculturable, the microorganisms can be transferred to the plant by any one or a combination of the following methods: grafting, explant insertion, aspiration, electroporation, injury, root pruning, inducing stomatal opening, or any physical, chemical, or biological treatment that provides an opportunity for the microorganisms to enter the plant cells or intercellular spaces. Many alternative techniques are readily apparent to those skilled in the art.

[0403] In one embodiment, the microorganisms infiltrate parts of the plant (e.g., roots, stems, leaves, and / or propagating plant parts (becoming endophytes)), and / or grow on the surface of roots, stems, leaves, and / or propagating plant parts (becoming epiphytes) and / or grow in the plant rhizosphere. In one embodiment, the microorganisms form a symbiotic relationship with the plant.

[0404] In some embodiments, this disclosure also relates to the finding that treating seeds with the compositions of this disclosure prior to sowing or planting can enhance desired plant traits, such as plant growth, plant health, and / or plant insect resistance.

[0405] Therefore, in some embodiments, this disclosure teaches the use of the compositions of this disclosure as seed treatment agents. A seed treatment agent can be a seed coating applied directly to untreated and “naked” seeds. However, a seed treatment agent can be a seed coating applied to seeds that have already been coated with one or more previous seed coatings or seed treatment agents. Previous seed treatment agents may contain one or more active compounds (chemical or biological) and one or more inert ingredients.

[0406] The term "seed treatment" generally refers to the application of material to seeds before or during planting in soil. Seed treatment with the compositions of this disclosure has the advantage of delivering the treatment agent to the area where seeds are planted shortly before seed germination and seedling emergence.

[0407] In other embodiments, this disclosure also teaches that the use of seed treatment agents can minimize the amount of the composition of this disclosure required for successful plant treatment and further limit worker contact with the composition compared to application techniques such as spraying over soil or emerging seedlings.

[0408] Furthermore, in some embodiments, this disclosure teaches that the compositions disclosed herein are important for enhancing plant life in the early stages (e.g., within the first thirty days after seedling emergence). Therefore, in some embodiments, delivering the compositions of this disclosure in the form of a seed treatment agent allows them to be placed on the site of action at a time when the composition's activity is important.

[0409] In some embodiments, the compositions disclosed herein are formulated as seed treatment agents. In some embodiments, it is envisioned that one or more layers of the compositions disclosed herein can be applied substantially uniformly to seeds using treatment agent application equipment specially designed and manufactured to apply seed treatment products precisely, safely, and effectively, using conventional mixing, spraying methods, or combinations thereof.

[0410] Such equipment utilizes various types of coating technologies, such as rotary coaters, drum coaters, fluidized bed technology, fountain beds, rotary sprayers, or combinations thereof. Liquid seed treatment agents (such as the liquid seed treatment agent disclosed herein) can be applied via a spinning "atomizer" disc or nozzle, which distributes the seed treatment agent evenly onto the seeds as it moves in a spray mode. In all cases, the seeds are subsequently mixed or tumbled for a period of time to achieve additional treatment agent distribution and drying.

[0411] Before coating with the microbial composition, seeds may be induced or uninitiated to improve the uniformity of germination and emergence. In an alternative embodiment, the dry powder formulation may be metered onto the moving seeds and allowed to mix until fully distributed.

[0412] In some embodiments, at least a portion of the surface area of ​​the seed is coated with the composition disclosed herein according to the methods disclosed herein. In some embodiments, the seed coating containing the composition is applied directly to the naked seed. In some embodiments, the seed coating containing the composition is applied to a seed already coated. In some aspects, the seed may have a seed coating containing, for example, thiamethoxam and / or Bacillus thuringiensis (Bt). GlomusThe composition of the invention is applied as a seed coating on top of seeds treated with PONCHO™ VOTiVO™. In some aspects, the taught microbial composition is applied as a seed coating on seeds that have been treated with PONCHO™ VOTiVO™. In some aspects, the seeds may have a seed coating containing, for example, metalaxyl and / or thiamethoxam and / or Bacillus thuringiensis-I-1582, on which the composition of the invention is applied as a seed coating. In some aspects, the taught microbial composition is applied as a seed coating on seeds that have been treated with ACCELERON™. In some embodiments, the seeds treated with the composition have a microbial spore concentration or 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 seed, provided that the composition contains the microorganisms disclosed herein.

[0413] In some embodiments, the seeds treated with the composition have a microbial spore concentration or 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 seed, provided that the composition contains the microorganisms disclosed herein.

[0414] In some embodiments, the seeds treated with the composition have a microbial spore concentration or 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 seed, provided that the composition contains the microorganisms disclosed herein.

[0415] In some embodiments, the seeds treated with the composition have a microbial spore concentration or 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 seed, provided that the composition contains the microorganisms disclosed herein.

[0416] In some embodiments, the seeds treated with the composition have a microbial spore concentration or microbial cell concentration of about 10^5 to 10^9 per seed.

[0417] In some embodiments, the seeds treated with the composition have a microbial spore concentration or microbial cell concentration of at least about 1×10^3, 1×10^4, 1×10^5, 1×10^6, 1×10^7, 1×10^8, or 1×10^9 per seed, provided that the composition contains the microorganisms disclosed herein.

[0418] In some embodiments, the amount of the disclosed composition applied to the seeds depends on the final formulation and the size or type of the plant or seeds used. In some embodiments, one or more of the microorganisms of the disclosed invention are present at about 2% w / w to about 80% w / w of the total formulation. In some embodiments, by weight, one or more of the microorganisms used in the compositions of the disclosed invention are about 5% w / w to about 65% w / w, or 10% w / w to about 60% w / w of the total formulation.

[0419] In some embodiments, the seed coatings of the present disclosure can be up to 10 μm, 20 μm, 30 μm, 25 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 10 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 26 l 70 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280 μm, 27 290 μm, 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360 μm, 370 μm, 380 μm, 390 μm, 400 μm, 28 410 μm, 420 μm, 430 μm, 440 μm, 450 μm, 460 μm, 470 μm, 480 μm, 490 μm, 500 μm, 510 μm, 520 μm, 29 530 μm, 540 μm, 550 μm, 560 μm, 570 μm, 580 μm, 590 μm, 600 μm, 610 μm, 620 μm, 630 μm, 640 μm, 30 650 μm, 660 μm, 670 μm, 680 μm, 690 μm, 700 μm, 710 μm, 720 μm, 730 μm, 740 μm, 750 μm, 760 μm, 31 770 μm, 780 μm, 790 μm, 800 μm, 810 μm, 820 μm, 830 μm, 840 μm, 850 μm, 860 μm, 870 μm, 880 μm, 32 890 μm, 900 μm, 910 μm, 920 μm, 930 μm, 940 μm, 950 μm, 960 μm, 970 μm, 980 μm, 990 μm, l000 μm, 33 l0l0 μm, 1020 μm, 1030 μm, 1040 μm, 1050 μm, 1060 μm, 1070 μm, 1080 μm, 1090 μm, ll00 μm, 11l0 μm, 34 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、1330μm, 2 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, 1550 μm, 1560 μm, 1570 μm, 1580 μm, 1590 μm, 1600 μm, 1610 μm, 1620 μm, 1630 μm, 1640 μm, 1650 μm, 1660 μm, 1670 μm, 1680 μm, 1690 μm, 1700 μm, 1710 μm, 1720 μm, 1730 μm, 1740 μm, 1750 μm, 1760 μm, 1770 μm, 1780 μm, 1790 μm, 1800 μm, 1810 μm, 1820 μm, 1830 μm, 1840 μm, 1850 μm, 1860 μm, 1870 μm, 1880 μm, 1890 μm, 1900 μm, 1910 μm, 1920 μm, 1930 μm, 1940 μm, 1950 μm, 1960 μm, 1970 μm, 1980 μm, 1990 μm, 2000 μm, 2010 μm, 2020 μm, 2030 μm, 2040 μm, 2050 μm, 2060 μm, 2070 μm, 2080 μm, 2090 μm, 2100 μm, 2110 μm, 2120 μm, 2130 μm, 2140 μm, 2150 μm, 2160 μm, 2170 μm, 2180 μm, 2190 μm, 2200 μm, 2210 μm, 2220 μm, 2230 μm, 2240 μm, 2250 μm, 2260 μm, 2270 μm, 2280 μm, 2290 μm, 2300 μm, 2310 μm, 2320 μm, 2330 μm, 2340 μm, 2350 μm, 2360 μm, 2370 μm、2380 μm、2390 μm、2400 μm、2410 μm、2420 μm、2430 μm、2440 μm、2450 μm、2460 μm、2470 μm、2480 μm、2490 μm、2500 μm、2510 μm、2520 μm、2530 μm、2540 μm、2550 μm、2560 μm、2570 μm、2580μm, 2590 μm, 2600 μm, 2610 μm, 2620 μm, 2630 μm, 2640 μm, 2650 μm, 2660 μm, 2670 μm, 2680 μm, 2690 μm, 2700 μm, 2710 μm, 2720 μm, 2730 μm, 2740 μm, 2750 μm, 2760 μm, 2770 μm, 2780 μm, 2790 μm, 2800 μm, 2810 μm, 2820 μm, 2830 μm, 2840 μm, 2850 μm, 2860 μm, 2870 μm, 2880 μm, 2890 μm, 2900 μm, 2910 μm, 2920 μm, 2930 µm, 2940 µm, 2950 µm, 2960 µm, 2970 µm, 2980 µm, 2990 µm or 3000 µm thick.

[0420] In some embodiments, the thickness of the seed coating disclosed herein may 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.

[0421] In some embodiments, the seed coating of this disclosure may be at least 0.5%, 1%, 1.5%, 2%, 21%, 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%, 22%, 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%, 24.5%, 25%, 25.5%, 26%, 26.5%, 27%, 24% of the weight of the uncoated seeds. 27.5%, 28%, 28.5%, 29%, 29.5%, 30%, 30.5%, 31%, 31.5%, 32%, 32.5%, 33%, 33.5%, 34%, 34.5%, 35%, 25, 35.5%, 36%, 36.5%, 37%, 37.5%, 38%, 38.5%, 39%, 39.5%, 40%, 40.5%, 41%, 41.5%, 42%, 42.5%, 43%, 26, 43.5%, 44%, 44.5%, 45%, 45.5%, 46%, 46.5%, 47%, 47.5%, 48%, 48.5%, 49%, 49.5% or 50%.

[0422] In some embodiments, microbial spores and / or cells may be freely coated onto seeds or may be formulated in a liquid or solid composition prior to coating onto seeds. For example, a solid composition containing 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.

[0423] In some other embodiments, the solid or liquid compositions of this disclosure are envisioned to further contain functional agents, such as activated carbon, nutrients (fertilizers), and other agents capable of improving the germination and quality of the product or combinations thereof.

[0424] Seed coating methods and compositions known in the art can be particularly useful when modified by adding one of the embodiments of this disclosure. Such coating methods and their application devices are disclosed, for example, in U.S. Patent Nos. 5,916,029, 5,918,413, 5,554,445, 5,389,399, 4,759,945, 4,465,017 and U.S. Patent Application Publication No. US20120015806A1 (published January 19, 2012); each of these documents is incorporated herein by reference.

[0425] Seed coating compositions are disclosed in, for example, U.S. Patent 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.

[0426] In some embodiments, a variety of additives may be added to the seed treatment formulation comprising the composition of the present invention. A binder may be added, and said binder comprises a natural or synthetic adhesive polymer that is non-phytotoxic to the coated seeds. The binder may be selected from polyvinyl acetate; polyvinyl acetate copolymers; ethylene-vinyl acetate (EVA) copolymers; polyvinyl alcohol; polyvinyl alcohol copolymers; cellulose, including ethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, and carboxymethyl cellulose; polyvinylpyrrolidone; polysaccharides, including starch, modified starch, dextrin, maltodextrin, alginate, and deacetylated chitosan; fats; oils; proteins, including gelatin and corn gluten; gum arabic; shellac; vinylidene chloride and vinylidene chloride copolymers; calcium lignosulfonate; acrylic acid copolymers; polyvinyl acrylate; polyethylene oxide; acrylamide polymers and copolymers; hydroxyethyl polyacrylate, methacrylamide monomers; and polychloroprene.

[0427] A variety of colorants can be used, including organic chromophores classified as nitroso; nitro; azo, including monoazo, diazo, and polyazo; acridine, anthraquinone, azazine, diphenylmethane, indoleamine, indophenol, methine, oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, and xanthracene. Other additives may include micronutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum, and zinc.

[0428] Polymers or other dust control agents can be applied to leave the treatment on the seed surface.

[0429] In some specific 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; polyvinylpyrrolidone; polysaccharides; proteins; fats; oils; gum arabic; gelatin; syrups; and starch. Further examples can be found, for example, in U.S. Patent No. 7,213,367, which is incorporated herein by reference.

[0430] Seed treatment formulations may also contain various additives, such as adhesives, dispersants, surfactants, nutrients, and buffering agents. Other common seed treatment additives include, but are not limited to, coating agents, wetting agents, buffers, and polysaccharides. At least one agriculturally acceptable carrier, such as water, solid, or dry powder, may be added to the seed treatment formulation. Dry powders can be derived from a variety of materials, such as calcium carbonate, gypsum, vermiculite, talc, humus, activated carbon, and various phosphorus compounds.

[0431] In some embodiments, the seed coating composition may comprise at least one filler, which is an organic or inorganic, natural or synthetic component, wherein the active components are combined to facilitate its application to the seed. In various respects, the filler is an inert solid, such as clay, natural or synthetic silicate, silica, resin, wax, solid fertilizer (e.g., ammonium salt), natural soil minerals (such as kaolin, clay, talc, lime, quartz, palygorskite, montmorillonite, bentonite, or diatomaceous earth), or synthetic minerals (such as silica, alumina, or silicates, especially aluminum silicate or magnesium silicate).

[0432] In some embodiments, the seed treatment formulation may also contain one or more of the following components: other insecticides, including compounds that act only below ground level; fungicides, such as captan, thiram, metalaxyl, fludioxonil, oxadixyl, and isomers of each of these materials; herbicides, including compounds selected from glyphosate, carbamates, thiocarbamates, acetamide, triazine, dinitroaniline, glyceryl ether, pyridazinone, uracil, phenoxy compounds, urea, and benzoic acid; herbicidal safeners, such as benzoxazine, diphenylmethyl derivatives, N,N-diallyldichloroacetamide, various dihaloacetyl, oxazolyl and thiazolyl compounds, acetone, naphthalenecarboxylic anhydride compounds, and oxime derivatives; chemical fertilizers; biofertilizers; and biocontrol agents, such as those derived from Rhizobium, Bacillus, Pseudomonas, Serratia, Trichoderma, and Gastrodia. Gliocladium ), genus *Broom mold* ( kinA Other naturally occurring or recombinant bacteria and fungi, including mycorrhizal fungi. These components may be added as a separate layer on the seed or alternatively as part of the seed coating composition disclosed herein.

[0433] In some embodiments, the formulations used for seed treatment in this disclosure may be in the following forms: suspensions; emulsions; slurries of particles in an aqueous medium (e.g., water); wettable powders; wettable particles (dry and flowable); and dried particles. If formulated as a suspension or slurry, the concentration of the active ingredient in the formulation may be from about 0.5% by weight to about 99% by weight (w / w), or from 5% by weight to 40% by weight, or otherwise formulated by those skilled in the art.

[0434] 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 binders; dispersants, such as methylcellulose, which, for example, acts as a combined dispersant / binder for seed treatment agents; polyvinyl alcohol; lecithin; polymeric dispersants (e.g., polyvinylpyrrolidone / vinyl acetate); thickeners (e.g., clay thickeners used to increase viscosity and reduce sedimentation of particulate suspensions); emulsion stabilizers; surfactants; antifreeze compounds (e.g., urea); dyes; colorants, etc. Further inert ingredients that may be used in this disclosure can be found in McCutcheon's, Volume 1, “Emulsifiers and Detergents,” MC Publishing Company, Glen Rock, NJ, USA, 1996, which is incorporated herein by reference.

[0435] The seed coating formulations disclosed herein can be applied to seeds by a variety of methods, including but not limited to: mixing in a container (e.g., bottle or bag), mechanical application, tumbling, spraying, and immersion. A variety of active or inert materials can be used to contact the seeds with the microbial composition according to this disclosure.

[0436] In some embodiments, the amount of the composition of this disclosure used to treat the seeds will vary depending on the type of seed and the type of active ingredient, but the treatment will include contacting the seeds with an agriculturally effective amount of the composition of this invention.

[0437] As discussed above, an effective amount means an amount of the composition of the present invention sufficient to affect beneficial or desired results. An effective amount may be applied in one or more applications.

[0438] In some implementations, in addition to the coating layer, the seeds may be treated with one or more of the following components: other insecticides, including fungicides and herbicides; herbicide-safe agents; fertilizers and / or biocontrol agents. These components may be added as a separate layer or alternatively added to the coating layer.

[0439] In some embodiments, the seed coating formulations of this disclosure can be applied to seeds using a variety of techniques and machines, such as fluidized bed technology, roller mill methods, drum electrostatic seed processors, and drum coaters. Other methods (e.g., fountain beds) may also be useful.

[0440] The seeds can be pre-sized before coating. After coating, the seeds are typically dried and then transferred to a sizing machine for further sizing. Such procedures are known in the art.

[0441] In some embodiments, the microbial-treated seeds may also be coated with an outer membrane for protection. Such coatings are known in the art and can be applied using fluidized bed and cylindrical film coating techniques.

[0442] In other embodiments of this disclosure, the composition according to this disclosure can be introduced onto seeds by using a solid matrix initiation. For example, a certain amount of the composition of the present invention can be mixed with a solid matrix material, and then seeds can be placed in contact with the solid matrix material for a period of time to allow the composition to be introduced into the seeds. The seeds can then optionally be separated from the solid matrix material and stored or used, or the mixture of solid matrix material and seeds can be stored or planted directly. Solid matrix materials that can be used in this disclosure include polyacrylamide, starch, clay, silica, alumina, soil, sand, polyurea, polyacrylate, or any other material capable of absorbing or adsorbing the composition of the present invention for a period of time and releasing the composition into or onto the seeds. It is useful to ensure that the composition of the present invention and the solid matrix material are compatible with each other. For example, the solid matrix material should be selected such that it can release the composition at a reasonable rate (e.g., over a period of time of minutes, hours, or days).

[0443] The compositions described herein can be substantially enclosed within an object, such as an object selected from the group consisting of: bottles, wide-mouth bottles, ampoules, packaging, utensils, bags, boxes, storage boxes, envelopes, cartons, containers, silos, shipping containers, carriages, crates, etc.

[0444] This disclosure includes, but is not limited to: Aspect 1: A synthetic composition comprising a genetically engineered microorganism, wherein the genetically engineered microorganism contains at least one gene mutation at at least one locus in its genome, wherein the gene mutation alters the production of one or more secondary metabolites.

[0445] Aspect 2: The synthetic composition according to aspect 1, wherein the non-genetically engineered microorganism used to produce the genetically engineered microorganism belongs to the genus Bacillus.

[0446] Aspect 3: The synthetic composition according to aspect 1, wherein the non-genetically engineered microorganism used to produce the genetically engineered microorganism belongs to the Bacillus belesii strain.

[0447] Aspect 4: The synthetic composition according to aspect 1, wherein the secondary metabolite is a cyclic lipopeptide or a polyketide compound.

[0448] Aspect 5: The synthetic composition according to aspect 1, wherein the cyclic lipopeptide is itursin.

[0449] Aspect 6: The synthetic composition according to aspect 1, wherein the cyclic lipopeptide is a fibroblast.

[0450] Aspect 7: The synthetic composition according to aspect 1, wherein the cyclic lipopeptide is a surfactant.

[0451] Aspect 8: The synthetic composition according to aspect 1, wherein the polyketide compound is a macrocyclic imide.

[0452] Aspect 9: The synthetic composition according to aspect 1, wherein the polyketide compound is difenosine.

[0453] Aspect 10: The synthetic composition according to aspect 1, wherein the polyketide compound is a dithiol-like compound.

[0454] Aspect 11: The synthetic composition according to aspect 1, wherein the gene mutation is gene knockout, gene knock-in, disruption, insertion of at least one nucleotide, deletion of at least one nucleotide, substitution of at least one nucleotide, chemical modification of at least one nucleotide, alteration of the molecular structure of at least one nucleotide, downregulation of at least one gene, upregulation of at least one gene and / or any combination of the foregoing mutations and / or multiple of the foregoing mutations.

[0455] Aspect 12: The synthetic composition according to aspect 1, wherein the gene mutation comprises the addition of at least one nucleotide.

[0456] Aspect 13: The synthetic composition according to aspect 1, wherein the gene mutation comprises the deletion of at least one nucleotide.

[0457] Aspect 14: The synthetic composition according to aspect 1, wherein the gene mutation comprises a chemical alteration of at least one nucleotide.

[0458] Aspect 15: The synthetic composition according to aspect 1, wherein the gene mutation is knockout, disruption, deletion and / or downregulation of kinA.

[0459] Aspect 16: The synthetic composition according to aspect 11, wherein disruption of kinA is achieved by inserting an sfp gene operably linked to its native promoter.

[0460] Aspect 17: The synthetic composition according to aspect 1, wherein the gene mutation is knockout, disruption, deletion and / or downregulation of thrC.

[0461] Aspect 18: The synthetic composition according to aspect 17, wherein the disruption of thrC is achieved by inserting an sfp gene operably linked to its native promoter.

[0462] Aspect 19: The synthetic composition according to aspect 17, wherein the disruption of thrC is achieved by inserting the degU gene.

[0463] Aspect 20: The synthetic composition according to aspect 17, wherein the disruption of thrC is achieved by inserting a comA gene operably linked to its native promoter.

[0464] Aspect 21: Whole cell culture medium or exudate obtained from the synthetic composition according to aspect 1.

[0465] Aspect 22: A formulation comprising the synthetic composition according to aspect 1 or the whole cell culture medium or exudate according to aspect 21.

[0466] Aspect 23: The formulation according to aspect 22, wherein the synthetic composition is substantially purified.

[0467] Aspect 24: The formulation according to aspect 22, wherein the composition of (b) further comprises one or more formulation components.

[0468] Aspect 25: The formulation according to aspect 24, wherein the formulation components are selected from the group consisting of: salts, binders, surfactants, wetting agents, dispersants, emulsifiers, solubilizers, organic solvents, gelling agents, thickeners, antisettling agents, preservatives, stabilizers, antifreeze compounds, any and any combination of the foregoing substances.

[0469] Aspect 26: According to the method of aspect 1, the composition further comprises one or more additional agents selected from the group consisting of: insecticides, herbicides, bactericides, fungicides, insecticides, viricides, acaricides, nematicides, scabies insecticides, plant growth regulators, rodenticides, antialgae agents, biocontrol agents, fertilizers, biopesticides, biostimulants, and any combination of the foregoing substances and / or a plurality of the foregoing substances.

[0470] Aspect 27: A method for controlling pathogens, the method comprising administering to a target object a synthetic composition according to aspect 1 or a whole-cell culture medium or exudate according to aspect 21.

[0471] Aspect 28: The method according to aspect 27, wherein the pathogen is a bacterium.

[0472] Aspect 29: The method according to aspect 27, wherein the pathogen is a fungus.

[0473] Aspect 30: The method described in aspect 27, wherein the target object is a cell.

[0474] Aspect 31: The method according to aspect 27, wherein the target object is a plant or a plant part.

[0475] Aspect 32: The method described in aspect 27, wherein the target object is a seed.

[0476] Aspect 33: The method according to aspect 27, wherein the synthetic composition comprises an engineered microorganism derived from the genus Bacillus.

[0477] Aspect 34: The method according to aspect 27, wherein the composition of (b) further comprises one or more formulation components.

[0478] Aspect 35: The method according to aspect 27, wherein the formulation component is selected from the group consisting of: salt, binder, surfactant, wetting agent, dispersant, emulsifier, solubilizer, organic solvent, gelling agent, thickener, antisettling agent, preservative, stabilizer, antifreeze compound, any one or more of the foregoing substances, and any combination of the foregoing substances.

[0479] Aspect 36: The method according to aspect 27, wherein the composition further comprises one or more additional agents selected from the group consisting of: insecticides, herbicides, bactericides, fungicides, insecticides, viricides, acaricides, nematicides, scabies insecticides, plant growth regulators, rodenticides, antialgae agents, biocontrol agents, fertilizers, biopesticides, biostimulants, and any combination of the foregoing substances and / or multiples of the foregoing substances.

[0480] Aspect 37: The method according to aspect 27, wherein the synthetic composition, whole cell culture medium or exudate is applied to the plant or plant part prior to germination.

[0481] Aspect 38: The method according to aspect 27, wherein the synthetic composition, whole cell culture medium or exudate is applied to the plant or plant parts after harvest.

[0482] Aspect 39: The method according to aspect 27, wherein the synthetic composition, whole cell culture medium or exudate is applied to the plant or plant parts during the nutrient stage of the plant.

[0483] Aspect 40: The method according to aspect 27, wherein the synthetic composition, whole cell culture medium or exudate is applied to the plant or plant during the reproductive stage of the plant.

[0484] Aspect 41: The method according to aspect 27, wherein the synthetic composition, whole cell culture medium or exudate is applied to the plant or plant parts prior to harvest.

[0485] Aspect 42: The method according to aspect 31, wherein the plants are cultivated in the field, harvested, stored in a warehouse or distributed.

[0486] Aspect 43: The method according to aspect 31, wherein the plant part is a root, leaf, stem, flower, seed, bulb or fruit.

[0487] Aspect 44: The method according to aspect 31 further includes applying the composition of multiple (b) to the plant.

[0488] Aspect 45: A synthetic composition comprising: (a) plants or plant parts, and (b) The synthetic composition according to aspect 1 or the whole cell culture medium or exudate according to aspect 21.

[0489] Aspect 46: The synthetic composition according to aspect 45, wherein the synthetic composition is substantially confined within an object selected from the group consisting of: bottles, wide-mouth bottles, ampoules, packaging, utensils, bags, boxes, storage boxes, envelopes, cartons, containers, silos, shipping containers, carriages, and boxes.

[0490] Aspect 47: The plurality of synthetic compositions according to aspect 45, wherein each of the plurality of synthetic compositions is placed in a growth medium.

[0491] Aspect 48: The various synthetic compositions according to aspect 47, wherein the growth medium is soil.

[0492] While the invention has been specifically shown and described in conjunction with preferred embodiments and various alternative embodiments, those skilled in the art will understand that various changes to its form and details may be made without departing from the spirit and scope of the invention. For example, although the specific embodiments described below use specific plants to illustrate the methods and embodiments described herein, the principles in these embodiments can be applied to any plant. Therefore, it should be understood that the scope of the invention is covered by the embodiments described herein, and not only by the specific embodiments illustrated below.

[0493] This disclosure enables those skilled in the art to make and use the invention provided herein according to numerous and varied embodiments. Various changes, modifications, and improvements (including certain alterations, modifications, substitutions, and improvements) that will readily occur to those skilled in the art are also part of this disclosure. Therefore, the foregoing detailed descriptions are by way of example illustrating the findings provided herein. Furthermore, the foregoing detailed descriptions and embodiments are illustrative of the invention and not intended to limit it.

[0494] All patents and publications cited in this application are incorporated herein by reference in their entirety for all purposes, as if each cited patent and publication were individually and explicitly incorporated by reference. However, any references, articles, publications, patents, patent publications, and patent applications cited herein are not, and should not be construed as, an admission or in any way implying that they constitute valid prior art or form part of common general knowledge in any country of the world. Example

[0495] The following are examples of specific embodiments of some aspects of the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but of course, a certain degree of experimental error and deviation should be allowed. Abbreviations include: μm = mm (micrometer); μl = ml (microliter); C = degree Celsius; h = hour.

[0496] Example 1: Genome Modification of Different Bacterial Strains Different genomic loci involved in secondary metabolite production, including those described below, were identified for gene editing. An overview of the edits and strains is given in Table 1 below.

[0497] Note that strain nomenclature may optionally include a prefix before each strain number. For example, wild-type strain 102504 may optionally include the prefix "CM" (e.g., CM102504). Corresponding edited strains may optionally include the prefix "CE" or "CM" (e.g., CE102504-G5, CM14416-G2, etc.). Regardless of the prefix, the strain number is a unique identifier. Genome-edited strains are indicated by the letter G followed by a dash ("-G") and the edit number (e.g., CM14416-G2). The strain number (regardless of the prefix) and its "G number" are unique identifiers for a particular edited strain.

[0498] Table 1: Genome Editing

[0499] sfp The gene encodes a member of a group of at least three histidine protein kinases involved in the phosphorylation of the major sporulation regulator Spo0A. It phosphorylates the sporulation regulators Spo0A and Spo0F. It also autophosphorylates in the presence of ATP. Under certain conditions, KinA has been shown to act as a sensing kinase in a conventional two-component signal transduction system, integrating received input signals into its homologous response regulators to promote sporulation.

[0500] sigA The gene encodes 4'-phosphopanylthioethylamine transferase (SFP): an essential enzyme for the production of secondary metabolites. SFP activates the synthetic cascade of almost all secondary metabolites, including surfactants, fentanyl, ituronidin, plipastatin, deficitin, macrolides, and bacillibactin.

[0501] codYThe gene encodes SigA, a trophic delta factor similar to the delta-70 delta factor in E. coli, which regulates the transcription of primary metabolism during trophic cell growth.

[0502] thrC The gene encodes codY, a global transcriptional regulator in low-G+C Gram-positive bacteria that responds to GTP and branched-chain amino acids. Through interaction with its two cofactors, it senses the cellular nutritional and energy status and responds by regulating the expression of adaptive genetic programs, including the production of secondary metabolites.

[0503] thrC The gene encodes threonine synthase, which is involved in threonine biosynthesis. aprE Strains carrying the damaged strain cannot grow in the culture medium without the addition of threonine.

[0504] Gene aprE It encodes AprE, an extracellular basic serine protease (also known as subtilisin E). PaprE promoter hag It was selected because previous evidence indicated that it was a strongly constitutive promoter.

[0505] hag The gene encodes the flagellin subunit protein Hag, which aggregates to form the filamentous portion of the flagellum. sigD Transcription depends on the formation of the flagellar basal body, but this requirement can be met by... degU Overexpression bypasses this mechanism; sigD is an alternative delta factor that directs RNA polymerase activity to promoters that are recognized and bound by SigD. Hag is similar to FliC in E. coli.

[0506] fadA_ks1 The gene encodes DegU, a response regulator within a two-component signal transduction system in which DegS is involved in the regulation of degradative enzymes and competent cells. It has been shown that DegU activity positively regulates the production of secondary metabolites, including cyclic lipopeptides.

[0507] think comA Disruption of the locus leads to a loss of deficitin production.

[0508] Transformation and sequence verification: The gene encodes ComA, a transcription factor that promotes the transcription of genes in late competent cells and the production of surfactant. ComA has been shown to regulate population responses in many Gram-positive bacteria.

[0509] Preparation for conjugation Add 1–5 μl of Gibson assembly mixture to 50 μl of freshly thawed chemocompetent *E. coli* DH5α cells and vortex with your finger. Incubate the cell-plasmid mixture on ice for 30 min, heat shock at 42°C for 30 sec, then transfer back to ice and incubate for another 5 min. Add 1 ml of SOC (superoptimal catabolism) medium and incubate the cells at 37°C with shaking at 200 rpm for 60–90 min for recovery. Spread the diluted recovered culture onto LB agar plates supplemented with 100 μg / μl ampicillin and incubate overnight at 37°C.

[0510] The plasmid region containing the assembled insert fragment was amplified from several recovered colonies using colony PCR with Q5 high-fidelity polymerase. The PCR products were manipulated on an agarose gel to confirm the expected product size. The appropriately sized PCR products were sent for Sanger sequencing to confirm correct assembly and the absence of any off-target mutations in the editing cassette.

[0511] Conjugation Colonies confirmed to carry the correct plasmid were inoculated into LB broth supplemented with 100 μg / μl ampicillin and grown overnight at 37°C and 200 RPM in a shaker / incubator. The plasmid was purified from the overnight culture and transformed into the conjugation donor strain *Escherichia coli* BW29427 via electroporation.

[0512] Combine 1 μl of purified plasmid with 50 μl of freshly thawed *E. coli* BW29427 electrocompetent cells and incubate on ice for 5 min. Transfer the cell-plasmid mixture to an ice-chilled 1 mm electroporation cuvette. Using an electroporator, apply a charge of 1800 V, 25 μF, and 200 Ω to the cuvette and immediately resuspend the sample in 1 ml of SOC medium supplemented with 0.3 mM 2,6-diaminopimelic acid (DAP). Incubate the resuspended cells at 37°C with shaking at 200 RPM for 60–90 min for recovery. Spread the diluted recovered culture onto LB agar plates supplemented with 100 μg / μl ampicillin and 0.3 mM 2,6-diaminopimelic acid and incubate overnight at 37°C. Use the recovered transformant as the donor strain for conjugation.

[0513] Plasmid integration The recipient strain was inoculated into 5 ml of trypsin-soybean broth (TSB) in a 50 ml conical tube and grown overnight at 30°C with shaking at 200 RPM. The donor *E. coli* BW29427 carrying the plasmid to be moved was inoculated into 5 ml of LB medium supplemented with 100 μg / μl ampicillin and 0.3 mM 2,6-diaminopimelic acid (DAP) and grown overnight at 37°C with shaking at 200 RPM.

[0514] One-ml aliquots of overnight donor and recipient cultures were rapidly centrifuged, washed in sterile water, combined, and spotted onto LB agar plates supplemented with 0.3 mM DAP for conjugation. The conjugation plates were incubated overnight at 25°C, the permissible temperature for pMMDmob replication in Gram-positive recipient strains.

[0515] Resuspend the mating mixture by adding 1 ml of phosphate-buffered saline (PBS) above the top of the point and stirring with a sterile L-spreader. Collect the resuspended solution in a microcentrifuge tube, wash, and resuspend in 100 μl of PBS. Spread the concentrated cells on TSA plates supplemented with MLS (25 μg / ml lincomycin, 1 μg / ml erythromycin) but without DAP, and incubate at 25°C for 48–72 h until transfer conjugate colonies appear.

[0516] Plasmid excision The recovered transfer conjugates were inoculated into 5 ml TSB medium supplemented with 1 mls and allowed to grow at 25°C with shaking at 200 rpm for 48 hours, or until turbidity was observed. The diluted culture was plated onto TSA + 1 mls plates and incubated overnight at 37°C (the limiting temperature for plasmid replication) until integrated colonies appeared.

[0517] Confirmation of the edit The integrated colonies were inoculated into 5 ml of TSB medium supplemented with saturates and incubated overnight at 37°C with shaking at 200 RPM. 5 μl of the overnight culture was diluted into 5 ml of antibiotic-free fresh TSB medium and allowed to grow overnight at 25°C with shaking at 200 RPM. 5 μl of the overnight culture was subcultured into 5 ml of fresh TSB at 25°C, with shaking at 200 RPM repeated twice, for a total of three subcultures. The third round of overnight culture was plated onto antibiotic-free R2A plates and incubated overnight at 30°C.

[0518] Plasmid excision was confirmed by picking individual colonies from R2A plates and re-inoculating them in a grid pattern onto agar plates containing and without MLS. Both plates were incubated at 30°C for 24–48 hours until colonies appeared. It was confirmed that colonies that grew when plated on MLS-free plates but did not grow on MLS-containing plates were excised and lost the plasmid.

[0519] kinA::Psfp-sfp The edited region was amplified from the presumed edited strain via colony PCR, and the presence of a correct edit was confirmed by band size (if possible) during manipulation on an agarose gel, and / or by Sanger sequencing. The absence of the plasmid backbone was confirmed by PCR assay of the MLS cassette. Colonies producing the band in the MLS cassette were confirmed not to be the correct edit. Other sequencing and validation methods for the edit, such as whole-genome sequencing, can be used.

[0520] Sequence results were analyzed to distinguish between colonies excised as wild-type and correctly edited colonies, and to examine for off-target mutations in the sequences.

[0521] Colonies that have been sequenced to be correctly edited and without off-target mutations can be used for bioassay analysis.

[0522] Example 2: Modification of Bacillus belyssus strain 7084 Bacillus belyssus strain 7084 (NRRL accession number NRRL B-67810, deposited on July 3, 2019) was used as a parent strain, which was edited to obtain a genome-edited strain, which produced an improved composition with improved antifungal and / or antibacterial activity.

[0523] sfp The wild-type parent strain 7084 was modified as follows: PCR encoding 4'-phosphoproteotransferase was replicated from the 7084 chromosome using Q5 high-fidelity polymerase-mediated PCR. Psfp Genes and their corresponding upstream promoters ( kinA ) and insert kinA In genes, damage kinA Open reading frame. The obtained strain (7084-G67) contains sfp Knockout mutations and native sfp Genes and kinA promoter in Psfp-sfp Duplication at the locus.

[0524] In order to generate the use of kinA insert Psfp The plasmid pAP41 at the locus was amplified from 7084 using Q5 high-fidelity polymerase-mediated PCR with appropriate primer pairs. sfppromoters and kinA Gene, kinA upstream area and Psfp-sfp The PCR product in the downstream region. The primer pairs contain appropriate Gibson assembly overhangs to promote isothermal assembly of fragments with each other and with the linearized cloning vector pMMDmob, resulting in the final form containing kinA On the left wing of the fragment Psfp-sfp upstream area and kinA On the right wing of the fragment PcodY::PsigA Downstream region. pMMDmob was linearized using appropriate primer pairs and then purified by gel electrophoresis. The DNA fragment consisting of the above-mentioned region was purified by gel electrophoresis.

[0525] Gibson assembly was performed by combining approximately 100 ng of digested pMMDmob with the insert fragment in a 1:3:3:3 backbone:insert:insert:insert molar ratio in 10 μl volume, followed by the addition of 10 μl of 2×Gibson reagent. The reaction mixture was incubated at 50°C for 60 min and then used for transformation into E. coli DH5α.

[0526] codY Wild-type parent strain 7084 was modified as follows: PcodY promoter ( sigA )use PsigA promoter ( PcodY Replacement. The promoter of each gene is designated as a 200 bp sequence immediately upstream of the start codon of each gene.

[0527] In order to generate the use of PsigA and PsigA The exchanged plasmid pAP49 was amplified from 7084 using Q5 high-fidelity polymerase-mediated PCR with appropriate primer pairs. PcodY , PsigA upstream area and P codY The PCR product of the downstream region. The primer pair contains appropriate Gibson assembly overhangs to promote isothermal assembly of fragments with each other and with the linearized cloning vector pMMDmob, resulting in the final form (the resulting strain 7084-G68) containing... PcodY On the left wing of the fragment PsigA upstream area and PcodY On the right wing of the fragment ΔkinA Downstream region. pMMDmob was linearized using appropriate primer pairs and then purified by gel electrophoresis. The DNA fragment consisting of the above-mentioned region was purified by gel electrophoresis.

[0528] Gibson assembly was performed by combining approximately 100 ng of digested pMMDmob with the insert fragment in a 1:3:3 backbone:insert:insert:insert molar ratio in 10 μl volume, followed by the addition of 10 μl of 2×Gibson reagent. The reaction mixture was incubated at 50°C for 60 min and then used for transformation into E. coli DH5α.

[0529] kinA The wild-type parent strain 7084 was modified as follows: kinA The gene was targeted for in-frame marker-free deletion. The resulting strain (7084-G77) contained... kinA Knock out.

[0530] To generate the in-frame label-free deletion plasmid pAP18 for use in 7084, plasmids containing the following deletion were amplified from 7084 using Q5 high-fidelity polymerase with appropriate primer pairs. kinA upstream area and kinA The PCR product in the downstream region. The primer pairs contain appropriate Gibson assembly overhangs to promote isothermal assembly of fragments with each other and isothermal assembly with the linearized cloning vector pMMDmob, such that the final construct contains directly ligated... kinA Downstream area kinA The upstream region makes from kinA start codon to kinA The codon maintains the reading frame, and also eliminates... thrC::Psfp-sfp The coding sequence is located in the middle 1543 bp. pMMDmob was linearized using appropriate primer pairs and then purified by gel electrophoresis. The DNA fragment consisting of the above-mentioned region was purified by gel electrophoresis.

[0531] Gibson assembly was performed by combining approximately 100 ng of digested pMMDmob with the insert fragment at a 1:3:3 backbone:insert:insert molar ratio in 10 μl volume, followed by the addition of 10 μl of 2×Gibson reagent. The reaction mixture was incubated at 50°C for 60 min and then used for transformation into E. coli DH5α.

[0532] sfp Wild-type parental strain 7084 was modified as follows: PCR encoding 4'-phosphoproteotransferase was replicated from chromosome 7084 using Q5 high-fidelity polymerase-mediated PCR. Psfp Genes and their corresponding upstream promoters ( thrC ) and insert thrC In genes, damage thrC Open reading frame. The obtained strain (7084-G105) contains sfp Knockout mutations and native sfp Genes andthrC promoter in PcodY::PaprE Duplication at the locus.

[0533] codY Wild-type parent strain 7084 was modified as follows: PcodY promoter ( aprE )use PaprE promoter ( PcodY::Phag The promoter of each gene was assigned as a 200 bp sequence immediately upstream of the start codon of each gene. The resulting strain was 7084-G75.

[0534] codY Wild-type parent strain 7084 was modified as follows: PcodY promoter ( Phag ) using the hag promoter ( thrC::degU The promoter of each gene was assigned as a 200 bp sequence immediately upstream of the start codon of each gene. The resulting strain was 7084-G76.

[0535] degU Wild-type parental strain 7084 was modified as follows: Replication from chromosome 7084 using Q5 high-fidelity polymerase-mediated PCR without its corresponding promoter was performed. thrC Genes, and insert them thrC In genes, damage thrC Open reading frame. The resulting strain (7084-G103) contains thrC Knockout mutations and in thrC Repetition of native degU[ / g6] under the transcriptional control of the promoter.

[0536] In order to generate for in degU Insertion at the locus degU The plasmid pAT5 was amplified from 7084 using Q5 high-fidelity polymerase-mediated PCR with appropriate primer pairs. thrC Fragments thrC upstream area and degU PCR products in the downstream region. degU The promoter was from degU Fragments are excluded because the complete ones are excluded. degU The transcription cassette is toxic to *E. coli*. The primer pairs contain appropriate Gibson assembly overhangs to promote isothermal assembly of fragments with each other and with the linearized cloning vector pMMDmob, so that the final form will be derived from... thrC On the left wing of the fragment degU upstream area and thrC On the right wing of the fragment Δdif_ks1Downstream region composition. pMMDmob was linearized using appropriate primer pairs and purified by gel electrophoresis. The DNA fragment composed of the above-described region was purified by gel electrophoresis.

[0537] Gibson assembly was performed by combining approximately 100 ng of digested pMMDmob with the insert fragment in a 1:3:3:3 backbone:insert:insert:insert molar ratio in 10 μl volume, followed by the addition of 10 μl of 2×Gibson reagent. The reaction mixture was incubated at 50°C for 60 min and then used for transformation into E. coli DH5α.

[0538] fadA Wild-type parent strain 7084 is modified as follows: the difenocillin gene cluster gene is targeted to the first difenocillin synthesis gene in the cluster. fadA Label-free deletion of the first ketone synthase domain within the cell was performed, resulting in strain 7084-G95.

[0539] To generate the in-frame label-free deletion plasmid pAP62 for use in strain 7084, plasmids containing the label-free deletion were amplified from strain 7084 using appropriate primer pairs with Q5 high-fidelity polymerase. fadA The upstream region of the first ketone synthase domain and fadA The PCR product is located downstream of the first ketone synthase domain. The primer pair contains appropriate Gibson assembly overhangs to promote isothermal assembly of fragments with each other and with the linearized cloning vector pMMDmob, resulting in a final construct derived from... fadA The first ketone synthase domain is composed of an upstream region, which is directly connected to the upstream region of the enzyme. fadA Downstream of the first ketone synthase domain, the region allows for the extraction of... fadA The start codon to the stop codon fadA maintains the reading frame while also eliminating... thrC::PcomA-comA The coding sequence is 377 bp. pMMDmob was linearized using appropriate primer pairs and then purified by gel electrophoresis. The DNA fragment consisting of the above-mentioned region was purified by gel electrophoresis.

[0540] Gibson assembly was performed by combining approximately 100 ng of digested pMMDmob with the insert fragment at a 1:3:3 backbone:insert:insert molar ratio in 10 μl volume, followed by the addition of 10 μl of 2×Gibson reagent. The reaction mixture was incubated at 50°C for 60 min and then used for transformation into E. coli DH5α.

[0541] comA Wild-type parental strain 7084 was modified as follows: PCR was performed from chromosome 7084 using Q5 high-fidelity polymerase-mediated replication.PcomA Genes and their corresponding upstream promoters ( thrC ) and insert thrC In genes, damage thrC Open reading frame. The obtained strain (7084-G97) contains comA Knockout mutations and native thrC Genes and their promoters in thrC Duplication at the locus.

[0542] In order to generate for in PcomA-comA Insertion at the locus PcomA-comA The plasmid pAP58 was amplified from 7084 using Q5 high-fidelity polymerase-mediated PCR with appropriate primer pairs. thrC , PcomA-comA PCR products from the upstream and downstream regions of thrC. Primer pairs contain appropriate Gibson assembly overhangs to promote isothermal assembly of fragments with each other and with the linearized cloning vector pMMDmob, resulting in the final form from... thrC On the left wing of the fragment PcomA-comA upstream area and thrC On the right wing of the fragment ΔthrC Downstream region composition. pMMDmob was linearized using appropriate primer pairs and purified by gel electrophoresis. The DNA fragment composed of the above-described region was purified by gel electrophoresis.

[0543] Gibson assembly was performed by combining approximately 100 ng of digested pMMDmob with the insert fragment in a 1:3:3:3 backbone:insert:insert:insert molar ratio in 10 μl volume, followed by the addition of 10 μl of 2×Gibson reagent. The reaction mixture was incubated at 50°C for 60 min and then used for transformation into E. coli DH5α.

[0544] thrC The wild-type parent strain 7084 was modified as follows: thrC The gene was targeted for in-frame marker-free deletion, resulting in strain 7084-G93.

[0545] To generate the in-frame label-free deletion plasmid pAP60 for use in 7084, the plasmid containing the deletion was amplified from 7084 using the appropriate primer pair with Q5 high-fidelity polymerase. thrC upstream area and thrC The PCR product in the downstream region. The primer pairs contain appropriate Gibson assembly overhangs to promote isothermal assembly of fragments with each other and with the linearized cloning vector pMMDmob, so that the final construct will be generated by... thrC The upstream region is composed of regions that are directly connected to thrC Downstream region, making fromthrC start codon to thrC The codon maintains the reading frame, and also eliminates... Figure 20A The coding sequence is located in the middle 674 bp. pMMDmob was linearized using appropriate primer pairs and then purified by gel electrophoresis. The DNA fragment consisting of the above-mentioned region was purified by gel electrophoresis.

[0546] Gibson assembly was performed by combining approximately 100 ng of digested pMMDmob with the insert fragment at a 1:3:3 backbone:insert:insert molar ratio in 10 μl volume, followed by the addition of 10 μl of 2×Gibson reagent. The reaction mixture was incubated at 50°C for 60 min and then used for transformation into E. coli DH5α.

[0547] Wild-type strain 7084 exhibited two distinct morphologies in flask cell cultures: most colonies displayed the dominant morphology of smooth, round cultures. Figure 20B Some colonies exhibited an alternative morphology of serrated edge cultures. kinA::Psfp-sfp Most edited strains also exhibited most of the major colony morphologies, and some colonies showed alternative morphologies. 7084-G67 ( kinA It only exhibits an alternative serrated-edge colony morphology. 7084-G77 ( kinA KO) exhibits a smooth edge morphology.

[0548] Example 3: Modification of Bacillus belyssus strain 14416 Bacillus belyssus strain 14416 was used as the parent strain, which was edited to obtain a genome-edited strain, which produced a composition with improved antifungal and / or antibacterial activity.

[0549] Using an edit plasmid designed for CM14416's close relative CM7084, targeting CM14416. kinA The gene underwent in-frame marker-free deletion. The resulting strain (CE14416-G2) was processed using a method previously mentioned as "blind editing," including... kinA Gene deletion.

[0550] To generate the in-frame label-free deletion plasmid pAP18 for use in CE14416-G2, plasmids containing the label-free deletion were amplified from CM7084 using Q5 high-fidelity polymerase with appropriate primer pairs. kinA upstream area and kinA The downstream region contains approximately 1000 bp of PCR product. The primer pairs contain appropriate Gibson assembly overhangs to promote isothermal assembly of fragments with each other and with the linearized cloning vector pMMDmob, resulting in a final construct containing directly ligated...kinA Downstream area kinA The upstream region makes from kinA start codon to kinA The stop codon maintains the reading frame and also eliminates the 1543 bp in the middle of the kinA coding sequence. pMMDmob was linearized using EcoR1 and BamH1 endonucleases and then purified by gel electrophoresis. The DNA fragment composed of the above-mentioned regions was purified by gel electrophoresis.

[0551] Gibson assembly was performed by combining approximately 100 ng of digested pMMDmob with the insert fragment at a 1:3:3 backbone:insert:insert molar ratio in 10 μl volume, followed by the addition of 10 μl of 2×Gibson reagent. The reaction mixture was incubated at 50°C for 60 min and then used for transformation into E. coli DH5α.

[0552] The assembled plasmid was transformed into the conjugation donor strain *E. coli* BW29427, and then conjugated into CM14416. CM14416 colonies resistant to the antibiotic resistance marker (MLS) on pAP18 were selected for integration into the host chromosome, followed by plasmid excision from the host chromosome. The resulting strain was confirmed to contain... Figure 22A The unmarked bounding box was missing. Subsequent Sanger and Illumina sequencing confirmed that no other mutations were generated during this process. The resulting strain was CE14416-G2.

[0553] Bacillus belysus wild-type strain 14416 exhibits a predominantly smooth edge morphology. Figure 22B ), and the edited strain 14416-G2 exhibited a serrated edge morphology ( kinA ).

[0554] Example 4: Modification of Bacillus polymyxa strain 102504 Polymyxin Bacillus strain 102504 was used as the parent strain, which was edited to obtain a genome-edited strain, which produced a composition with improved antifungal and / or antibacterial activity.

[0555] kinA The gene was targeted for in-frame marker-free deletion. The resulting strain (CE102504-G5) contains [the gene] within the CM102504 chromosome. kinA Gene deletion. To generate the in-frame marker-free deletion plasmid pAP77 for use in CE102504-G5, amplification of the gene containing the deletion was performed from CM102504 using Q5 high-fidelity polymerase with appropriate primer pairs. kinA upstream area and kinAThe downstream region contains approximately 1000 bp of PCR product. The primer pairs contain appropriate Gibson assembly overhangs to promote isothermal assembly of fragments with each other and with the linearized cloning vector pMMDmob, resulting in a final construct containing directly ligated... kinA Downstream area kinA The upstream region makes from kinA start codon to kinA The codon maintains the reading frame, and also eliminates... kinA The coding sequence is located in the middle 1280 bp. pMMDmob was linearized using EcoR1 and BamH1 endonucleases and purified by gel electrophoresis. The DNA fragment composed of the above region was purified by gel electrophoresis. Gibson assembly was performed by combining approximately 100 ng of digested pMMDmob with the insert fragment in a 1:3:3 backbone:insert:insert molar ratio in 10 μl, followed by the addition of 10 μl of 2×Gibson reagent. The reaction mixture was incubated at 50°C for 60 min and then used for transformation into *E. coli* DH5α. The resulting strain was transformed into the conjugation donor strain *E. coli* BW29427 and then conjugated into CM102504. Colonies of CM102504 resistant to the antibiotic resistance marker (MLS) on pAP77 were selected for integration into the host chromosome and subsequent excision of the plasmid from the host chromosome. The resulting strain was confirmed to contain... Retention time (min) The unmarked bounding box was missing. Subsequent Sanger and Illumina sequencing confirmed that no other mutations were generated during this process. The resulting strain was CE102504-G5.

[0556] Example 5: Culture and analysis of genome-edited bacteria Primary cultures of wild-type and edited strains of *Bacillus belye* were grown overnight in 5 mL LB at 30°C and 200 RPM. Subcultures were then grown in flasks in 50 mL FM4 at 30°C and 200 RPM for 5 days. Samples of each culture were obtained for purity and HPLC determination.

[0557] Metabolite profiles of the supernatant samples of the culture of interest were generated by separating the samples using high-performance liquid chromatography (HPLC) using the methods summarized in Tables 2A (Cyclic Lipopeptides) and 3B (Polyketones). Reference retention times for peak identification are listed in Tables 2A (Cyclic Lipopeptides) and 3B (Polyketones).

[0558] Table 2A: HPLC parameters of cyclic lipopeptides (CLP)

[0559] Table 2B: HPLC parameters of polyketides (PK)

[0560] Table 3A: Retention Time of Cyclic Lipopeptides Composition ID ​ 4.6 to 10.3 Ikusmin 10.3 - 17.3 Fengyuansu 17.3 to the end Surfactants

[0561] Table 3B: Retention Time of Polyketides Retention time (minutes) Composition ID 4.8 dithiols 5.6 Macrolide 1 5.7 Macrolide 2 6.2 Macrolide 3 6.6 Macrolide 4 8.2 Macrolide 5 12.8 Diffusion 1 13.1 Diffusion 2 13.6 Diffusion 3

[0562] Cyclic lipopeptide profiles of wild-type and genome-edited strain 7084 Figure 1A and 1B The polyketide profiles of wild-type and genome-edited strains are given in [the table / reference]. Figure 2A-2D The results are given in Figure 3-12. The genome-edited strain 7084-G67 produced two different spectra in 11 different HPLC runs. Eight of the 11 runs produced a “high-yield” variant or variant 1 (e.g., Figure 3A (As shown by the magenta line). Three out of 11 runs produced "low-yield" variants or variant 2 (as shown by the magenta line). Figure 3A (As shown by the royal blue line). Variant 2 also exhibits a shift in the retention time of the surfactant peak, such as... Figure 3B As shown. A comparison of the composition produced by two different variants of strains G67 and WT is shown in the figure. Figure 3C (Cyclic lipopeptides) and Figure 3D (In polyketide compounds).

[0563] CLP in the supernatants of strains 7084, 7084-G2, 14416, and 14416-G2 was observed. Figure 23 middle. kinA The knockout-edited strains produced more CLP than the wild-type strains.

[0564] Polyketide compound production demonstration Figure 24 In comparison to each corresponding wild-type strain, kinA The knockout strain showed increased production of macrolides. Compared to its wild-type strain 7084, the edited strain 7084-G77 showed increased production of difenocil.

[0565] Evaluate the bioavailability of the composition. Bioavailability is the ratio of the amount in the soluble supernatant to the amount in the whole cell culture medium (including precipitated solids). Only bioavailable compounds are responsible for producing activity. High bioavailability indicates that all generated metabolites are active in a laboratory / field setting. Results showed... Figure 21 In the wild-type and wild-type of Bacillus belye kinA The bioavailability percentages of ituronidin, cytosine, surfactant, and total cyclic lipopeptides in the KO strain were shown in [data missing]. Figure 25middle.

[0566] In each wild type and kinA Total survival count (TVC, estimated total number of microorganisms) and sporulation were assessed in the knockout edited strains. Results showed... Figure 26 middle.

[0567] A summary of the composition production of the 7084 genome editing strain compared to the wild-type strain is given in Table 3C.

[0568] Table 3C: Relative Peak Areas of Secondary Metabolites

[0569] Example 6: In vitro antifungal activity of genome-edited microorganisms As described in Table 4, the supernatant material of each culture was tested against six different fungal pathogens. Each supernatant was prepared and tested at different dilutions.

[0570] Table 4: Tested fungal pathogens Taxonomy spores / pores Incubation time (days) Fusarium graminearum 500 3-9 Penicillium 500 3-4 Fusarium oxysporum tomato-specific strain 250 3 Fusarium oxysporum specialized lettuce 250 3-9 Penicillium finger 500 3 Staphylococcus aureus 500 6 Type of vaccination Ultimate mold Agar plug 2-3

[0571] As described above, supernatant material was obtained from each of the WT and genome-edited strains. Briefly, the strains were grown in FM4 medium at 30°C for 4 days. The samples were centrifuged at 15,000 RPM for 20 minutes, and the supernatant was collected in new test tubes. Appropriate dilutions (1:5, 1:10, 1:20, 1:50, 1:100, or 1:200) were prepared using sterile RO water.

[0572] Pipe samples at 200 μl / well into the wells of a sterile 48-well plate in a BSC. Pipe 200 μl of PDA containing antibiotics (penn / strep) (still liquid and warm, but not hot) into each well. Gently rotate each plate while pipetting to mix the agar with the supernatant and hold for up to 30 minutes to allow the agar to solidify. Add an appropriate amount (5–10 μl) of fungal spore suspension to each well. Seal the plate with sealing film and incubate for up to 5 days. Starting from 2 dpi (day post-inoculation), visually and microscopically score the plates. Rate activity using a 0–5 rating scale as defined in Table 5.

[0573] Table 5: Antifungal Activity Classification Table score describe 5 Fungal growth was completely inhibited. 4 Fungal growth is severely inhibited, and very little growth can be observed. 3 Small fungal patches can be seen, but the pores are still mostly clear. 2 Large and numerous fungal patches and local inhibition were observed. 1 The fungal growth was extremely rampant, with only minor differences compared to the water control. 0 Fungal growth was not inhibited and there was no difference compared to the water control.

[0574] Strain 7084 wild type (WT) and Δ kinA (KO) WT strain 7084 and Δ were inoculated with each pathogen. kinA Results for strain 7084-G77 and the untreated control (average across 3 wells) Figures 13A-13B (Fusarium graminearum at 4 dpi (days post-inoculation)) Figures 14A-14B (Extended Penicillium at 4 dpi) Figures 15A-15B (Fusarium oxysporum tomato-specific strain, 3 dpi) Figures 16A-16B (3 dpi Fusarium oxysporum lettuce-specific strain) and Figure 16C (5 dpi Fusarium oxysporum lettuce-specific strain) Figures 17A-17B (Penicillium fingering at 3 dpi) and Figures 18A-18B (Gray Staphylococcus aureus at 6 dpi) is given.

[0575] Undiluted (UD), 1:5, and 1:10 dilutions of CM7084 and CE7084-G77 equally inhibited the growth of Fusarium graminearum. At dilutions of 1:20, 1:50, and 1:100, CE7084-G77 showed significantly stronger activity than CM7084. At 1:50, the activity of CE7084-G77 was significantly enhanced compared to CM7084.

[0576] Undiluted, 1:10, and 1:20 dilutions of CM7084 and CE7084-G77 equally inhibited the growth of *Penicillium expansum*. At a 1:50 dilution, CE7084-G77 showed enhanced activity compared to CM7084.

[0577] Undiluted and 1:10 diluted supernatants of CM7084 and CE7084-G77 inhibited the growth of Fusarium oxysporum equally. At dilutions of 1:20 and 1:50, CE7084-G77 showed enhanced activity compared to CM7084. Activity decreased sharply at dilutions of 1:100 and 1:200, but was still superior to the water control.

[0578] Undiluted and 1:10 diluted supernatants of CM7084 and CE7084-G77 equally inhibited the growth of Fusarium oxysporum lettuce-specific strain. At a 1:20 dilution, CE7084-G77 significantly reduced mycelial growth compared to CM7084.

[0579] Undiluted, 1:10, and 1:20 dilutions of CM7084 and CE7084-G77 equally inhibited the growth of Penicillium fingerlings. At 1:50 and 1:100 dilutions, CE7084-G77 significantly reduced mycelial growth compared to CM7084.

[0580] Undiluted, 1:10, 1:20, and 1:50 dilutions of CM7084 and CE7084-G77 equally inhibited the growth of *Botrytis cinerea*. At a 1:100 dilution, CE7084-G77 showed enhanced activity compared to CM7084.

[0581] Strain 14416 wild type (WT) and Δ kinA WT strain 14416 and Δ were inoculated with each pathogen. kinA Results of strain 14416-G2 and the untreated control (average across replicate wells) Figures 27A-27C (Fusarium graminearum at 3 and 9 dpi (days post-inoculation)) Figures 28A-28C (Fusarium oxysporum at 3 and 9 dpi) and Figure 29 As given in (Ultimate Pythium).

[0582] Undiluted, 1:10, 1:20, and 1:50 dilutions of CM14416 and CE14416-G2 inhibited the growth of Fusarium graminearum equally. At 1:100 and 1:200 dilutions, CE14416-G2 showed significantly stronger activity than CM14416. At 1:200, the activity of CE14416-G2 was significantly enhanced compared to CM14416.

[0583] Undiluted and 1:10 diluted supernatants of CM14416 and CE14416-G2 inhibited the growth of Fusarium oxysporum equally. At 1:100 and 1:200 dilutions, CE14416-G2 showed significantly stronger activity than CM14416. At 1:200, the activity of CE14416-G2 was significantly enhanced compared to CM14416.

[0584] Undiluted supernatants of CM14416 and CE14416-G2 completely inhibited mycelial growth of *Pythium oxysporum*. At dilutions of 1:10, 1:20, and 1:50, CE14416-G2 showed superior reduction in mycelial growth of *Pythium oxysporum* compared to CM14416.

[0585] Strain 102504 wild type (WT) and kinA knockout (KO) WT strain 102504 and Δ were inoculated with each pathogen. kinA Results of strain 102504-G5 and the untreated control (average across replica...

Claims

1. A synthetic composition comprising a genetically engineered microorganism, wherein the genetically engineered microorganism contains at least one gene mutation at at least one locus in its genome, wherein the gene mutation alters the production of one or more secondary metabolites, and wherein the gene mutation confers the genetically engineered microorganism the ability to control at least one pathogen.

2. The synthetic composition according to claim 1, wherein the at least one locus is the kinA gene.

3. The synthetic composition according to claim 1, wherein the at least one locus is the thrC gene.

4. The synthetic composition according to claim 1, wherein the non-genetically engineered microorganism used to obtain the genetically engineered microorganism belongs to the genus Bacillus.

5. The synthetic composition according to claim 1, wherein the non-genetically engineered microorganism used to obtain the genetically engineered microorganism belongs to the genus Paenibacillus.

6. The synthetic composition according to claim 1, wherein the secondary metabolite is a cyclic lipopeptide or a polyketide.

7. The synthetic composition according to claim 6, wherein the cyclic lipopeptide is selected from the group consisting of: iturin, fengycin, surfactant, any one or more of the foregoing substances, and any combination of the foregoing substances.

8. The synthetic composition according to claim 6, wherein the polyketide compound is selected from the group consisting of: macrolides, difficidin, baccilaene, any one or more of the foregoing substances, and any combination of the foregoing substances.

9. The synthetic composition according to claim 1, wherein the pathogen is a fungus.

10. The synthetic composition according to claim 9, wherein the fungus belongs to a genus selected from the group consisting of: Fusarium, Penicillium, Botrytis, and Pythium.

11. The synthetic composition according to claim 1, wherein the gene mutation is a gene knockout, gene knock-in, disruption, insertion of at least one nucleotide, deletion of at least one nucleotide, substitution of at least one nucleotide, chemical modification of at least one nucleotide, alteration of the molecular structure of at least one nucleotide, downregulation of at least one gene, upregulation of at least one gene and / or any combination of the foregoing mutations and / or multiples of the foregoing mutations.

12. A whole-cell culture medium or exudate obtained from the synthetic composition according to claim 1, wherein the whole-cell culture medium or exudate contains an increased level of at least one secondary metabolite compared to a whole-cell culture medium or exudate obtained from a wild-type microorganism of the same strain as the synthetic composition.

13. A formulation comprising the synthetic composition according to claim 1 or the whole cell culture medium or exudate according to claim 12.

14. The formulation according to claim 13, wherein the synthetic composition is substantially purified.

15. The formulation of claim 13, wherein the composition further comprises one or more formulation components selected from the group consisting of: salts, binders, surface-active agents, surfactants, wetting agents, dispersants, emulsifiers, solubilizers, organic solvents, gelling agents, thickeners, antisettling agents, preservatives, stabilizers, antifreeze compounds, any and any combination of the foregoing substances.

16. A method for controlling pathogens, the method comprising administering to a subject the synthetic composition according to claim 1 or the whole-cell culture medium or exudate according to claim 12.

17. The method of claim 16, wherein the pathogen is bacteria.

18. The method of claim 16, wherein the pathogen is a fungus.

19. The method of claim 18, wherein the fungus belongs to a genus selected from the group consisting of Fusarium, Penicillium, Botrytis, and Pythium.

20. The method of claim 16, wherein the synthetic composition further comprises one or more reagents selected from the group consisting of: insecticides, herbicides, bactericides, fungicides, insecticides, viricides, acaricides, nematicides, scabies insecticides, plant growth regulators, rodenticides, antialgae agents, biocontrol agents, fertilizers, biopesticides, biostimulants, and any combination of the foregoing substances and / or a plurality of the foregoing substances.

21. The method of claim 16, wherein the object is a plant or a plant part.

22. The method of claim 21, wherein the plant part is a root, leaf, stem, flower, seed, bulb, or fruit.

23. The method of claim 16, wherein the composition further comprises one or more formulation components selected from the group consisting of: salts, binders, surfactants, wetters, dispersants, emulsifiers, solubilizers, organic solvents, gelling agents, thickeners, antisettling agents, preservatives, stabilizers, antifreeze compounds, any and any combination of the foregoing substances.

24. The method of claim 16, wherein the composition further comprises one or more additional agents selected from the group consisting of: insecticides, herbicides, bactericides, fungicides, insecticides, viricides, acaricides, nematicides, scabies insecticides, plant growth regulators, rodenticides, antialgae agents, biocontrol agents, fertilizers, biopesticides, biostimulants, and any combination of the foregoing substances and / or a plurality of the foregoing substances.

25. The method of claim 21, wherein the synthetic composition, whole-cell culture medium, or exudate is applied to the plant or plant part prior to germination.

26. The method of claim 21, wherein the synthetic composition, whole-cell culture medium, or exudate is applied to the plant or plant part after harvesting.

27. The method of claim 21, wherein the synthetic composition, whole-cell culture medium, or exudate is applied to the plant or plant parts during the nutrient stage of the plant.

28. The method of claim 21, wherein the synthetic composition, whole-cell culture medium, or exudate is applied to the plant or plant during the reproductive stage of the plant.

29. The method of claim 21, wherein the synthetic composition, whole-cell culture medium, or exudate is applied to the plant or plant part prior to harvest.

30. The method of claim 21, wherein the plant or a portion thereof is cultivated in a field, harvested, stored in a warehouse, and / or distributed.

31. The method of claim 21, further comprising applying the plurality of compositions to the plant or plant parts.

32. A method for reducing or inhibiting the growth of fungal hyphae, the method comprising: a. Modifying Bacillus or Bacillus-like strains by knocking out, reducing, and / or downregulating the activity of the kinA gene to produce modified strains. b. Introduce the modified strain into fungi.

33. A method for reducing or inhibiting fungal spore germination, the method comprising: a. Modifying Bacillus or Bacillus-like strains by knocking out, reducing, and / or downregulating the activity of the kinA gene to produce modified strains. b. Introduce the modified strain into fungi.

34. A synthetic composition comprising: a. plants or parts of plants, and b. The synthetic composition according to claim 1 or the whole cell culture medium or exudate according to claim 21.

35. The synthetic composition of claim 34, wherein the synthetic composition is substantially confined within an object selected from the group consisting of: bottles, wide-mouth bottles, ampoules, packaging, utensils, bags, boxes, storage boxes, envelopes, cartons, containers, silos, shipping containers, carriages, and crates.

36. A plurality of synthetic compositions according to claim 34, wherein some of the plurality of synthetic compositions are placed in a growth medium.

37. The various synthetic compositions according to claim 36, wherein the growth medium is soil.

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