Methods and compositions for increasing soybean yield

Abstract

This disclosure provides a method and composition for increasing soybean yield. More specifically, it provides a yield-enhancing composition comprising Methylbacterium, a method for increasing soybean yield, and a method for preparing said composition. Isolated soybean yield-enhancing Methylbacterium is also provided.

Description

[0001] This application is a divisional application of the application filed on May 22, 2020, with application number 202080085077.7 and invention title "Method and Composition for Increasing Soybean Yield".

[0002] Cross-reference to related applications

[0003] This patent application claims the benefit of US 62 / 884,423, filed August 8, 2019, which is incorporated herein by reference in its entirety.

[0004] Sequence list declaration

[0005] The sequence listing containing a file named “53907_197268_ST25.txt” (which is 24,090 bytes (measured in MS-Windows®) and created on May 7, 2020) contains 79 nucleotide sequences and is provided by the EFS system of the U.S. Patent and Trademark Office, and is incorporated herein by reference in its entirety. Background Technology

[0006] One-carbon organic compounds, such as methane and methanol, are widely found in nature and are used as carbon sources by bacteria classified as methanogenic and methyltrophic bacteria. Methanotrophic bacteria include genera and species from the genera *Methylobacter*, *Methylomonas*, *Methylomicrobium*, *Methylococcus*, *Methylosinus*, *Methylocystis*, *Methylosphaera*, *Methylocaldum*, and *Methylocella* (Lidstrom, 2006). Methanogenic bacteria possess methane monooxygenase, which incorporates an oxygen atom from O2 into methane to form methanol. All methanogenic bacteria are obligate one-carbon users and cannot utilize compounds containing carbon-carbon bonds. On the other hand, methyltrophic bacteria can also utilize more complex organic compounds, such as organic acids, higher alcohols, and sugars. Therefore, methyltrophic bacteria are facultative methyltrophic bacteria. Methyltrophic bacteria include genera such as *Methylobacterium*, *Hyphomicrobium*, *Methylophilus*, *Methylobacillus*, *Methylophaga*, *Aminobacter*, *Methylorhabdus*, *Methylopila*, *Methylosulfonomonas*, *Marinosulfonomonas*, and *P. paracoccus*. Species of the genera *Arachoccus*, *Xanthobacter*, *Ancylobacter* (also known as *Microcyclus*), *Thiobacillus*, *Rhodopseudomonas*, *Rhodobacter*, *Acetobacter*, *Bacillus*, *Mycobacterium*, *Arthobacter*, and *Nocardia* (Lidstrom, 2006).

[0007] Most methyltrophic bacteria in the genus *Methylbacteria* are pink. They are commonly referred to as PPFM bacteria and are pink facultative methyltrophic bacteria. Green (2005, 2006) identified 12 confirmed species in the genus *Methylbacteria*, specifically *M. aminovorans*, *M. chloromethanicum*, *M. dichloromethanicum*, *M. extorquens*, *M. Fujisawaense*, *M. mesophilicum*, *M. organophilum*, *M. radiotolerans*, *M. rhodesianum*, *M. rhodinum*, *M. thiocyanatum*, and *M. zatmanii*. However, *M. nidulans* is a non-PPFM nitrogen-fixing methylbacterium (Sy et al., 2001). Methylbacteria are found in soil, dust, fresh water, sediment, and leaf surfaces, as well as in industrial and clinical environments (Green, 2006). Summary of the Invention

[0008] This document provides a method for increasing soybean plant yield, the method comprising: (a) applying a composition to soybean plants or portions thereof, wherein the composition comprises one or more of (i) Methylbacterium NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), NLS0610 (ISO26) or variants thereof, or (ii) a combination of Methylbacterium isolate NLS0109 (NRRL B-67340) or variants thereof and Methylbacterium isolate NLS0017 (B-50931) or NLS0610 (ISO26) or variants thereof; wherein the composition further comprises at least one additional component selected from the group consisting of additional active ingredients, agriculturally acceptable adjuvants, and agriculturally acceptable excipients; and (b) allowing the soybean plants to mature, thereby increasing soybean plant yield. In some embodiments of the method, the composition is applied to soybean seeds. In some embodiments of the above methods, the composition comprises a solid material on which Methylobacterium grows and adheres, or an emulsion in which Methylobacterium grows. In some embodiments of any of the above methods, the composition comprises a titer of about 1 × 10⁻⁶ for solid compositions. 6CFU / gm to approximately 1×10 14 CFU / gm or a titer of approximately 1×10⁻⁶ for liquid compositions 6 CFU / mL to approximately 1×10⁻⁶ 11CFU / mL of Methylobacterium. In some embodiments of the above methods, the composition comprises NLS0934 (NRRL B-67341), NLS1181 (ISO25), or a variant thereof. In some embodiments of any of the above methods, the Methylobacterium variant is glyphosate-resistant or glufosinate-resistant. In some embodiments of the above methods, the applied composition is applied to or partially applied to soybean plants or a portion thereof. In some embodiments of the above methods, the composition is applied to the leaves of soybean plants. In some embodiments of the above methods, the composition further comprises a fungicide. In some embodiments of the above methods, the composition is applied at about VE to about R6 developmental stages. In some embodiments of the above methods, the composition is applied at about V2 to about V3 developmental stages, about V3 to V4, or V3 developmental stages. In some embodiments of the above methods, the method further includes the step of harvesting seeds from mature soybean plants. In some embodiments of the above methods, the yield of harvested seeds is increased compared to the yield of harvested seeds obtained from control soybean plants that have never received application of Methylobacterium. In some embodiments of the above methods, the composition is applied by spraying, coating, partially coating, impregnating, and / or wetting the soybean plant or a portion thereof. In some embodiments of the above methods, the applied composition is coated or partially coated onto the soybean plant or a portion thereof, wherein partial coating comprises coating at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 99.5% of the surface area of ​​the soybean plant or a portion thereof. In some embodiments of the above methods, the soybean plant portion is a seed. In some embodiments of the above methods, the composition comprises one or more of the following Methylobacterium isolates: NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), and NLS0610 (ISO26), or a combination of Methylobacterium isolates: NLS0109 (NRRL B-67340) and NLS0017 (B-50931) or NLS0610 (ISO26). In some embodiments of the above methods, the composition further comprises an additional active ingredient. In some embodiments of the above methods, the additional active ingredient is selected from the group consisting of fungicides, insecticides, nematicides, and biological agents. In some embodiments of the above methods, the biological agent is a biocontrol agent.In some embodiments of the above method, the additional active ingredient is selected from the group consisting of clothianidin, Bacillus firmus, abamectin, thiamethoxam, imidacloprid, azoxystrobin, fluopyram, fluoxastrobin, ipconazole, mefenoxam, metalaxyl, penflufen, prothioconazole, pyraclostrobin, and sedaxane. In some embodiments of the above method, the composition comprises (i) methylbacteria, wherein the chromosomal genomic DNA is combined with NLS0934 (NRRL). (ii) A combination of one or more of the following chromosomal genomic DNAs: B-67341, NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), or NLS0610 (ISO26) having at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity; or (ii) a combination of Methylobacterium isolates, wherein the chromosomal genomic DNA of the first isolate has at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity. The chromosomal genomic DNA of A has at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity with NLS0109 (NRRLB-67340), and the chromosomal DNA of the second isolate has at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity with NLS0017 (B-50931) or NLS0610 (ISO26).In some embodiments of the above method, the composition comprises (i) *Methylobacterium*, wherein the assembled genomic DNA sequence of *Methylobacterium* has an average nucleotide identity (ANI) score of at least 99.00 when compared with the assembled genomic DNA sequences of NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), or NLS0610 (ISO26), or (ii) a combination of *Methylobacterium* isolates, wherein the composition comprises (i) *Methylobacterium*, wherein the assembled genomic DNA sequence of *Methylobacterium* has an ANI score of at least 99.00 when compared with the assembled genomic DNA sequences of NLS0109 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), or NLS0610 (ISO26), or (ii) a combination of *Methylobacterium* isolates, wherein the composition comprises (i) *Methylobacterium*, NLS0109 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), or NLS0610 (ISO26), ... When compared with the assembled genomic DNA sequence of B-67340, the assembled genomic DNA sequence of the first isolate has an average nucleotide identity (ANI) score of at least 99.00, and when compared with the assembled genomic DNA sequence of NLS0017 (B-50931) or NLS0610 (ISO26), the assembled genomic DNA sequence of the second isolate has an average nucleotide identity (ANI) score of at least 99.00. In some embodiments of the above method, the composition comprises one or more of NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), NLS0610 (ISO26), or variants thereof, and the composition further comprises selected from ISO01 (NRRL B-50929), ISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO04 (NRRL B-50932), ISO05 (NRRL B-50933), ISO06 (NRRL B-50934), ISO07 (NRRL B-50935), ISO08 (NRRL B-50936), ISO09 (NRRL B-50937), ISO10 (NRRL B-50938), and ISO11 (NRRL B-50938). The group consisting of B-50939, ISO12 (NRRL B-50940), ISO13 (NRRL B-50941), ISO14 (NRRL B-50942) and ISO16 (NRRL B-67340) is an additional methylbacterium.

[0009] This document also provides soybean plants or soybean plant portions coated or partially coated with a composition comprising Methylobacterium, wherein Methylobacterium is one or more of the following: (i) NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), NLS0610 (ISO26) or variants thereof, or (ii) a combination of Methylobacterium isolate NLS0109 (NRRL B-67340) or variants thereof and Methylobacterium isolate NLS0017 (B-50931) or variants thereof. In some embodiments, the composition further comprises at least one additional component selected from the group consisting of additional active ingredients, agriculturally acceptable adjuvants, and agriculturally acceptable excipients. In some of the above embodiments, the composition comprises a titer of about 1 × 10⁻⁶ for solid compositions. 6 CFU / gm to approximately 1×10 14 CFU / gm or a titer of approximately 1×10⁻⁶ for liquid compositions 6 CFU / mL to approximately 1×10⁻⁶ 11CFU / mL of Methylobacterium. In some of the above embodiments, the Methylobacterium is NLS0934 (NRRL B-67341) or NLS1181 (ISO25). In some embodiments of any of the above compositions, the soybean plant part is selected from the group consisting of seeds, stems, flowers, leaves, petioles, pods, and axillary buds. In some of the above embodiments, the composition comprises one or more of Methylobacterium NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), and NLS0610 (ISO26), or a combination of Methylobacterium isolates NLS0109 (NRRL B-67340) and NLS0017 (B-50931) or NLS0610. In some of the above embodiments, the composition comprises additional active ingredients. In some of the above embodiments, the additional active ingredient is selected from the group consisting of fungicides, insecticides, nematicides, and biological agents. In some of the above embodiments, the biological agent is a biocontrol agent. In some of the above embodiments, the additional active ingredient is selected from the group consisting of thiamethoxam, Bacillus subtilis, abamectin, thiamethoxam, imidacloprid, pyraclostrobin, fluopyram, fluopyram, cymoxanil, metalaxyl, metalaxyl-M, fluopyram, prothioconazole, pyraclostrobin, and fluoxastrobin. In some of the above embodiments, the composition comprises one or more Methylobacteria, wherein the chromosomal genomic DNA has at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity with the chromosomal genomic DNA of NLS0934 (NRRLB-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), or NLS0610 (ISO26), or (ii) Methylobacteria The composition comprises a combination of isolates wherein the chromosomal genomic DNA of a first isolate has at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity with the chromosomal genomic DNA of NLS0109 (NRRLB-67340), and the chromosomal DNA of a second isolate has at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity with the chromosomal genomic DNA of NLS0017 (B-50931) or NLS0610 (ISO26). In some embodiments, the composition comprises (i) a methylbacterium having genomic DNA comprising one or more polynucleotide marker fragments of at least 50, 60, 100, 120, 180, 200, 240, or 300 nucleotides of SEQ ID NO: 1-15.In some of the above embodiments, the composition further comprises an additional methylbacterium selected from the group consisting of: ISO01 (NRRL B-50929), ISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO04 (NRRL B-50932), ISO05 (NRRL B-50933), ISO06 (NRRL B-50934), ISO07 (NRRL B-50935), ISO08 (NRRL B-50936), ISO09 (NRRL B-50937), ISO10 (NRRL B-50938), ISO11 (NRRL B-50939), ISO12 (NRRL B-50940), ISO13 (NRRL B-50941), and ISO14 (NRRL B-50942); or a methylbacterium having genomic DNA containing SEQ ID. NO: 61-79 is one or more polynucleotide marker fragments of at least 50, 60, 100, 120, 180, 200, 240 or 300 nucleotides.

[0010] This document also provides isolated Methylobacterium strains NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), NLS0610 (ISO26), or variants thereof. Compositions comprising one or more of Methylobacterium NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), NLS0610 (ISO26), or variants thereof are also provided. In some embodiments, the Methylobacterium strains or compositions are applied or partially applied to soybean plants or parts thereof. In some embodiments, the compositions are applied or partially applied to soybean seeds. In some embodiments, the composition for coating or partially coating soybean plants, soybean seeds or other parts of soybean plants comprises one or more of NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), NLS0610 (ISO26) or variants thereof. Detailed Implementation

[0011] definition

[0012] As used herein, the term “and / or” is considered to be a specific disclosure of each of two specified features or components, with or without the other. Therefore, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0013] Where a term is provided in singular form, embodiments including the plural form of the term are also provided.

[0014] As used herein, the terms “include, include, and including” should be interpreted as having at least the features they refer to or covering the items they refer to, without excluding any additional unspecified features or items.

[0015] As used herein, the phrases “adhere to” and “adhesive” refer to methylbacteria that associate with solid matter by growing on or already growing on it.

[0016] As used herein, the phrase “active ingredient” refers to a biological agent or pesticide in a composition used to treat plants and / or plant parts.

[0017] As used herein, the term "biologic" refers to a component of a composition for treating a plant or plant part composed of or derived from microorganisms. Biologics include biocontrol agents, other beneficial microorganisms, microbial extracts, natural products, plant growth activators, or plant defense agents. Non-limiting examples of biocontrol agents include bacteria, fungi, beneficial nematodes, and viruses. In some compositions, a biologic may include a single culture or co-culture of Methylobacterium, or a combination of individually cultured Methylobacterium strains or isolates.

[0018] As used herein, the phrase "agriculturally acceptable adjuvant" refers to a substance that enhances the performance of a biological agent or pesticide in a composition used to treat plants and / or plant parts. In some compositions, the biological agent may include a single culture or co-culture of methylbacterium.

[0019] As used herein, the phrase "agriculturally acceptable excipient" refers to a substantially inert substance that can be used as a diluent and / or carrier for biological agents or pesticides in compositions used to treat plants and / or plant parts. In some compositions, the biological agent may include a single culture or co-culture of Methylobacterium.

[0020] As used herein, the term “strain” should include all isolates of such strains.

[0021] As used herein, the phrase "control plant" refers to a plant that has not been treated with yield-enhancing methylbacterium or a composition containing thereof at any subsequent stage of seed or control plant development. In some embodiments, the control plant may be a plant treated with an additional active ingredient or yield-neutral methylbacterium.

[0022] As used herein, the phrase “co-culture of Methylbacteria” refers to a culture of Methylbacteria containing at least two strains of Methylbacteria or at least two species of the genus Methylbacteria.

[0023] As used herein, the phrase “contaminating microorganism” refers to a microorganism in a culture, fermentation broth, fermentation product, or composition that has not been identified prior to its introduction into the culture, fermentation broth, fermentation product, or composition.

[0024] As used herein, when applied in the context of Methylobacterium isolates, a “variant” refers to any isolate having chromosomal genomic DNA with at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity with the chromosomal genomic DNA of the deposited Methylobacterium isolates provided herein. Variants of isolates can be obtained from a variety of sources, including soil, plants or plant material, and water, particularly water associated with plants and / or agriculture. Variants include derivatives obtained from deposited isolates. Methylobacterium isolates or strains can be sequenced using sequence analysis tools such as BLAST (as taught by Altschul et al. (1990)) or clustalw (https: / / www.ebi.ac.uk / Tools / msa / clustalw2 / ) (e.g., as taught by Sanger et al. (1977), Bentley et al. (2008), or Caporaso et al. (2012)) and genome-scale comparisons of sequences can be performed (Konstantinidis et al. (2005)).

[0025] As used herein, when applied in the context of Methylobacterium isolates, “derived strain” means any Methylobacterium obtained from the deposited Methylobacterium isolates provided herein. Derivatives of Methylobacterium isolates include, but are not limited to, derivatives obtained by selection, derivatives obtained by mutagenesis and selection, and genetically transformed Methylobacterium obtained from Methylobacterium isolates. A “derived strain” can be identified, for example, based on genetic identity with the strain or isolate from which it was obtained, and will generally be a chromosomal genomic DNA exhibiting at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity with the chromosomal genomic DNA from which it is derived.

[0026] As used herein, when evaluating whether a particular Methylobacterium strain is a variant or derivative of the Methylobacterium strain presented herein, “sequence identity” refers to a measure of nucleotide-level genomic similarity between coding regions of two genomes. Sequence identity between coding regions of bacterial genomes can be calculated, for example, using FastANI (Jain et al., “High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries,” *Nature Communications* 9, 5114 (2018)) and Han et al. (“ANI tools web: a web tool or fast genome comparison within multiple bacterial strains”; *Database*, 2016, 1–5) to determine the average nucleotide identity (ANI) score.

[0027] As used herein, the term "emulsion" refers to a colloidal mixture of two immiscible liquids, wherein one liquid is a continuous phase and the other is a dispersed phase. In some embodiments, the continuous phase is an aqueous liquid and the dispersed phase is a liquid that is immiscible or partially miscible in the aqueous liquid.

[0028] As used herein, the phrase “substantially free of contaminating microorganisms” means a culture, fermentation broth, fermentation product, or composition wherein at least about 95% of the microorganisms present in the culture, fermentation broth, fermentation product, or composition in terms of quantity or type are the desired methylbacteria or other desired microorganisms with predetermined characteristics.

[0029] As used in this article, the phrase "inanimate solid substance" refers to a substance that is insoluble in or partially soluble in water or aqueous solution and is part of a still-living organism from which it is not derived.

[0030] As used in this article, the phrase "single culture of Methylobacterium" refers to a culture of Methylobacterium consisting of a single strain of Methylobacterium.

[0031] As used in this article, "pesticide" refers to insecticides, fungicides, nematicides, bactericides, or any combination thereof.

[0032] As used in this article, the phrase "bacteriostatic agent" refers to an agent that inhibits the growth of bacteria without killing them.

[0033] As used herein, the phrase "a pesticide that substantially does not inhibit the growth of the methylbazinobacterium" means any pesticide that, when provided in a composition comprising a fermentation product (which comprises a solid substance in which monocultures or cocultures of methylbazinobacterium are adhered), results in no more than 50% inhibition of methylbazinobacterium growth when the composition is applied to a plant or plant part, compared to a composition lacking the pesticide. In some embodiments, the pesticide results in no more than 40%, 20%, 10%, 5%, or 1% inhibition of methylbazinobacterium growth when the composition is applied to a plant or plant part, compared to a composition lacking the pesticide.

[0034] As used herein, the term “methylbacterium” refers to genera and species within the family Methylbacteriaceae, including bacterial species within the genus Methylbacterium and proposed genus Methylbacterium (Green and Ardley (2018)). Methylbacteria include pink facultative methyltrophs (PPFM) and also encompass non-pink nodular Methylbacterium, as well as colorless mutant strains of Methylbacterium isolates. For example, but not limited to, "methylobacterium" refers to bacteria of the following species and any new genus or species of methylobacterium that has not yet been reported or described, which can be characterized as methylobacterium or Methylorubrum based on phylogenetic analysis: Methylobacterium adhaesivum; Methylobacterium oryzae; Methylobacterium aerolatum; Methylobacterium oxalidis; Methylobacterium aquaticum; Methylobacterium pericinum; Methylobacterium brachiatum; Methylobacterium phyllosphaerae; Methylobacterium brachythecii; Methylobacterium phyllostachyos; Methylobacterium bullatum; Methylobacterium... platani); Methylobacterium cerastii; Methylobacterium pseudosasicola; Methylobacterium currus; Methylobacterium radiotolerans; Methylobacterium dankookense; Methylobacterium soli; Methylobacterium frigidaeris; Methylobacterium specialis; Methylobacterium fujisawaense; Methylobacterium tardum;Methylobacterium gnaphalii; Methylobacterium tarhaniae; Methylobacterium goesingense; Methylobacterium thuringiense; Methylobacterium gossipiicola; Methylobacterium trifolii; Methylobacterium gregans; Methylobacterium variabile; Methylobacterium haplocladii; Methylobacterium aminovorans (Methylorubrum aminovorans); Methylobacterium hispanicum; Methylobacterium extorquens (Methylorubrum extorquens); Methylobacterium Methylobacterium indicum; Methylobacterium podarium; Methylobacterium iners; Methylobacterium populi; Methylobacterium isbiliense; Methylobacterium pseudosasae; Methylobacterium jeotgali; Methylobacterium rhodesianum; Methylobacterium komagatae.Methylobacterium rhodinum; Methylobacterium longum; Methylobacterium salsuginis; Methylobacterium marchantiae; Methylobacterium suomiense; Methylobacterium mesophilicum; Methylobacterium thiocyanatum; Methylobacterium nodosa; Methylobacterium zatmanii; Methylobacterium organophilum.

[0035] As used in this article, the phrase "solid substance" refers to a substance that is insoluble or partially soluble in water or aqueous solutions.

[0036] As used in this article, the phrase “solid phase that can be suspended therein” refers to a solid substance that can be distributed throughout the liquid by stirring.

[0037] As used herein, the term “non-renewable” refers to a plant part or processed plant product that cannot be regenerated into a whole plant.

[0038] As used herein, the phrase “substantially all the solid phase is suspended in the liquid phase” means that at least 95%, 98%, or 99% of the solid material constituting the solid phase is distributed throughout the liquid by agitation.

[0039] As used herein, the phrase “substantially all the solid phase is not suspended in the liquid phase” means that less than 5%, 2%, or 1% of the solid is in the form of particles distributed throughout the medium by stirring.

[0040] It should be understood that the foregoing definitions will be used in this document when they are inconsistent with those provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference listed herein, or any patent or non-patent reference found elsewhere.

[0041] Yield-enhancing Methylobacterium, compositions containing yield-enhancing Methylobacterium, methods of use and preparation thereof.

[0042] This document provides various yield-enhancing Methylobacterium isolates, compositions comprising these Methylobacterium isolates, methods for using said compositions to increase soybean plant yield, and methods for preparing said compositions. In some embodiments, the yield-enhancing Methylobacterium isolates are selected from NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), NLS0610 (ISO26) and their variants, as well as combinations of Methylobacterium isolate NLS0109 (NRRL B-67340) or its variants with Methylobacterium isolate NLS0017 (B-50931) or NLS0610 (ISO26) or its variants. The amount of the composition comprising yield-enhancing Methylobacterium sufficient to provide increased soybean plant yield can be determined by measuring any or all changes in yield relative to untreated plants or plant parts. In some embodiments, yield can be assessed by measuring seed yield on a unit area basis (i.e., bushels per acre, kilograms per hectare, etc.), wherein plants treated with yield-enhancing methylbacterium or plants grown from methylbacterium-treated seeds grow at approximately the same density as control plants. In some embodiments, yield can be assessed by measuring the yield of the yield-enhancing methylbacterium-treated plants on a per-plant or per-plant-part basis (grains of seeds per plant, grams of seeds per pod, number of pods per plant, number of seeds per pod, etc.).

[0043] This document provides isolated yield-enhancing methylbacteria. In some embodiments, the methylbacteria are selected from the group consisting of *Methylobacterium gravidarum*, *Methylobacterium dansnezoffii*, *Methylobacterium radiation-resistant*, *Methylobacterium coumarin*, and *Methylobacterium vesicularis*. In some embodiments, the yield-enhancing methylbacteria isolates are selected from the group consisting of NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), NLS0610 (ISO26), their variants, and combinations thereof. In some embodiments, the yield-enhancing methylbacteria isolates are selected from the group consisting of NLS0934 (NRRL B-67341), NLS1181 (ISO25), and their variants. In some embodiments, the yield-enhancing methylbacteria are combinations of methylbacteria isolates NLS0109 and NLS0017 (NRRL B-50931) or NLS0610 (ISO26). In some embodiments, the yield-enhancing methylbacterium isolate can increase yield when applied to soybean seeds, during the vegetative stage of soybean development, or during the reproductive stage of soybean development. In some embodiments, the yield-enhancing methylbacterium has chromosomal genomic DNA with at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity with NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), or NLS0610 (ISO26); or the yield-enhancing methylbacterium is a combination of a methylbacterium variant of NLS0109 and a variant of NLS0017 (NRRL B-50931) or NLS0610 (ISO26), wherein the methylbacterium variant of NLS0109 has sequence identity with NLS0109 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), or NLS0610 (ISO26). The chromosomal genomic DNA of NLS0017 (NRRL B-50931) or NLS0610 (ISO26) has at least 99%, 99.9%, 99.8%, 99.7%, 99.6% or 99.5% sequence identity with the chromosomal genomic DNA of NLS0017 (NRRL B-50931) or NLS0610 (ISO26), and the variants of NLS0017 (NRRL B-50931) or NLS0610 (ISO26) have chromosomal genomic DNA with at least 99%, 99.9%, 99.8%, 99.7%, 99.6% or 99.5% sequence identity with the chromosomal genomic DNA of NLS0017 (NRRL B-50931) or NLS0610 (ISO26).In some embodiments, compared to untreated control plants or plants grown from untreated seeds, *Methylobacterium yield-enhancing* provides an increase in yield of at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, or at least about 15% in the treated plants or plants grown from treated seeds. In some embodiments, compared to untreated control plants or plants grown from untreated seeds, *Methylobacterium yield-enhancing* provides an increase in yield of at least about 2% or at least about 5% to at least about 10% or at least about 20% in the treated plants or plants grown from treated seeds.

[0044] In some embodiments, when applied to seeds, *Methylobacterium yield-enhancing* provides increased yield. In some embodiments, when applied during the vegetative stage of soybean development, *Methylobacterium yield-enhancing* provides increased yield. In some embodiments, when applied just before or during the reproductive stage of soybean development, *Methylobacterium yield-enhancing* provides increased yield. In some embodiments, the *Methylobacterium* is applied more than once, wherein such application may be applied to soybean seeds and soybean plants at one or more developmental stages, or may be applied to soybean plants more than once at two or more developmental stages. In some embodiments of any of the above compositions, the composition comprises a solid substance to which a single culture or co-culture of *Methylobacterium* is adhered. In some embodiments in which the *Methylobacterium* is adhered to a solid substance, the composition comprises a colloid formed from a solid substance to which a single culture or co-culture of *Methylobacterium* is adhered and a liquid. In some embodiments, the colloid is a gel. In some embodiments of some of the above compositions, the composition is an emulsion without solid substance. In some embodiments of any of the above compositions, the yield-enhancing methylbacteria are selected from the group consisting of NLS0934 (NRRLB-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), NLS0610 (ISO26), and their variants. In some embodiments of any of the above compositions, the yield-enhancing methylbacteria are a combination of methylbacteria NLS0109 and NLS0017 or NLS0610. In some embodiments, the yield-enhancing methylbacterium has chromosomal genomic DNA with at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity with NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), or NLS0610 (ISO26); or the yield-enhancing methylbacterium is a combination of a methylbacterium variant of NLS0109 and a methylbacterium variant of NLS0017 or NLS0610, wherein the methylbacterium variant of NLS0109, NLS0017, or NLS0610 has a sequence identity with NLS0109 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), or NLS0610 (ISO26); Chromosomal genomic DNA of B-50931 or NLS0610 with at least 99%, 99.9%, 99.8%, 99.7%, 99.6% or 99.5% sequence identity.

[0045] In some embodiments, the isolated yield-enhancing methylbacteria can be identified by treating plants, seeds, soil in which said plants or plants produced from said seeds grow, or other plant growth media in which said plants or plants produced from said seeds grow and measuring the increased yield.

[0046] In some embodiments, soybean seeds or soybeans in the developmental vegetative stage are treated with yield-enhancing Methylobacterium. The vegetative stages of soybean are as follows: VE (emergence), VC (cotyledon stage), V1 (first ternate leaf), V2 (second ternate leaf), V3 (third ternate leaf), V4 (fourth ternate leaf), up to V(n) (nth ternate leaf, where the final number of ternate leaves depends on the soybean variety and environmental conditions). A description of the soybean vegetative stages can be found on the World Wide Web (Internet) at extension.agron.iastate.edu / soybean / production_growthstages.html and in “Soybean Growth and Development,” Pedersen, P., Iowa State University Extension and Outreach publication, PM 1945, December 2009. In some embodiments, yield-enhancing methylbacterium is applied during the developmental stages from about VE to about V4, V5, V6, or Vn, where n is the number of trilobites present before entering the reproductive stage of development. In some embodiments, yield-enhancing methylbacterium is applied during the developmental stages from about VC, V1, V2, or V3 to about V4, V5, V6, or Vn, where n is the number of trilobites present before entering the reproductive stage of development. In some embodiments, the yield-enhancing methylbacterium applied to seeds or during the vegetative stage is selected from the group consisting of NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), NLS0610 (ISO26), and their variants.

[0047] In some embodiments, yield-enhancing methylbacterium is applied to herbicide-resistant, insect-resistant, or disease-resistant transgenic soybean plants. In some embodiments, yield-enhancing methylbacterium is applied before, during, or after glyphosate-resistant transgenic soybean plants. Commercially available glyphosate formulations that may be used include, but are not limited to: RoundupOriginal MAX®, Roundup PowerMAX®, Roundup UltraMax®, or RoundUp WeatherMAX® (Monsanto Co., St. Louis, Missouri, USA); Touchdown IQ® or Touchdown Total® (Syngenta, Wilmington, Delaware, USA); Glyphomax®, Glyphomax Plus®, or Glyphomax XRT® (Dow Agrosciences LLC, Indianapolis, USA). Soybean plants are typically sprayed with glyphosate at approximately the V3-V4 vegetative development stage. In some embodiments, the yield-enhancing methylbacterium applied before, during, or after glyphosate application is a derivative of glyphosate-resistant selected strains such as NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), or NLS0610 (ISO26). The described selection criteria for glyphosate-resistant bacteria (Comai et al., Science 221(4608):370-371) can be adapted for the selection of glyphosate-resistant yield-enhancing methylbacterium. This article provides information on the selection and use of glyphosate-resistant yield-enhancing methylbacteria from mutagenized or other methylbacterial populations such as NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25) or NLS0610 (ISO26) and their variants.

[0048] In some embodiments, soybean seeds or soybeans in the late vegetative to reproductive stages of development are treated with yield-enhancing Methylobacterium. The late vegetative stage of soybeans is from V5 or V6 to V(n) (the nth ternate leaf, where the final number of ternate leaves depends on the soybean variety and environmental conditions). The reproductive stages of soybean development are: R1 (beginning of flowering - at least one flower at any node); R2 (fully flowering - a fully opened flower at one of the two uppermost nodes); R3 (beginning of pod formation - pods 5 mm long at one of the four uppermost nodes); R4 (fully formed pods - pods 2 cm long at one of the four uppermost nodes); R5 (beginning of seed formation - seeds 3 mm long in pods at one of the four uppermost nodes on the main stem); R6 (fully formed seeds - pods containing green seeds, filling the pod volume at one of the four uppermost nodes on the main stem); R7 (beginning of maturity - a normal pod on the main stem has reached its mature pod color); and R8 (fully mature - 95% of the pods have reached their fully mature color). In some embodiments, the yield-enhancing Methylobacterium applied to the late vegetative or reproductive stages is selected from NLS0934 (NRRL). The group consists of B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), NLS0610 (ISO26) and their variants, as well as combinations of Methylbacterium NLS0109 and NLS0017 or NLS0610 and their variants.

[0049] The various methylbacterial isolates provided in this article are disclosed in Table 1.

[0050]

[0051]

[0052] 1In accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, the accession number of the strain deposited at the Agricultural Research Service Culture Collection (NRRL) of the National Center for Agricultural Utilization Research, Agricultural Research Service, US Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604 USA. Pursuant to 37 CFR §1.808(b), upon the grant of any patent derived from this patent application, all restrictions imposed by the depositor on public access to the deposited material will be irrevocably lifted.

[0053] The variant strains of *Methylobacterium* isolates listed in Table 1 include derivative strains obtained through genetic transformation, mutagenesis, and / or insertion of heterologous sequences. In some embodiments, such variant strains are identified by the presence of chromosomal genomic DNA having at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity with the chromosomal genomic DNA from which it is derived. In some embodiments, derivative strains are distinguished from other variant strains by the presence of one or more single DNA sequences, said single DNA sequences including: (i) single sequences of SEQ ID NO: 1-15 and SEQ ID NO: 61-79. Specific assays and primer sequences using such sequences for the specific detection of *Methylobacterium* strains from isolated DNA or from various samples of treated plants are described in the examples herein and in WO2020010264.

[0054] All commonly assigned patents or patent applications are incorporated herein by reference in their entirety, disclosing additional specific uses of certain Methylobacterium strains listed in Table 1, such as: increasing maize yield (US20160295868); improving lettuce cultivation (USPN 10,212,939); improving tomato growth (US10,368,547); increasing soybean yield (US2016 / 0302423); increasing fruit yield (USPN 10,111,438); controlling maize rootworms (US20170238553); controlling root-knot nematodes (US10,448,645); controlling root-knot nematodes (USPN 10,098,353); and controlling fungal diseases (US20180295841 and US20190364905).

[0055] This document also provides a method for increasing soybean yield, the method comprising applying to a plant or plant part any of the above-described compositions provided herein in an amount equal to the infection of a control plant, plant part, or plant obtained therefrom that has not received the application of the composition, providing an increased soybean yield in the plant, plant part, or plant obtained therefrom. In some embodiments, the plant part is selected from the group consisting of leaves, stems, flowers, roots, pods, coleoptiles, and seeds. In some embodiments, the method further comprises the step of harvesting at least one plant part selected from the group consisting of leaves, stems, flowers, roots, pods, or seeds from the plant or plant part. In some embodiments of any of the above-described methods, the method further comprises obtaining a treated food or feed composition from the plant or plant part. In some embodiments, the treated food or feed composition is a powder or paste. In some embodiments of any of the above methods, the yield-enhancing methylbacteria are selected from the group consisting of NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), NLS0610 (ISO26), and their variants. In some embodiments of any of the above compositions, the yield-enhancing methylbacteria are a combination of methylbacteria NLS0109 and NLS0017 or NLS0610, or their variants.

[0056] A method for preparing a composition that can be used to increase soybean yield is also provided, the method comprising combining yield-enhancing Methylobacterium with an agriculturally acceptable excipient and / or with an agriculturally acceptable adjuvant. In some embodiments of the method, the Methylobacterium is adhered to a solid substance. In some embodiments of the method, the Methylobacterium is adhered to a solid substance, wherein the solid substance is combined with a liquid to form a colloidal composition. In some embodiments of the method, the colloidal substance is a gel. In some embodiments of the method, the Methylobacterium adhered to the solid substance is provided by culturing the Methylobacterium in the presence of the solid substance. In some embodiments of the method, the composition comprises an emulsion. In some embodiments of the method, the Methylobacterium is provided by culturing the Methylobacterium in the emulsion. In any of the embodiments of the above methods, the yield-enhancing Methylobacterium is selected from the group consisting of NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), NLS0610 (ISO26), and their variants. In some embodiments of any of the above compositions, the yield-enhancing methylbacterium is a combination of methylbacterium NLS0109 and NLS0017 or NLS0610 or variants thereof.

[0057] It has been found that culturing Methylobacterium in a two-phase medium containing both liquid and solid matter significantly increases the yield of Methylobacterium compared to culturing it in a separate liquid medium. In some embodiments, the method may include growing Methylobacterium in a liquid medium containing particulate solid matter, which may be suspended in the liquid by stirring under conditions suitable for Methylobacterium growth. In some embodiments where particulate solid matter is used, at least substantially all of the solid phase is thereby suspended in the liquid phase by stirring. Such particulate solid matter may comprise material with a length or diameter of about 1 mm or less. In some embodiments, the degree of stirring is sufficient to provide a uniform distribution of the particulate solid matter in the liquid phase and / or optimal culture aeration. However, in other embodiments provided herein, at least substantially all of the solid phase is not suspended in the liquid phase, or a portion of the solid phase is suspended in the liquid phase while a portion is not suspended. In some two-phase media where the solid phase is not suspended in the liquid phase, non-particulate solid matter may be used. Such non-particulate solid matter includes, but is not limited to, material with a length or diameter greater than about 1 mm. Such granular and non-granular solid materials also include, but are not limited to, porous, fibrous, or otherwise configured to provide an increased surface area for the adhesive growth of Methylobacterium. Two-phase culture media in which a portion of the solid phase is suspended in the liquid phase and a portion of the solid phase is not suspended in the liquid phase may contain a mixture of granular and non-granular solid materials. Such granular and non-granular solid materials used in any of the aforementioned two-phase culture media also include, but are not limited to, porous, fibrous, or otherwise configured to provide an increased surface area for the adhesive growth of Methylobacterium. In some embodiments, the culture medium comprises a colloid formed from a solid and a liquid phase. A colloid comprising both solid and liquid can be pre-formed and added to a liquid culture medium or can be formed in a culture medium containing both solid and liquid. A colloid comprising both solid and liquid can be formed by subjecting certain solid materials to chemical and / or thermal changes. In some embodiments, the colloid is a gel. In some embodiments, the liquid phase of the culture medium is an emulsion. In some embodiments, the emulsion comprises an aqueous liquid and a liquid that is immiscible or only partially miscible in the aqueous liquid. Liquids that are immiscible or only partially miscible in water include, but are not limited to, any of the following: (1) liquids that, at 25°C, have a miscibility in water equal to or less than that of pentanol, hexanol, or heptanol; (2) liquids comprising alcohols, aldehydes, ketones, fatty acids, phospholipids, or any combination thereof; (3) alcohols selected from the group consisting of aliphatic alcohols and sterols containing at least five carbon atoms; (4) animal oils, microbial oils, synthetic oils, vegetable oils, or combinations thereof; and / or (5) vegetable oils selected from the group consisting of corn, soybean, cotton, peanut, sunflower, olive, flax, coconut, palm, rapeseed, sesame, safflower, or combinations thereof. In some embodiments, the immiscible or partially immiscible liquid may constitute at least about 0.02% to about 20% by mass of the liquid phase.In some embodiments, the method may include obtaining a two-phase culture medium comprising liquid, solid, and Methylobacterium, and culturing the culture under conditions provided for the growth of Methylobacterium. The two-phase culture medium comprising liquid, solid, and Methylobacterium can be obtained by a variety of methods, including but not limited to any of the following: (a) inoculating a two-phase culture medium comprising liquid and solid matter with Methylobacterium; (b) inoculating a solid matter with Methylobacterium and then introducing the solid matter containing Methylobacterium into a liquid culture medium; (c) inoculating a solid matter with Methylobacterium, incubating Methylobacterium on the solid matter, and then introducing the solid matter containing Methylobacterium into a liquid culture medium; or (d) any combination of (a), (b), or (c). Methods and compositions for growing Methylobacterium in a two-phase culture medium comprising liquid and solid matter are disclosed in commonly assigned U.S. Patents 9,181,541 and 9,845,462 (which are incorporated herein by reference in their entirety) and commonly assigned International Patent Publication WO2013181610, published December 5, 2013 (which is incorporated herein by reference in its entirety).

[0058] It has also been found that culturing Methylbacillus in a medium containing an emulsion significantly increases the yield of Methylbacillus compared to culturing it in a separate liquid medium. In some embodiments, methods for preparing the compositions provided herein may include growing yield-enhancing Methylbacillus in an emulsion under conditions provided for its growth. A variety of methods can be used to obtain a medium containing an emulsion and yield-enhancing Methylbacillus, including but not limited to any of the following: (a) inoculating a medium containing an emulsion with Methylbacillus; (b) inoculating an aqueous liquid with Methylbacillus, introducing a non-aqueous liquid, and mixing to form an emulsion; (c) inoculating an aqueous liquid with Methylbacillus, introducing a non-aqueous liquid, and mixing to form an emulsion; or (d) any combination of (a), (b), or (c). In some embodiments, the emulsion comprises an aqueous liquid and a liquid that is immiscible or only partially miscible in the aqueous liquid. Non-aqueous liquids that are immiscible or only partially miscible in water include, but are not limited to, any of the following: (1) liquids whose miscibility in water at 25°C is equal to or less than that of n-pentanol, n-hexanol, or n-heptanol; (2) liquids comprising alcohols, aldehydes, ketones, fatty acids, phospholipids, or any combination thereof; (3) alcohols selected from the group consisting of aliphatic alcohols and sterols containing at least 5, 6, or 7 carbons; (4) animal oils, microbial oils, synthetic oils, vegetable oils, or combinations thereof; and / or (5) vegetable oils selected from the group consisting of corn, soybean, cotton, peanut, sunflower, olive, flax, coconut, palm, rapeseed, sesame, safflower, or combinations thereof. In some embodiments, the immiscible or partially immiscible non-aqueous liquid may constitute at least about 0.02% to about 20% by mass of the emulsion. In some embodiments, the immiscible or partially immiscible non-aqueous liquid may comprise, by weight, at least about 0.05%, 0.1%, 0.5%, or 1% of the emulsion to about 3%, 5%, 10%, or 20%. Methods and compositions for growing Methylobacteria in a culture medium containing an emulsion are disclosed in commonly assigned U.S. Patent 10,287,544 and International Patent Publication WO2014194189, published December 4, 2014 (which is incorporated herein by reference in its entirety).

[0059] In some embodiments, the compositions or methods disclosed herein may include one or more additional components. In some embodiments, the additional component may be an additional active ingredient, such as a pesticide or a second biological agent. The pesticide may be, for example, an insecticide, fungicide, herbicide, or nematicide. The second biological agent may be a biocontrol agent.

[0060] Non-limiting examples of insecticides and nematicides include carbamates, diamides, macrolides, neonicotinoids, organophosphates, phenylpyrazoles, pyrethroids, spinosads, synthetic pyrethroids, terfenicol, and tetramic acid. In specific embodiments, insecticides and nematicides include abamectin, aldicarb, aldoxycarb, bifenthrin, carbofuran, chlorantraniliporle, chlothianidin, cyfluthrin, cyhalothrin, cypermethrin, deltamethrin, dinotefuran, emamectin, ethiprole, fenamiphos, fipronil, and flubendiamide. diamide, fosthiazate, imidacloprid, ivermectin, lambda-cyhalothrin, milkemectin, nitenpyram, oxamyl, permethrin, tioxazafen, spinosad, spirodichlofen, spirotetramat, tefluthrin, thiacloprid, thiamethoxam, and thiodicarb.

[0061] Non-limiting examples of useful fungicides include aromatic hydrocarbons, benzimidazoles, benzothiadiazoles, formamides, carboxylic acid amides, morpholines, benzamides, phosphonates, quinone external inhibitors (e.g., strobilurin), thiazolidinyl ethers, thiophanates, thiophene formamides, and triazoles. Specific examples of fungicides include acibenzolar-S-methyl, azoxystrobin, benalaxyl, bixafen, boscalid, carbendazim, cyproconazole, dimethomorph, epoxiconazole, fluopyram, fluoxastrobin, flutianil, flutolanil, fluxapyroxad, fosetyl-Al, ipconazole, isopyrazam, and kremoxydim. Soxim-methyl, mefenoxam, metalaxyl, metconazole, myclobutanil, orysastrobin, penflufen, penthiorad, picoxystrobin, propiconazole, prothioconazole, pyraclofos-methyl, sedaxane, silthiofam, tebuconazole, thifluzamide, thiophanate-methyl, tolclofos-methyl, trifloxystrobin, and triticonazole.

[0062] Non-limiting examples of herbicides include ACC enzyme inhibitors, acetanilide, AHAS inhibitors, carotenoid biosynthesis inhibitors, EPSPS inhibitors, glutamine synthase inhibitors, PPO inhibitors, PS II inhibitors, and synthetic auxins. Specific examples of herbicides include acetochlor, clethodim, dicamba, propyzoxystrobin, flumetsulam, glyphosate, glufosinate, mesotrione, quizalofop-P-ethyl, saflufenacil, sulfadiazine, and 2,4-D.

[0063] In some embodiments, the compositions or methods disclosed herein may include additional active ingredients, which may be additional biological agents. Additional biological agents may be biocontrol agents, other beneficial microorganisms, microbial extracts, natural products, plant growth activators, or plant defense agents. Non-limiting examples of biocontrol agents include bacteria, fungi, beneficial nematodes, and viruses.

[0064] In some embodiments, the additional biological agent may be Methylobacterium. In some embodiments, the additional biological agent is Methylobacterium or a variant thereof listed in Table 1. Therefore, the compositions or methods included herein may contain two or more of NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), NLS0610 (ISO26), or variants thereof. In some embodiments, the adjunctive biological agent is selected from the group consisting of ISO01 (NRRL B-50929), ISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO04 (NRRL B-50932), ISO05 (NRRL B-50933), ISO06 (NRRL B-50934), ISO07 (NRRL B-50935), ISO08 (NRRL B-50936), ISO09 (NRRL B-50937), ISO10 (NRRL B-50938), ISO11 (NRRL B-50939), ISO12 (NRRL B-50940), ISO13 (NRRL B-50941), ISO14 (NRRL B-50942), ISO16 (NRRL B-67340) and their variants. In some embodiments, the aforementioned variants may be those having a similarity to ISO01 (NRRL B-50929). B-50929), ISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO04 (NRRL B-50932), ISO05 (NRRL B-50933), ISO06 (NRRL B-50934), ISO07 (NRRL B-50935), ISO08 (NRRL B-50936), ISO09 (NRRLB-50937), ISO10 (NRRL B-50938), ISO11 (NRRL B-50939), ISO12 (NRRL B-50940), ISO13 (NRRL B-50941), ISO14 (NRRL B-50942) or ISO16 (NRRL Methylbacteria (B-67340) possessing chromosomal genomic DNA with at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity. In some embodiments, the additional biological agent may be a methylbacterium selected from *Methylobacterium gravidarum*, *Methylobacterium radiation-resistant*, *Methylobacterium truncatum*, *Methylobacterium quorum*, *Methylobacterium oviductum*, *Methylobacterium armatum*, and *Methylobacterium coccineum*.

[0065] In some embodiments, the additional biological agent may be bacteria of the following genera: Actinomycetes, Agrobacterium, Arthrobacter, Alcaligenes, Aureobacterium, Azobacter, Beijerinckia, Bacillus, Brevibacillus, Burkholderia, Chromobacterium, Clostridium, Clavibacter, Comomonas, Corynebacterium, Curtobacterium, Enterobacter. The genera *Bacillus*, *Flavobacterium*, *Gluconobacter*, *Hydrogenophaga*, *Klebsiella*, *Methylobacterium*, *Paenibacillus*, *Pasteuria*, *phingobacterium*, *Photorhabdus*, *Phyllobacterium*, *Pseudomonas*, *Rhizobium*, *Bradyrhizobium*, *Serratia*, *Stenotrophomonas*, *Variovorax*, or *Xenorhadbus* are listed.In certain embodiments, the bacteria were selected from the group consisting of: Bacillus amyloliquefaciens, Bacillus cereus, Bacillus firmus, Bacillus lichenformis, Bacillus pumilus, Bacillus sphaericus, Bacillus subtilis, Bacillus thuringiensis, Chromobacterium suttsuga, Pasteuria penetrans, Pasteuria usage, and Pseudomonas fluorescens.

[0066] In some embodiments, the additional biological agent may be one of the following fungal genera: Alternaria, Ampelomyces, Aspergillus, Aureobasidium, Beauveria, Colletotrichum, Coniothyrium, Gliocladium, Metarhizium, Muscodor, Paecilomyces, Trichoderma, Typhula, Ulocladium, or Verticillium. In specific embodiments, the fungus is Beauveria bassiana, Coniothyrium minitans, Gliocladium virens, Muscodor albus, Paecilomyces lilacinus, or Trichoderma polysporum.

[0067] In further embodiments, the adjunct biopharmaceutical may be a nodulation-associated factor, plant growth activator, or plant defense agent, including but not limited to harpin, Reynoutria sachalinensis, jasmonate, lipochitooligosaccharides (LCO), and isoflavones. In some embodiments, the adjunct biopharmaceutical component may include a combination of bacteria and nodulation-associated factors, plant growth activators, or plant defense agents. One such combination includes, but is not limited to, Optimize®, a combination of a slow-growing soybean rhizobium (Bradyrhizobium japonicum) and LCO.

[0068] In further embodiments, the additional biological agent may include, but is not limited to, various species of Bacillus sp., Pseudomonas sp., Coniothyrium sp., Pantoea sp., Streptomyces sp., and Trichodermas sp. The microbial biopesticide may be bacteria, fungi, viruses, or protozoa. Particularly useful biopesticide microorganisms include various strains of Bacillus subtilis, Bacillus thuringiensis, Bacillus pumilis, Pseudomonas syringae, Trichoderma harzianum, Trichoderma virens, and Streptomyces lydicus. Other added microorganisms may be genetically engineered or wild-type isolates, which may be used in pure culture form. In some embodiments, it is anticipated that the biological agent or biocontrol agent may be provided in the form of spores in the fermentation broth, fermentation product, or composition.

[0069] In some embodiments, the fermentation broth, fermentation product, or composition containing yield-enhancing Methylobacterium may further contain one or more additional active ingredients or microorganisms with predetermined characteristics other than Methylobacterium.

[0070] In some embodiments, liquid culture media are prepared from inexpensive and readily available components, including but not limited to inorganic salts such as potassium phosphate, magnesium sulfate, etc.; carbon sources such as glycerol, methanol, glutamic acid, aspartic acid, succinic acid, etc.; and amino acid blends such as peptone, tryptone, etc. Exemplary liquid culture media that can be used include, but are not limited to, ammonium mineral salt (AMS) medium (Whittenbury et al., 1970), Vogel-Bonner (VB) minimal culture medium (Vogel and Bonner, 1956), and LB medium (“Luria-Bertani medium”).

[0071] Typically, the solid material that can be used in the methods and compositions to provide efficient growth of Methylobacterium can be any suitable solid material that is insoluble in or only partially soluble in water or aqueous solutions. When the solid material is provided in a liquid culture medium, such suitable solid materials are also non-sterilizing or non-bacteriostatic compared to Methylobacterium-enhancing materials. In some embodiments, such suitable solid materials are also solid materials that are readily available in a sterile form or can be sterilized. Solid materials can be sterilized by any method that removes contaminated microorganisms and therefore includes, but is not limited to, methods such as autoclaving, radiation, chemical treatment, and any combination thereof. These solid materials include natural, artificial, or combinations of natural and artificial materials of animal, plant, microbial, fungal, or mineral origin. In some embodiments, the solid material is a non-living solid material. Non-living solid materials of animal, plant, microbial, or fungal origin can be obtained from animals, plants, microorganisms, or fungi that are incapable (i.e. no longer viable) or have become incapable of survival. Thus, diatom shells are non-living solid materials when previously associated diatoms have been removed or otherwise rendered incapable. Because diatom shells are inanimate solid materials, they are not considered photosynthetic organisms or photosynthetic microorganisms. In some embodiments, the solid material includes, but is not limited to, sand, silt, soil, clay, ash, charcoal, diatomaceous earth and other similar ores, frosted glass or glass beads, frosted ceramic materials, porcelain beads, bentonite, kaolin, talc, perlite, mica, vermiculite, silica, quartz powder, montmorillonite, and combinations thereof. In some embodiments, the solid material may be a polymer or polymer beads. Polymers that can be used as solid materials include, but are not limited to, various polysaccharides, such as cellulose polymers and chitin polymers that are insoluble or only partially soluble in water or aqueous solutions, agar (i.e., galactomannan), and combinations thereof. In some embodiments, the solid material may be insoluble or only partially soluble salt crystals. Salt crystals that can be used include, but are not limited to, insoluble or only partially soluble carbonates, chromates, sulfites, phosphates, hydroxides, oxides, and sulfides. In some embodiments, the solid material may be microbial cells, fungal cells, microbial spores, or fungal spores. In some embodiments, the solid material may be microbial cells or microbial spores, wherein the microbial cells or microbial spores are not photosynthetic microorganisms. In some embodiments, the microbial cells or microbial spores are not photosynthetic microorganisms, wherein the photosynthetic microorganisms are selected from the group consisting of: algae, cyanobacteria, diatoms, *Botryococcus braunii*, *Chlorella*, *Dunaliella tertiolecta*, *Gracilaria*, *Pleurochrysis carterae*, *Sargassum*, and *Ulva*.In other embodiments, the solid material may be inactivated (i.e., non-viable) microbial cells, fungal cells, microbial spores, or fungal spores. In other embodiments, the solid material may be quiescent (i.e., viable but not actively dividing) microbial cells, fungal cells, microbial spores, or fungal spores. In other embodiments, the solid material may be cell debris of microbial origin. In other embodiments, the solid material may be particulate matter from any part of a plant. Plant parts that can be used to obtain the solid material include, but are not limited to, rachis, bark, shell, leaves, roots, flowers, stems, bark, seeds, and combinations thereof. Products obtained from treated plant parts may also be used, including but not limited to bagasse, wheat bran, soybean meal, crushed seed cake, straw, etc. Such plant parts, treated plants, and / or treated plant parts may be ground to obtain a usable granular form of solid material. In some embodiments, wood or wood products may be used, including but not limited to wood pulp, sawdust, shavings, etc. In some embodiments, the solid material may be particulate matter from animals, including but not limited to bone meal, gelatin, ground or powdered shells, hair, softened hides, etc.

[0072] In some embodiments, the solid matter is provided in particulate form to provide distribution of the solid matter in the culture medium. In some embodiments, the solid matter consists of particles with an average length or average diameter of about 2 micrometers to about 1000 micrometers. In some embodiments, the solid matter consists of particles with an average length or average diameter of about 1 micrometer to about 1000 micrometers. In some embodiments, the solid matter is particles with an average length or average diameter of about 1, 2, 4, 10, 20, or 40 micrometers to about 100, 200, 500, 750, or 1000 micrometers. Ideally, the particles used in the methods and compositions provided herein should have suitable wettability so that they remain suspended throughout the culture medium when stirred.

[0073] In some embodiments, a solid substance is provided in the culture medium in colloidal form, wherein the continuous phase is liquid and the dispersed phase is solid. Suitable solids that can be used to form colloids in liquid culture media for growing yield-enhancing Methylobacterium include, but are not limited to, a variety of solids referred to as hydrocolloids. Such hydrocolloids used in the culture media, methods, and compositions provided herein can be hydrophilic polymers of plant, animal, microbial, or synthetic origin. The hydrocolloid polymers used in the methods may contain multiple hydroxyl groups and / or may be polyelectrolytes. The hydrocolloid polymers used in the compositions and methods provided herein include, but are not limited to, agar, alginate, arabinoxylan, carrageenan, carboxymethyl cellulose, cellulose, curdlan, gelatin, gellan gum, β-glucan, guar gum, gum arabic, locust bean gum, pectin, starch, xanthan gum, and mixtures thereof. In some embodiments, the colloids used in the culture media, methods, and compositions provided herein may comprise an aqueous colloidal polymer and one or more proteins.

[0074] In some embodiments, the solid material can provide for the adhesive growth of yield-enhancing methylbacteria on the solid material. The yield-enhancing methylbacteria adhering to the solid material are methylbacteria that cannot be substantially removed by simply washing the solid material with growth medium containing adhesive yield-enhancing methylbacteria, while non-adhesive methylbacteria can be substantially removed by washing the solid material with liquid growth medium. Herein, “substantially removed” means that at least about 30%, 40%, 50%, 60%, 70%, or 80% of the present methylbacteria are removed when the solid material is washed with three volumes of liquid growth medium. Such washing can be achieved by a variety of methods, including but not limited to decanting liquid from the washed solid phase or passing the liquid through a filter over a solid phase that allows bacteria in the liquid to flow through. In some embodiments, the adhesive yield-enhancing methylbacteria associated with the solid can include methylbacteria directly attached to the solid and / or methylbacteria indirectly attached to the solid material. Methylbacteria indirectly attached to the solid material include, but are not limited to, methylbacteria attached to another methylbacteria or another microorganism attached to the solid material, methylbacteria attached to the solid material through attachment to another substance (which is attached to the solid material), etc. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% of the methylbacteria in the fermentation broth, fermentation product, or composition are methylbacteria that adhere to the solid material. In some embodiments, methylbacteria with enhanced adhesion yield may be present on the surface of the solid material in the fermentation broth, fermentation product, or composition at the following densities: at least about 1 methylbacterium / 20 square micrometers, at least about 1 methylbacterium / 10 square micrometers, at least about 1 methylbacterium / 10 square micrometers, at least about 1 methylbacterium / 5 square micrometers, at least about 1 methylbacterium / 2 square micrometers, or at least about 1 methylbacterium / square micrometer. In some embodiments, the adhesive yield-enhancing methylbacteria may be present on the surface of solid matter in the fermentation broth, fermentation product, or composition at densities of at least about 1 methylbacterium / 20 square micrometers to about 1 methylbacterium / square micrometer, at least about 1 methylbacterium / 10 square micrometers to about 1 methylbacterium / square micrometer, at least about 1 methylbacterium / 10 square micrometers to about 1 methylbacterium / square micrometer, at least about 1 methylbacterium / 5 square micrometers to about 1 methylbacterium / square micrometer, or at least about 1 methylbacterium / 2 square micrometers to about 1 methylbacterium / square micrometer.In some embodiments, the adhesive yield-enhancing methylbacteria may be present on the surface of solid matter in the fermentation broth, fermentation product, or composition at densities of at least about 1 methylbacteria / 20 μm² to about 1 methylbacteria / 2 μm², at least about 1 methylbacteria / 10 μm² to about 1 methylbacteria / 2 μm², at least about 1 methylbacteria / 10 μm² to about 1 methylbacteria / 2 μm², or at least about 1 methylbacteria / 5 μm² to about 1 methylbacteria / 2 μm². The two-phase fermentation broth provided herein may comprise a liquid phase containing non-adhesive methylbacteria. In some embodiments, the titer of non-adhesive methylbacteria in the liquid phase may be less than about 100,000, 10,000, or 1,000 colony-forming units (CFU) / ml. In some embodiments of any of the above compositions, the yield-enhancing Methylbacterium is selected from the group consisting of NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), NLS0610 (ISO26), and their variants. In some embodiments of any of the above compositions, the yield-enhancing Methylbacterium is selected from the group consisting of NLS0934 (NRRL B-67341), NLS1181 (ISO25), and their variants. In some embodiments of any of the above compositions, the yield-enhancing Methylbacterium is a combination of Methylbacterium NLS0109 and NLS0017, or their variants.

[0075] This article provides values ​​greater than approximately 5 × 10 8 Titer of colony-forming units / mL, greater than approximately 1 × 10⁻⁶ 9 Titer of colony-forming units / mL, greater than approximately 1 × 10⁻⁶ 10 A titer of colony-forming units per milliliter, at least approximately 3 × 10⁻⁶. 10 Fermentation broth and composition for enhancing the yield of *Methylobacterium* at a titer of colony-forming units / mL. In some embodiments, the fermentation broth and composition provided herein may contain a titer of at least about 5 × 10⁻⁶. 8 From 1 colony-forming unit / mL to at least about 3 × 10⁻⁶ 10 Colony forming units / mL, at least approximately 5 × 10⁻⁶ 8 From 1 colony-forming unit / mL to at least about 4 × 10⁻⁶ 10 1 colony-forming unit / mL, or at least about 5 × 10⁻⁶ 8 From 1 colony-forming unit / mL to at least about 6 × 10⁻⁶ 10 Production-enhancing Methylobacterium: 1 colony-forming unit / mL. In some embodiments, the fermentation broth provided herein may contain a titer of at least about 1 × 10⁻⁶. 9 From 1 colony-forming unit / mL to at least about 3 × 10⁻⁶10 Colony forming units / mL, at least approximately 1×10⁻⁶ 9 From 1 colony-forming unit / mL to at least about 4 × 10⁻⁶ 10 Colony forming units / mL, or at least about 1×10⁻⁶ 9 From 1 colony-forming unit / mL to at least about 6 × 10⁻⁶ 10 Production-enhancing Methylobacterium: 1 colony-forming unit / mL. In some embodiments, the fermentation broth provided herein will contain a titer of at least about 1 × 10⁻⁶. 10 From 1 colony-forming unit / mL to at least about 3 × 10⁻⁶ 10 Colony forming units / mL, at least approximately 1×10⁻⁶ 10 From 1 colony-forming unit / mL to at least about 4 × 10⁻⁶ 10 Colony forming units / mL, or at least about 1×10⁻⁶ 10 From 1 colony-forming unit / mL to at least about 6 × 10⁻⁶ 10 Production-enhancing Methylobacterium: 1 colony-forming unit / mL. In some embodiments, the fermentation broth provided herein will contain a titer of at least about 3 × 10⁻⁶. 10 From 1 colony-forming unit / mL to at least about 4 × 10⁻⁶ 10 1 colony-forming unit / mL, or at least about 3 × 10⁻⁶ 10 From 1 colony-forming unit / mL to at least about 6 × 10⁻⁶ 10 Yield-enhancing Methylbacteria per colony-forming unit / mL. In some embodiments of any of the above compositions, the yield-enhancing Methylbacteria are selected from the group consisting of NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), NLS0610 (ISO26), and their variants. In some embodiments of any of the above compositions, the yield-enhancing Methylbacteria are a combination of Methylbacteria NLS0109 and NLS0017, or their variants.

[0076] Yield-enhancing Methylobacterium can be obtained as a fermentation product and can be used to prepare various compositions that can be used to treat plants or plant parts to increase soybean yield. Methylobacterium compositions can be applied to plants or plant parts in various forms, including, for example, as a liquid composition or as a dried composition, such as a dried powder. Dry powders can be obtained by various methods, including, for example, spray drying, freeze drying, vacuum drying, air drying, and fluidized bed drying. Thus, plants or plant parts that have been at least partially coated or coated with a fermentation broth product or composition containing yield-enhancing Methylobacterium are provided. In some embodiments, the plant part is a seed. Partial coating of plants, plant parts such as seeds includes, but is not limited to, coating at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 99.5% of the surface area of ​​the plant, plant part, or plant seed. Treated plant products comprising a fermentation broth product or composition having yield-enhancing Methylobacterium or adhesive yield-enhancing Methylobacterium are also provided. In some embodiments, solid substances having adhesive yield-enhancing Methylobacterium can be used to prepare various compositions specifically for treating plant seeds. Therefore, seeds at least partially coated with a fermentation product or composition are provided. Partial coating of the seeds includes, but is not limited to, coating at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 99.5% of the seed surface area. Treated seed products are also provided, including, but not limited to, powders, feeds, and flakes containing the fermentation product or composition provided herein. In some embodiments, the treated plant product will be non-renewable (i.e., will not develop into a plant). In some embodiments, the solid material used in the fermentation product or composition to at least partially coat the plant or plant part, or contained in the treated plant or plant part product, includes solid material and associated or adhesive yield-enhancing methylbacteria, which can be readily identified by comparing treated and untreated plants or plant parts or their treated products. In some embodiments, the yield-enhancing methylbacteria are selected from the group consisting of NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), NLS0610 (ISO26), and their variants. In some embodiments, the yield-enhancing methylbacteria are selected from the group consisting of NLS0934 (NRRL B-67341) and / or NLS1181 (ISO25). In some embodiments of any of the above compositions, the yield-enhancing methylbacteria are a combination of methylbacteria NLS0109 and NLS0017 or NLS0610, or their variants.

[0077] Compositions containing yield-enhancing Methylobacterium, which can be used to treat plants or plant parts, may further comprise additional components, including active ingredients, agriculturally acceptable adjuvants, or agriculturally acceptable excipients. Agriculturally acceptable adjuvants or excipients are generally components that do not cause undue phytotoxicity or other side effects when exposed to plants or plant parts. In some embodiments, the solid substance used in the fermentation broth may itself be an agriculturally acceptable adjuvant or agriculturally acceptable excipient, provided that it does not have a bactericidal or bacteriostatic effect on Methylobacterium. In some embodiments, agriculturally acceptable adjuvants and / or excipients are added to Methylobacterium to increase stability and / or shelf life. In other embodiments, the composition further comprises at least one of agriculturally acceptable adjuvants or agriculturally acceptable excipients.

[0078] Any of the above compositions may further comprise additional active ingredients. In some embodiments, the additional active ingredient is a pesticide used in the composition that does not substantially inhibit the growth of Methylobacterium. Since Methylobacterium is a Gram-negative bacterium, suitable bactericides used in the composition may include, but are not limited to, bactericides exhibiting activity against Gram-positive bacteria rather than Gram-negative bacteria. The compositions provided herein may also comprise bacteriostatic agents that do not substantially inhibit the growth of Methylobacterium. Suitable bacteriostatic agents in the compositions provided herein include, but are not limited to, bacteriostatic agents exhibiting activity against Gram-positive bacteria rather than Gram-negative bacteria. Any of the above compositions may also be substantially dry products (i.e., having a water content of about 5% or less), mixtures of compositions with emulsions, or suspensions.

[0079] Agriculturally acceptable adjuvants used in compositions containing yield-enhancing Methylobacterium include, but are not limited to, components that enhance product efficacy and / or products that enhance product applicability. Adjuvants that enhance product efficacy may include various wetting / dispersing agents (which promote adhesion and diffusion of the composition onto plant parts), adhesives that promote adhesion to plant parts, penetrants that promote contact between the active agent and internal tissues, fillers that increase the half-life of the active agent by inhibiting environmental degradation, and humectants that increase the density or drying time of the spray composition. Wetting / dispersing agents used in the composition may include, but are not limited to, nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, organosilicon surfactants, and / or acidifying surfactants. Adetrants used in the composition may include, but are not limited to, latex-based substances, terpenes / pine resin dienes, and pyrrolidone-based substances. Penetrants may include mineral oils, vegetable oils, esterified vegetable oils, organosilicon surfactants, and acidifying surfactants. Fillers used in the composition may include, but are not limited to, ammonium sulfate or menthene-based substances. Humectants used in the composition may include, but are not limited to, glycerol, propylene glycol, and diethylene glycol. Adjuvants that improve the ease of application of the product include, but are not limited to, acidifiers / buffers, defoamers / antifoamers, compatibility agents, offset reducers, dyes, and water conditioners. Defoamers / antifoamers used in the composition may include, but are not limited to, dimethicone. Compatibility agents used in the composition may include, but are not limited to, ammonium sulfate. Offset reducers used in the composition may include, but are not limited to, polyacrylamide and polysaccharides. Water conditioners used in the composition may include, but are not limited to, ammonium sulfate.

[0080] This document also provides methods for treating plants and / or plant parts with fermentation broths, fermentation products, and compositions containing *Methylobacterium for Yield Enhancement*. The treated plants and the treated plant parts obtained therefrom include, but are not limited to, soybean. As used herein, the term soybean includes, but is not limited to, all varieties, subspecies, and cultivars of soybean (Glycine max). Soybean subspecies include, but are not limited to, soybean L. ssp. Max and soybean ssp. Formosana. The treated plant parts include, but are not limited to, leaves, stems, flowers, roots, seeds, pods, etc. The seeds or other propagules of any of the aforementioned plants may be treated with the fermentation broths, fermentation products, fermentation products, and / or compositions provided herein.

[0081] In some embodiments, plants and / or plant parts are treated by applying a fermentation broth, fermentation product, fermentation product, and composition containing *Methylobacterium yield-enhancing* as a spray. Such spray application includes, but is not limited to, treating a single plant part or any combination of plant parts. Spraying can be achieved using any device that disperses the fermentation broth, fermentation product, fermentation product, and composition onto the plant and / or plant part. Useful spraying devices include cantilever sprayers, manual or backpack sprayers, crop dusters (i.e., air sprayers), etc. Spraying devices and / or methods for applying the fermentation broth, fermentation product, fermentation product, and composition to any one or both of the adjacent and / or abaxial surfaces can also be used. This document also provides plants and / or plant parts at least partially coated with any one of a two-phase fermentation broth, fermentation product, fermentation product, or composition, said two-phase fermentation broth, fermentation product, fermentation product, or composition comprising a solid substance with *Methylobacterium yield-enhancing* adhering to it. In some embodiments, the plant part is a seed. Partial coating of plants or plant parts includes, but is not limited to, coating at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 99.5% of the surface area of ​​the plant or plant part. In some embodiments, the plant part is a seed, and partial coating includes, but is not limited to, coating at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 99.5% of the surface area of ​​the seed. This document also provides treated plant products comprising a solid substance attached with yield-enhancing methylbazinobacterium.

[0082] In some embodiments, seeds are treated by exposing them to a fermentation broth, fermentation product, fermentation product, and composition containing *Methylobacterium yield-enhancing*. Seeds can be treated with the fermentation broth, fermentation product, and composition provided herein by methods including, but not limited to, wetting, coating, and spraying. Seed treatment can be performed using continuous and / or batch seed processors. In some embodiments, coated seeds can be prepared by pulping and air-drying the resulting product with a coating composition containing a fermentation broth or fermentation product containing *Methylobacterium yield-enhancing*. Air-drying can be performed at any temperature that is harmless to the seeds or *Methylobacterium*. In some embodiments, air-drying is performed at a temperature not exceeding 30°C. The proportion of the coating containing *Methylobacterium yield-enhancing* is, but is not limited to, 0.1-25% of the seed weight, 0.5-5% of the seed weight, and 0.5-2.5% of the seed weight. In some embodiments, the solid material used in seed coating or treatment is coated with *Methylobacterium yield-enhancing*. In some embodiments, the solid material used for seed coating or treatment will be associated with *Methylobacterium yield-enhancing* and will be present in the fermentation broth, fermentation product, or composition obtained by the methods provided herein. Various seed treatment compositions and methods disclosed in U.S. Patent Nos. 5,106,648, 5,512,069, and 8,181,388 are incorporated herein by reference in their entirety and may be adapted for use with activators comprising the fermentation broth, fermentation product, or composition provided herein. In some embodiments, the composition for treating seeds may contain agriculturally acceptable excipients, including but not limited to sawdust, clay, activated carbon, diatomaceous earth, fine-grained inorganic solids, calcium carbonate, etc. Clays and inorganic solids that may be used with the fermentation broth, fermentation product, or composition provided herein include, but are not limited to, calcium bentonite, kaolin, porcelain clay, talc, perlite, mica, vermiculite, silica, quartz powder, montmorillonite, and mixtures thereof.Agriculturally acceptable adjuvants that promote adhesion to seeds may be used, including but not limited to polyvinyl acetate, polyvinyl acetate copolymers, hydrolyzed polyvinyl acetate, polyvinylpyrrolidone-vinyl acetate copolymers, polyvinyl alcohol, polyvinyl alcohol copolymers, polyvinyl methyl ether, polyvinyl methyl ether-maleic anhydride copolymers, waxes, latex polymers, cellulose (including ethyl cellulose and methyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxymethylpropyl cellulose), polyvinylpyrrolidone, alginate, dextrin, maltodextrin, polysaccharides, fats, oils, proteins, gum arabic, guar gum, tragacanth gum, polysaccharide gum, gum paste, gum arabic, shellac, vinylidene chloride polymers and copolymers, soybean-based protein polymers and copolymers, lignosulfonates, acrylic acid copolymers, starch, polyvinyl acrylate, corn protein, gelatin, carboxymethyl cellulose, polyglucosamine, polyethylene oxide, acrylamide polymers and copolymers, hydroxyethyl polyacrylate, methacrylamide monomer, alginate, ethyl cellulose, polychloroprene, and syrups or mixtures thereof. Other suitable agriculturally acceptable adjuvants that can promote coating include, but are not limited to, polymers and copolymers of vinyl acetate, polyvinylpyrrolidone-vinyl acetate copolymers, and water-soluble waxes. Various surfactants, dispersants, anti-caking agents, foam control agents, and dyes disclosed herein and in U.S. Patent No. 8,181,388 may be adapted for use with active agents comprising the fermentation broth, fermentation product, or composition provided herein.

[0083] This invention provides compositions comprising yield-enhancing methylbacterium, which provide increased soybean plant yield relative to untreated plants, plant parts, and plants derived therefrom that have not been exposed to the composition. In some embodiments, plant parts, including but not limited to seeds, leaves, flowers, stems, roots, pods, or coleoptiles, may be treated with the compositions provided herein to increase soybean plant yield. Treatment or application may include, but is not limited to, spraying, coating, partially coating, immersing, and / or wetting plants or plant parts with the compositions provided herein. Partial coating of soybean plants or soybean plant parts includes, but is not limited to, coating at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 99.5% of the surface area of ​​soybean plants or soybean plant parts. In some embodiments, the partially coated soybean plant part is a soybean seed. In some embodiments, seeds, leaves, flowers, stems, roots, pods, or coleoptiles may be immersed and / or wetted with a liquid, semi-liquid, emulsion, or slurry of the compositions provided herein. Compared to untreated soybean plants or soybean plants grown from untreated seeds, such seed immersion or soaking is sufficient to provide increased yield in treated soybean plants or soybean plants grown from treated seeds. In some embodiments, plant seeds may be immersed and / or soaked for at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, such immersion and / or soaking may be carried out at temperatures that will not be harmful to plant seeds or methylbacterium. In some embodiments, seeds may be treated at a temperature of about 15°C to about 30°C or about 20°C to about 25°C. In some embodiments, seed soaking and / or soaking may be carried out with gentle agitation. In some embodiments, soybean seeds or soybean plants at the VE stage are exposed to the composition by providing the composition in a furrow. Providing the composition in a furrow represents one of several methods provided herein for applying the composition to soybean seeds or soybean plants at approximately the VE stage of soybean plant development.

[0084] Therefore, the compositions and related methods containing yield-enhancing methylbacterium provided herein are intended to be used to increase the yield of a variety of soybean plants, including but not limited to all varieties, subspecies and cultivars of soybean (Glycine max).

[0085] In some embodiments, the amount of the composition provided herein sufficient to provide increased soybean yield may be a composition having a yield-enhancing methylbacterium, the titer of which is at least about 1 × 10⁻⁶. 6 Colony forming units / mL, at least approximately 5 × 10⁻⁶ 6 Colony forming units / mL, at least approximately 1×10⁻⁶ 7 Colony forming units / mL, at least approximately 5 × 10⁻⁶ 8 Colony forming units / mL, at least approximately 1×10⁻⁶9 Colony forming units / mL, at least approximately 1×10⁻⁶ 10 1 colony-forming unit / mL or at least about 3 × 10⁻⁶ 10 Colony forming units / mL. In some embodiments, the amount of the composition provided herein sufficient to provide increased soybean yield for the plant or plant part may be a composition having a yield-enhancing methylbacterium, the titer of which is at least about 1 × 10⁻⁶. 6 Colony forming units / mL, at least approximately 5 × 10⁻⁶ 6 Colony forming units / mL, at least approximately 1×10⁻⁶ 7 1 colony-forming unit / mL, or at least about 5 × 10⁻⁶ 8 From 1 colony-forming unit / mL to at least about 6 × 10⁻⁶ 10 Colony-forming units per milliliter of liquid or emulsion. In some embodiments, the amount of the composition provided herein sufficient to provide increased soybean yield may be a fermentation broth product, wherein the solid phase yield of the product is enhanced by a Methylobacterium titer of at least about 1 × 10⁻⁶. 6 Colony forming units / mL, at least approximately 5 × 10⁻⁶ 6 Colony forming units / mL, at least approximately 1×10⁻⁶ 7 1 colony-forming unit / mL, or at least about 5 × 10⁻⁶ 8 From approximately 6 × 10⁶ colony-forming units / gram to at least 6 × 10⁶. 10 Methylobacterium colony-forming units per gram of solid phase. In some embodiments, the amount of the composition provided herein sufficient to provide increased soybean yield may be a composition having the following Methylobacterium titer: at least about 1 × 10⁻⁶ in a composition containing particles comprising solid matter. 6 1 colony-forming unit / gram of particles, at least approximately 5 × 10⁻⁶ 6 Colony forming units / gram of particles, at least approximately 1 × 10⁻⁶ 7 1 colony-forming unit / gram of particles, or at least about 5 × 10⁻⁶ 8 From 1 colony-forming unit / gram of particles to at least approximately 6 × 10⁻⁶ 10 Methylobacterium colony-forming units / gram of particles, wherein a single culture or co-culture of yield-enhancing methylobacterium is adhered to a solid material. In some embodiments, the amount of the composition provided herein sufficient to provide increased soybean yield to a plant or plant part may be a composition having the following methylobacterium titer: at least about 1 × 10⁻⁶ in a composition comprising an emulsion. 6 Colony forming units / mL, at least approximately 5 × 10⁻⁶ 6 Colony forming units / mL, at least approximately 1×10⁻⁶ 7 Colony forming units / mL, or at least about 5 × 10⁻⁶ 8 From 1 colony forming unit / mL to at least about 6 × 10⁻⁶10 Methylobacterium colony-forming units / mL, wherein a single culture or co-culture of yield-enhancing Methylobacterium adhering to a solid substance is provided in or grown in the composition. In some embodiments, the amount of the composition provided herein sufficient to provide increased soybean yield to a plant or plant part may be a composition having the following Methylobacterium titer: at least about 1 × 10⁻⁶ in a composition comprising an emulsion. 6 Colony forming units / mL, at least approximately 5 × 10⁻⁶ 6 Colony forming units / mL, at least approximately 1×10⁻⁶ 7 Colony forming units / mL, or at least about 5 × 10⁻⁶ 8 From 1 colony forming unit / mL to at least about 6 × 10⁻⁶ 10 Methylobacterium colony-forming units / mL, and single or co-cultures of Methylobacterium with enhanced yield are provided in or grown in the composition. In some embodiments of any of the above compositions, the Methylobacterium is selected from (i) NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), NLS0610 (ISO26) and their variants; and (ii) a combination of Methylobacterium isolate NLS0109 (NRRL B-67340) or its variants with Methylobacterium isolate NLS0017 (B-50931) or NLS0610 or its variants. In some embodiments of any of the above compositions, the composition is a lyophilized composition.

[0086] In some embodiments, the amount of composition provided herein sufficient to provide increased soybean yield may be a composition of Methylobacterium having a titer of at least about 1 × 10⁻⁶. 4 Colony forming units / mL, at least approximately 1×10⁻⁶ 5 Colony forming units / mL, at least approximately 1×10⁻⁶ 6 Colony forming units / mL, at least approximately 5 × 10⁻⁶ 6 Colony forming units / mL, at least approximately 1×10⁻⁶ 7 Colony forming units / mL, at least approximately 5 × 10⁻⁶ 8 Colony forming units / mL, at least approximately 1×10⁻⁶ 9 Colony forming units / mL, at least approximately 1×10⁻⁶ 10 1 colony-forming unit / mL, or at least about 3 × 10⁻⁶ 10 Colony forming units / mL. In some embodiments, the amount of composition provided herein sufficient to provide increased soybean yield may be a composition of Methylobacterium having a titer of at least about 1 × 10⁻⁶.4 Colony forming units / mL, at least approximately 1×10⁻⁶ 5 Colony forming units / mL, at least approximately 1×10⁻⁶ 6 Colony forming units / mL, at least approximately 5 × 10⁻⁶ 6 Colony forming units / mL, at least approximately 1×10⁻⁶ 7 1 colony-forming unit / mL, or at least about 5 × 10⁻⁶ 8 From 1 colony-forming unit / mL to at least about 6 × 10⁻⁶ 10 Colony-forming units per milliliter of liquid or emulsion. In some embodiments, the amount of the composition provided herein sufficient to provide increased soybean yield may be a fermentation broth product, wherein the solid phase of the product has a Methylobacterium titer of at least about 1 × 10⁻⁶. 4 Colony forming units / gram, at least approximately 1 × 10⁻⁶ 5 Colony forming units / gram, at least approximately 1 × 10⁻⁶ 6 Colony forming units / gram, at least approximately 5 × 10⁻⁶ 6 Colony forming units / gram, at least approximately 1 × 10⁻⁶ 7 Colony forming units / gram, or at least approximately 5 × 10⁻⁶ 8 From approximately 6 × 10⁶ colony-forming units / gram to at least 6 × 10⁶. 10 Methylobacterium colony-forming units / gram, at least approximately 1 × 10⁻⁶ 11 Methylobacterium colony-forming units / gram, at least approximately 1 × 10⁻⁶ 12 Methylobacterium colony-forming units / gram, at least approximately 1 × 10⁻⁶ 13 Methylobacterium colony-forming units / gram, or at least approximately 5 × 10⁻⁶. 13 Methylobacterium colony-forming units per gram of solid phase. In some embodiments, the amount of the composition provided herein sufficient to provide increased soybean yield may be a composition having the following Methylobacterium titer: at least about 1 × 10⁻⁶ in a composition containing particles comprising solid matter. 6 1 colony-forming unit / gram of particles, at least approximately 5 × 10⁻⁶ 6 Colony forming units / gram of particles, at least approximately 1 × 10⁻⁶ 7 1 colony-forming unit / gram of particles, or at least about 5 × 10⁻⁶ 8 From 1 colony-forming unit / gram of particles to at least approximately 6 × 10⁻⁶ 10 Methylobacterium colony-forming units / gram of particles, at least approximately 1 × 10⁻⁶ 11 Methylobacterium colony-forming units / gram of particles, at least approximately 1 × 10⁻⁶ 12 Methylobacterium colony-forming units / gram of particles, at least approximately 1 × 10⁻⁶ 13 Methylobacterium colony-forming units / gram of particles, or at least approximately 5 × 10⁻⁶ 13Methylobacterium colony-forming units per gram of particles, wherein a single culture or co-culture of Methylobacterium is adhered to a solid material. In some embodiments, the amount of the composition provided herein sufficient to provide increased soybean yield may be a composition having the following Methylobacterium titer: at least about 1 × 10⁻⁶ in the composition comprising the emulsion. 6 Colony forming units / mL, at least approximately 5 × 10⁻⁶ 6 Colony forming units / mL, at least approximately 1×10⁻⁶ 7 Colony forming units / mL, or at least about 5 × 10⁻⁶ 8 From 1 colony forming unit / mL to at least about 6 × 10⁻⁶ 10 Methylobacterium colony-forming units / mL, wherein a single culture or co-culture of Methylobacterium adhering to a solid substance is provided in or grown in the composition. In some embodiments, the amount of the composition provided herein sufficient to provide increased soybean yield may be a composition having the following Methylobacterium titer: at least about 1 × 10⁻⁶ in a composition comprising an emulsion. 6 Colony forming units / mL, at least approximately 5 × 10⁻⁶ 6 Colony forming units / mL, at least approximately 1×10⁻⁶ 7 Colony forming units / mL, or at least about 5 × 10⁻⁶ 8 From 1 colony forming unit / mL to at least about 6 × 10⁻⁶ 10 Methylobacterium colony-forming units / mL, wherein a single culture or co-culture of Methylobacterium is provided in or grown in the composition. In some embodiments of any of the above compositions, Methylobacterium is selected from (i) NLS0934 (NRRL B-67341), NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), NLS0610 (ISO26) and variants thereof; and (ii) a combination of Methylobacterium isolate NLS0109 (NRRL B-67340) or a variant thereof with Methylobacterium isolate NLS0017 (B-50931) or NLS0610 (ISO26) or a variant thereof.

[0087] Example

[0088] The following examples are included to illustrate illustrative, non-limiting embodiments of this disclosure. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques discovered by the applicant that work well in the practice of this invention. However, based on this disclosure, those skilled in the art will understand that various changes can be made to the specific embodiments disclosed without departing from the scope of this disclosure, while still obtaining the same or similar results.

[0089] Example 1:

[0090] Preparation of Methylobacterium

[0091] In preparation for the field trial, the Methylobacterium culture was grown for 4 days at 30°C in a peptone medium containing ammonium mineral salts (AMS) and a carbon source (AMS + carbon (15 g / L) + peptone (10 g / L)). To prepare 1 liter of liquid AMS medium + carbon + peptone (“AMS medium solution”), the following components were added to a total volume of one (1) liter of distilled water: 700 mg of anhydrous dipotassium hydrogen phosphate, 540 mg of anhydrous potassium dihydrogen phosphate, 1 g of magnesium sulfate heptahydrate, 500 mg of anhydrous ammonium chloride, 200 mg of calcium chloride dihydrate, 15 g of carbon source, and 10 g of peptone.

[0092] The AMS medium solution was sterilized by autoclaving. After 4 days of growth at 30°C, the culture was harvested by centrifugation at 5000 rpm for 10 minutes and then resuspended in AMS medium solution containing 20% ​​(v / v) glycerol as a cryoprotectant. The cultures were aliquoted and frozen at -80°C until thawed for use. The liquid Methylobacterium preparation was applied to commercial soybean seeds that had been treated with conventional fungicides and insecticides. The liquid Methylobacterium preparation was applied at approximately 1 × 10⁻⁶... 5 CFU / seed to approximately 1×10 6 CFU / seed rate is applied to the seed.

[0093] Example 2:

[0094] In a 2017 yield trial, soybean yield was increased by applying a methylbacterium composition.

[0095] Soybean microplot field trials were established at four sites across the Midwestern soybean-growing region to evaluate 67 methylbacterium isolates applied as seed treatments to commercial soybean seeds. Six controls untreated with methylbacterium isolates were included in the trials.

[0096] Micro-plot trials were conducted using a randomized complete block design. At each site, there were six replicates of each of the Methylobacterium isolates and controls. These trials were conducted at existing farmer field sites and managed according to local agronomic practices. All soybean varieties used were glyphosate-tolerant. Experiments were collected at physiological maturity using commercially available collection assemblies to obtain yield data.

[0097] result

[0098] The yield results were analyzed using JMP14 (SAS Institute), and the assumptions of normality and equal variance were tested before performing the analysis of variance (ANOVA). ANOVA was performed using the standard analytical-fit model approach. Under the null hypothesis that the differences in means were zero, a two-tailed t-test was used to compare the means of the Methylobacterium isolates with those of the six untreated controls, applied to the paired differences between the least squares means estimated from the ANOVA model.

[0099] A cross-location analysis of the four locations is performed based on the following model: [2]Y hijk = M + I i + S j + IS ij + L h + R(L) k(h) + LI hi + IR(L) ik(h) + LS hj + LIS hij + e hijk , where Y hijk In replication k, the yield of isolate i at stage j at location h is given by M, which represents the overall average. i It is the immobilization effect of isolate i, S j It is a fixed effect of stage j, IS ij It is a fixed effect of the interaction between isolate i and stage j, L h This is a random effect of location h, R(L). k(h) It is a random effect of repeated k nested within location h, LI hi IR(L) is a random effect of the interaction between location h and isolate i. ik(h) It is a random effect of the interaction between isolate i and replicate k nested at location h, LS hj It is a random effect of the interaction between location h and stage j, LIS hij It is a stochastic effect of the three-way interaction between location h, isolate i, and stage j, and e hijk It is random error.

[0100] The yield results of the isolates selected for further analysis are shown in Table 2 below.

[0101]

[0102]

[0103] *P-values ​​are derived from a two-tailed t-test that compares each treatment to the untreated check (UTC).

[0104] Example 3:

[0105] In a 2018 yield trial, soybean yield was increased by applying a methylbacterium composition.

[0106] Based on the results of the aforementioned 2017 field trial, strains were selected for the 2018 field trial, and cultures for inoculation were prepared as described in Example 1. Isolates that showed positive yield results in 2017 and several isolates that showed negative yield effects were selected. Soybean field trials were established at four locations throughout the Midwestern soybean growing region to evaluate Methylbacillus isolates applied as seed treatment to commercial soybean seeds. Controls untreated with Methylbacillus isolates were included in the trials.

[0107] These trials were conducted using a randomized complete block design, consisting of four rows of 30-inch, 40-foot lengths. Four replicates of each of the Methylobacterium isolates and controls were present at each site. These trials were conducted at existing farmer field trial sites and managed according to local agronomic practices. Harvesting was performed according to standard grower practices, and plot yields were determined using conventional metering combine harvesters. Plot trial weights were used to calculate the estimated yield per acre for each plot.

[0108] result

[0109] The yield results were analyzed as described above. Data for isolates showing increased soybean yield are provided in Table 3 below.

[0110]

[0111]

[0112] Example 4:

[0113] Increase soybean yield by applying a dry methyl bacillus composition

[0114] In preparation for the field trial, the Methylobacterium culture was grown for 4 days at 30°C in a peptone medium containing ammonium mineral salts (AMS) and a carbon source (AMS + carbon (15 g / L) + peptone (10 g / L)). To prepare 1 liter of liquid AMS medium + carbon + peptone (“AMS medium solution”), the following components were added to a total volume of one (1) liter of distilled water: 700 mg of anhydrous dipotassium hydrogen phosphate, 540 mg of anhydrous potassium dihydrogen phosphate, 1 g of magnesium sulfate heptahydrate, 500 mg of anhydrous ammonium chloride, 200 mg of calcium chloride dihydrate, 15 g of carbon source, and 10 g of peptone.

[0115] The AMS medium solution was sterilized by autoclaving. After growing at 30°C for 4 days, the culture was harvested by centrifugation at 5000 rpm for 10 minutes, and the cell pellet from the centrifugation was resuspended in one-tenth the original volume of AMS medium solution (“concentrated slurry”).

[0116] The concentrated slurry was mixed with commercial formulation additives, and the resulting mixture was frozen at -80°C. The frozen mixture was then freeze-dried using standard industrial freeze-drying conditions and ground into a powder. The powder was then thawed at approximately 1 × 10⁻⁶. 5 CFU / seed to approximately 1×10 6 CFU / seed rate is applied to commercial soybean seeds that have already been treated with conventional fungicides and insecticides.

[0117] Soybean field trials were established at four sites across the Midwestern soybean-growing region (nine sites in 2017 and eleven sites in 2018). These trials were conducted using a randomized complete block design, consisting of four rows of 30-inch, 40-foot lengths. Four replicates of each of the Methylobacterium isolates and controls were present at each site. The Methylobacterium S6 positive control contained one Methylobacterium isolate that had previously been shown to increase soybean yield (US20160302423). These trials were conducted within existing farmer field trial sites and managed according to local agronomic practices. Harvesting was performed according to standard grower practices, and plot yields were determined using conventional metering combine harvesters. Plot trial weights were used to calculate the estimated bu / acre yield for each plot. Data are reported in Table 4 below.

[0118]

[0119] Example 5: Detection and identification of Methylobacterium strains, variants, and derivatives

[0120] Assays for detecting or identifying specific Methylobacterium strains and closely related derivatives are disclosed. Strain-specific genomic DNA fragments of Methylobacterium strains were identified, and a qPCR-based locked nucleic acid (LNA) assay was developed.

[0121] BLAST analysis of approximately 300 bp fragments, using a sliding window of 1–25 nucleotides, compared the genomic DNA sequence of *Methylobacterium* strains with the whole genome sequences of over 1000 public and proprietary *Methylobacterium* isolates. Genomic DNA fragments were identified as having weak BLAST alignments, indicating approximately 60–95% identity with the corresponding fragments in the *Methylobacterium* of interest. Fragments from the genomes of target *Methylobacterium* strains corresponding to the identified weakly aligned regions were selected for assay development.

[0122] The target fragment sequences that distinguish NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25) and NLS0610 (ISO26) from the related Methylobacterium isolates are provided in Table 5 below.

[0123]

[0124]

[0125]

[0126]

[0127]

[0128] Regions in SEQ ID NO: 1-15 were selected, where corresponding regions in other Methylobacterial strains were identified as having one or more nucleotide mismatches with the target Methylobacterial strain sequence. qPCR primers were designed using Primer3 software (Untergasser et al. (2012), Koressaar et al. (2007)) to be located lateral to the mismatched regions, with a melting temperature (Tm) in the range of 55-60 degrees Celsius, producing a PCR DNA fragment of approximately 100 bp. Probe sequences were designed using 5' FAM reporter dye, 3' Iowa Black FQ quencher, and contained 1 to 6 LNA bases (Integrated DNA Technologies, Coralville, Iowa). At least one LNA base was located at the mismatched position, while the other LNA bases were used to increase the Tm. The target Tm for the probe sequence was 10 degrees Celsius higher than the Tm of the primers.

[0129] Primer and probe sequences for the specific detection of NLS0497 (ISO22), NLS0693 (ISO23), NLS1179 (ISO24), NLS1181 (ISO25), NLS0610 (ISO26) and their variants are provided as SEQ ID NO: 16-51 in Table 6. Each probe contains a 5' FAM reporter dye and a 3' Iowa Black FQ quencher.

[0130]

[0131]

[0132] Primer / probe sets were used on isolated DNA to detect specific Methylobacterium isolates and their variants, and compared with... Distinguishing related Methylobacterium isolates

[0133] Each 10 μL qPCR reaction contains 5 μL of Quantabio PerfeCTa qPCR ToughMix 2x Mastermix, Low ROX from VWR, 0.05 μL of 100 μM forward primer, 0.05 μL of 100 μM reverse primer, 0.05 μL of 50 μM probe, 2.85 μL of nuclease-free water, and 2 μL of DNA template. Approximately 1 ng of DNA template is used per reaction. Reactions are performed in a ThermoFisher QuantStudio. TM The qPCR was performed on a 6-Flex real-time PCR system using the following procedure: 95°C for 3 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 45 seconds. The analysis software on the PCR instrument calculated the threshold and Ct value for each sample. Each sample was run three times on the same qPCR plate. A positive result was defined as a ΔCt of at least 5 between the positive and negative controls.

[0134] Using the assays described above, the other methylbacterial isolates and their variants listed in Table 1 were identified by the presence of DNA fragments. Specific fragments used in this type of method are provided in Table 7.

[0135]

[0136]

[0137]

[0138]

[0139] References

[0140] Altschul SF, Gish W., Miller W., Myers EW, Lipman DJ (1990) “Basic local alignment search tool”. Journal of Molecular Biology 215:403–410

[0141] Bentley DR, Balasubramanian S., Swerdlow HP, Smith GP, Milton J., Brown DG, Hall KP, Evers DJ, Barnes CL, Bignell HR et al. (2008) "Accurate whole human genome sequencing using reversible terminator chemistry". Nature 456:53-59.

[0142] Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D., Huntley J., Fierer N., Owens SM, Betley J., Fraser L., Bauer M., Gormley N., Gilbert J.A., Smith G., Knight R. (2012) “Ultra-high-throughput microbial community analysis on the IlluminaHiSeq and MiSeq platforms”. Journal of the International Society for Microbial Ecology (ISME J) 6:1621-1624.

[0143] Comai et al. (1983) "An altered aroA gene product confers resistance to the herbicide glyphosate." Science 221(4608):370-371.

[0144] Green, PN 2005. “Methylobacterium”. In Brenner, DJ, NRKrieg and JT Staley (eds.), Bergey's Manual of Systematic Bacteriology, Volume two, “The Proteobacteria, Part C, The alpha-, beta-, delta-, andepsilonproteobacteria”, 2nd ed. Springer, New York, pp. 567-571.

[0145] Green, PN 2006. “Methylobacterium”. In Dworkin, M., S. Falkow, E. Rosenberg, K.-H. Schleifer and E. Stackebrandt (eds.), “The Prokaryotes”. A Handbook on the Biology of Bacteria, Vol. 5, “Proteobacteria: Alpha and Beta Subclasses”, 3rd ed., Springer, New York, pp. 257-265.

[0146] Green and Ardley (2018) “Review of the genus Methylobacterium and closely related organisms: a proposal that some Methylobacterium species be reclassified into a new genus, Methylorubrum gen.nov”. International Journal of Systematic and Evolutionary Microbiology 68:2727-2748.

[0147] Konstantinidis, KT and Tiedje JM (2005), Proceedings of the National Academy of Sciences (Proc. Nat. Acad. Sci. USA) 102:2567-2572.

[0148] Lidstrom, ME 2006. “Aerobic methylotrophic prokaryotes” in Dworkin, M., S. Falkow, E. Rosenberg, K.-H. Schleifer and E. Stackebrandt (eds.) “Handbook of Prokaryotes, Volume 2, Ecophysiology and Biochemistry”, 3rd ed. New York Springer. 618-634.

[0149] Sanger F, Nicklen S, Coulson AR (1977) "DNA sequencing with chain-terminating inhibitors". Proceedings of the National Academy of Sciences. 74(12):5463–5467

[0150] Sy, A., Giraud, E., Jourand, P., Garcia, N., Willems, A., De Lajudie, P., Prin, Y., Neyra, M., Gillis, M., Boivin-Masson, C., and Dreyfus, B. 2001. “Methylotrophic Methylobacterium Bacteria Nodulate and Fix Nitrogen in Symbiosis with Legumes”. Journal of Bacteriol. 183(1):214-220.

[0151] Vogel, HJ, and DM Bonner. 1956. "Acetylornithinase of Escherichia coli: Partial purification and some properties". Journal of Biol. Chem. 218:97-106.

[0152] Whittenbury, R., SL Davies, and JF Wilkinson. 1970. “Enrichment, isolation, and some properties of methane-utilizing bacteria.” Journal of General Microbiology, 61:205-218.

[0153] The principles of this disclosure have been explained and described, and it will be apparent to those skilled in the art that modifications to the configuration and details of this disclosure can be made without departing from such principles.

[0154] Although materials and methods have been described with reference to various embodiments and illustrative examples, it will be apparent to those skilled in the art that variations may be made to the materials and methods described herein without departing from the concept, spirit, and scope of this disclosure. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of this disclosure as defined by the appended claims.

Claims

1. A method for increasing soybean plant yield, the method comprising: (a) Applying the composition to soybean plants or parts thereof, wherein the composition comprises Methylbacterium (i) NLS0934 (NRRL B-67341), NLS0497 (NRRL B-67925), NLS0693 (NRRL B-67926), NLS1179 (NRRL B-67929), NLS1181 (NRRL B-67927) or NLS0610 (NRRL B-67892), or (ii) a combination of Methylbacterium isolate NLS0109 (NRRL B-67340) and Methylbacterium isolate NLS0017 (NRRL B-50931); or a combination of Methylbacterium isolate NLS0109 (NRRL B-67340) and Methylbacterium isolate NLS0610 (NRRL B-67892). The composition comprises a combination of B-67892; wherein the composition further comprises at least one additional component selected from the group consisting of additional active ingredients, agriculturally acceptable adjuvants and agriculturally acceptable excipients; and (b) Allow the soybean plant to grow to maturity, thereby increasing the yield of the soybean plant.

2. The method of claim 1, wherein the composition is applied to soybean seeds.

3. The method of claim 1, wherein the composition comprises a solid substance on which the methylbacterium grows and adheres, or an emulsion in which the methylbacterium grows.

4. The method of claim 1, wherein the composition comprises a titer of about 1 × 10⁻⁶ for solid compositions. 6 CFU / gm to approximately 1×10 14 CFU / gm or a titer of approximately 1×10⁻⁶ for liquid compositions 6 CFU / mL to approximately 1×10⁻⁶ 11 The methylbacterium, CFU / mL.

5. The method according to claim 1, wherein the composition comprises Methylobacterium NLS0934 (NRRL B-67341) or NLS1181 (NRRL B-67927).

6. The method according to claim 1, wherein the methylbacterium is glyphosate-resistant or glufosinate-resistant.

7. The method according to any one of claims 1 to 6, wherein the applied composition is applied to or partially applied to the soybean plant or a portion thereof.

8. The method according to any one of claims 1 to 6, wherein the composition is applied to the leaves of the soybean plant.

9. The method according to any one of claims 1 to 5, wherein the methylbacterium is a derivative of NLS0934 (NRRL B-67341), NLS0497 (NRRL B-67925), NLS0693 (NRRL B-67926), NLS1179 (NRRL B-67929), NLS1181 (NRRL B-67927) or NLS0610 (NRRL B-67892) selected for glyphosate resistance or glufosinate resistance.

10. The method of claim 6, wherein the methylbacterium is glyphosate resistant, wherein the soybean plant is a glyphosate-tolerant soybean plant, and wherein glyphosate is applied at approximately V2 to approximately V4 stages of soybean plant development.

11. The method according to any one of claims 1 to 6, further comprising the step of harvesting seeds from the mature soybean plant.

12. The method of claim 11, wherein the yield of harvested seeds is increased compared to the yield of harvested seeds obtained from control soybean plants that have never received application of methylbacterium.

13. The method of claim 1, wherein the composition is applied to soybean plants at about VE to about R6 stage of soybean plant development.

14. The method of claim 13, wherein the composition is applied at a developmental stage of about V2 to about V3, about V3 to about V4, or about V3.

15. The method of claim 1, wherein the composition is applied by spraying, coating, partially coating, immersing and / or wetting the soybean plant or plant parts with the composition.

16. The method of claim 15, wherein the applied composition is coated or partially coated on the soybean plant or a portion thereof, wherein partial coating comprises at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or about 99.5% of the surface area of ​​the soybean plant or a portion thereof.

17. The method of claim 16, wherein the soybean plant part is a seed.

18. The method according to any one of claims 1 to 6, 10, 12 to 17, wherein the composition comprises NLS0934 (NRRL B-67341), NLS0497 (NRRL B-67925), NLS0693 (NRRL B-67926), NLS1179 (NRRL B-67929), NLS1181 (NRRL B-67927) or NLS0610 (NRRL B-67892), or a combination of Methylobacterium isolate NLS0109 (NRRL B-67340) and Methylobacterium isolate NLS0017 (NRRL B-50931).

19. The method according to any one of claims 1 to 6, 10, 12 to 17, wherein the composition further comprises an additional active ingredient.

20. The method of claim 19, wherein the additional active ingredient is selected from the group consisting of fungicides, insecticides, nematicides, and additional biological agents.

21. The method of claim 20, wherein the additional biological agent is a biocontrol agent.

22. The method of claim 20, wherein the additional active ingredient: (a) The group consisting of thiamethoxam, Bacillus thuringiensis, abamectin, thiamethoxam, imidacloprid, pyraclostrobin, fluopyram, fluopyram, styraclostrobin, metalaxyl, metalaxyl, fluopyram, prothioconazole, pyraclostrobin and fluopyram. (b) Select from the group consisting of ISO01 (NRRL B-50929), ISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO04 (NRRL B-50932), ISO05 (NRRL B-50933), ISO06 (NRRL B-50934), ISO07 (NRRL B-50935), ISO08 (NRRL B-50936), ISO09 (NRRL B-50937), ISO10 (NRRL B-50938), ISO11 (NRRL B-50939), ISO12 (NRRL B-50940), ISO13 (NRRL B-50941), ISO14 (NRRL B-50942), and ISO16 (NRRL B-67340); and / or (c) are bacteria belonging to the following genera: Actinomycetes, Agrobacterium, Arthrobacterium, Alcaligenes, Chlorobacterium, Azotobacter, Bacillus, Bacillus, Bacillus, Bacillus, Burkholderia, Chromobacterium, Clostridium, Corynebacterium, Trichomonas, Corynebacterium, Brachybacterium, Enterobacter, Flavobacterium, Staphylococcus, Hydrogen-eating Bacteria, Klebsiella, Bacillus-like Bacteria, Pasteurella, Sphingobacterium, Luteobacterium, Chlorobacterium, Pseudomonas, Rhizobium, Chlorogenic Rhizobium, Serratia, Oligotrophozoites, Gluconobacterium, or Pathogenic Bacteria.

23. Use of a composition containing methylbacterium in the treatment of plants or plant parts, wherein, The plant or plant part is coated or partially coated with the composition containing methylbacterium, wherein the methylbacterium is selected from the group consisting of NLS0497 (NRRLB-67925), NLS0693 (NRRL B-67926), NLS1179 (NRRL B-67929), NLS1181 (NRRL B-67927) and NLS0610 (NRRL B-67892).

24. The use according to claim 23, wherein the composition further comprises at least one additional component selected from the group consisting of additional active ingredients, agriculturally acceptable adjuvants, and agriculturally acceptable excipients.

25. The use according to claim 23, wherein the composition comprises a titer of about 1 × 10⁻⁶ for solid compositions. 6 CFU / gm to approximately 1×10 14 CFU / gm or a titer of approximately 1×10⁻⁶ for liquid compositions 6 CFU / mL to approximately 1×10⁻⁶ 11 The methylbacterium, CFU / mL.

26. The use according to claim 23, wherein the methylbacterium is NLS1181 (NRRL B-67927).

27. The use according to any one of claims 23 to 26, wherein the plant part is selected from the group consisting of seeds, stems, flowers, leaves, petioles, pods and axillary buds.

28. The use according to any one of claims 23 to 26, wherein the composition comprises NLS0497 (NRRLB-67925), NLS0693 (NRRL B-67926), NLS1179 (NRRL B-67929), NLS1181 (NRRL B-67927) or NLS0610 (NRRL B-67892).

29. The use according to any one of claims 23 to 26, wherein the additional component is an additional active ingredient.

30. The use according to claim 29, wherein the additional active ingredient is selected from the group consisting of fungicides, insecticides, nematicides and additional biological agents.

31. The use according to claim 29, wherein the additional biological agent is a biocontrol agent.

32. The use according to claim 29, wherein the additional active ingredient: (a) The group consisting of thiamethoxam, Bacillus thuringiensis, abamectin, thiamethoxam, imidacloprid, pyraclostrobin, fluopyram, fluopyram, styraclostrobin, metalaxyl, metalaxyl, fluopyram, prothioconazole, pyraclostrobin and fluopyram. (b) Select from the group consisting of ISO01 (NRRL B-50929), ISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO04 (NRRL B-50932), ISO05 (NRRL B-50933), ISO06 (NRRL B-50934), ISO07 (NRRL B-50935), ISO08 (NRRL B-50936), ISO09 (NRRL B-50937), ISO10 (NRRL B-50938), ISO11 (NRRL B-50939), ISO12 (NRRL B-50940), ISO13 (NRRL B-50941), ISO14 (NRRL B-50942), and ISO16 (NRRL B-67340); and / or (c) are bacteria belonging to the following genera: Actinomycetes, Agrobacterium, Arthrobacterium, Alcaligenes, Chlorobacterium, Azotobacter, Bacillus, Bacillus, Bacillus, Bacillus, Burkholderia, Chromobacterium, Clostridium, Corynebacterium, Trichomonas, Corynebacterium, Brachybacterium, Enterobacter, Flavobacterium, Staphylococcus, Hydrogen-eating Bacteria, Klebsiella, Bacillus-like Bacteria, Pasteurella, Sphingobacterium, Luteobacterium, Chlorobacterium, Pseudomonas, Rhizobium, Chlorogenic Rhizobium, Serratia, Oligotrophozoites, Gluconobacterium, or Pathogenic Bacteria.

33. A composition comprising isolated Methylobacterium selected from the group consisting of NLS0497 (NRRL B-67925), NLS0693 (NRRL B-67926), NLS1179 (NRRL B-67929), NLS1181 (NRRL B-67927) and NLS0610 (NRRL B-67892), and at least one additional component selected from the group consisting of additional active ingredients, agriculturally acceptable adjuvants, and agriculturally acceptable excipients.

34. An isolated methylbacterium, said isolated methylbacterium being selected from the group consisting of NLS0497 (NRRL B-67925), NLS0693 (NRRL B-67926), NLS1179 (NRRL B-67929), NLS1181 (NRRL B-67927) and NLS0610 (NRRL B-67892).

35. A method of treating a plant or plant part with a composition comprising a yield-enhancing methyl bacillus selected from the group consisting of NLS0497 (accessed as NRRL B-67925), NLS0693 (accessed as NRRL B-67926), NLS1179 (accessed as NRRL B-67929), NLS1181 (accessed as NRRL B-67927) and NLS0610 (accessed as NRRL B-67892).

36. The method of claim 35, wherein the plant parts being treated comprise leaves, stems, flowers, roots, seeds, and pods.

37. The method according to claim 35 or 36, wherein the composition is applied as a spray to the plant or the plant part.

38. The method according to claim 35 or 36, wherein the composition is applied as a seed treatment.

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