Methylobacterium strains and methods related thereto for improving plant production and quality

Abstract

The present application relates to a strain of Methylobacterium and methods related thereto for improving plant production and quality. Provided herein are strains of the genus Methylobacterium (Methylobacterium strain) that enhance early plant growth and methods of using the same. Also provided are methods for identifying strains of the genus Methylobacterium that can be used to increase the content of one or more mineral nutrients and / or vitamins in green leaf plants. Related methods are also provided that provide green leaf plants having increased levels of one or more mineral nutrients and / or vitamins, as well as green leaf plants and green leaf items harvested from the plants having increased levels of one or more mineral nutrients and / or vitamins, as a result of treatment with a strain of the genus Methylobacterium as provided herein.

Description

[0001] This application is a divisional application of the application filed on June 2, 2021, with application number 202180043537.4 and entitled "Methylobacterium strains for improving plant production and quality and related methods thereof".

[0002] Cross-referencing related applications

[0003] This international patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 033,364, filed June 2, 2020, and U.S. Provisional Patent Application No. 63 / 088,837, filed October 7, 2020, the entire disclosure of which is incorporated herein by reference.

[0004] Sequence list declaration

[0005] The sequence listing contained in a file named “LeafyGreens_ST25.txt”, which is 22,944 bytes in size (measured in MS-Windows®) and created on May 25, 2021, contains 76 nucleic acid sequences provided with this document via the USPTO’s EFS system and is incorporated herein by reference in its entirety. Background Technology

[0006] Plants require certain macronutrients and micronutrients for growth and metabolism. These elements are typically present in the soil as salts and can be absorbed by plants as ions. In agriculture, the soil may deplete one or more of these nutrients, necessitating the addition of fertilizers to provide sufficient amounts for crop growth. In hydroponic systems, all nutrients must be supplied to the growing plants, and this is often the largest cost component of hydroponic plant production systems. Plants are an important part of a healthy diet, and the high vitamin and nutrient content, especially in leafy green vegetables, is well-known. Methods aimed at increasing yields by improving plant growth and / or increasing macronutrient and micronutrient levels are desired to benefit agricultural practices and human and animal nutrition.

[0007] 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 *Aracoccus*, *Xanthobacter*, *Ancylobacter* (also known as *Microcyclus*), *Thiobacillus*, *Rhodopseudomonas*, *Rhodobacter*, *Acetobacter*, *Bacillus*, *Mycobacterium*, *Arthobacter*, and *Nocardia* (Lidstrom, 2006).

[0008] Some 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 twelve confirmed species in the genus *Methylbacteria*: *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. nodulans* is a non-PPFM nitrogen-fixing methylbacterium (Sy et al., 2001). Methylbacteria are found in soil, dust, freshwater, sediment, and leaf surfaces, as well as in industrial and clinical environments (Green, 2006). Summary of the Invention

[0009] This document provides compositions comprising one or more strains of the genus *Methylobacterium* that enhance early plant growth, improve reproductive / transplant viability, increase nutrient uptake, improve seedling establishment, and / or improve stress tolerance. In some embodiments, the *Methylobacterium* genus in the composition is selected from the group consisting of LGP2022, LGP2023, and LGP2021. In some embodiments, the *Methylobacterium* genus in the composition is a variant of LGP2022, LGP2023, or LGP2021. In some embodiments, the plant is a leafy plant comprising miniature leafy vegetables and / or herbaceous plants. In some embodiments, the plant is a fruit or vegetable plant. In some embodiments, the plant is an inter-row crop. In some embodiments, the plant is grown in a greenhouse. In some embodiments, the plant is grown hydroponically or aeroponicly. Also provided are isolated Methylobacterium species selected from LGP2022, LGP2023 and LGP2021, including compositions of such Methylobacterium species isolates or variants thereof, and plants, plant parts or seeds at least partially coated with compositions including LGP2022, LGP2023, LGP2021 or variants thereof.

[0010] This document provides a method for improving plant production by applying one or more strains of the genus *Methylobacterium* to plants, plant parts, or seeds. In some embodiments, a composition comprising one or more strains of the genus *Methylobacterium* is applied such that the composition coats or partially coats a plant, plant part, or seed. In some embodiments, plant production is improved by enhancing early plant growth. In some embodiments, plant production is improved by increasing rooting of the plant. In some embodiments, plant production is improved by increasing the nutrient content present in the plant or plant part. In some embodiments, plant production is improved by enhancing propagation / transplanting vigor. In some embodiments, plant production is improved by enhancing seedling emergence. In some embodiments, plant production is improved by enhancing stress tolerance. In some embodiments, the *Methylobacterium* in the composition is selected from the group consisting of: LGP2009, LGP2002, LGP2019, LGP2022, LGP2023, and LGP2021. In some embodiments, the *Methylobacterium* spp. in the composition is selected from the group consisting of LGP2001, LGP2002, LGP2009, LGP2015, combinations of LGP2002 and LGP2015, and variants thereof, and the composition is applied such that it coats or partially coats a plant, plant part, or seed, said plant including rosemary, tarragon, basil, foxtail grass, and / or other herbaceous plants. In some embodiments, the *Methylobacterium* spp. in the composition is a variant of any of the *Methylobacterium* spp. isolates described above. In some embodiments, the plant is a leafy plant. In some embodiments, plant biomass is increased by treatment with one or more *Methylobacterium* strains as provided herein. In some embodiments, plant biomass is increased due to enhanced early growth caused by treatment with LGP2022, LGP2023, or LGP2021 or variants thereof. In some embodiments, enhanced early growth is assessed at the two-true-leaf stage of development. In some embodiments of the methods provided herein, the *Methylobacterium* composition is applied to the seeds of hydroponically grown plants, plant parts, or fruits or vegetables. In some embodiments, the Methylobacterium compositions provided herein are applied to the seeds of plants, plant parts, or leafy green vegetables. In some embodiments, such leafy green vegetables are grown hydroponically. In some embodiments, the plants are intercropping crops.

[0011] In some embodiments of the methods for improving plant production provided herein, the plants are leafy plants, and the plant improvements include enhanced early growth in the plant or plant parts, increased nutrient levels, improved propagation / transplanting vigor, improved seedling establishment, and / or improved stress tolerance, and the *Methylobacterium* genus is selected from LGP2009, LGP2022, LGP2023, or LGP2021 or variants thereof. In some embodiments, the leafy plants are selected from the group consisting of spinach, lettuce, beets, Swiss chard, watercress, kale, broccoli, endive, arugula, chicory, bok choy, and turnip. In some embodiments, the *Methylobacterium* genus is selected from combinations of LGP2001, LGP2002, LGP2009, LGP2015, LGP2002, and LGP2015 and variants thereof, and the leafy plants include rosemary, tarragon, basil, pennisetum, and / or other herbaceous plants. In some embodiments of the methods for improving plant production provided herein, the plant is a cannabis plant, and the plant improvement is selected from enhanced growth and / or rooting, reduced cycle time, and increased biomass or yield, and the genus *Methylobacterium* is selected from LGP2002, LGP2009, LGP2019, and variants thereof. In some embodiments, variants of LGP2002 have genomic DNA comprising one or more polynucleotide marker fragments having at least 50, 60, 100, 120, 180, 200, 240, or 300 nucleotides having SEQ ID NO: 13-15. In some embodiments, variants of LGP2009 have genomic DNA comprising one or more polynucleotide marker fragments having at least 50, 60, 100, 120, 180, 200, 240, or 300 nucleotides having SEQ ID NO: 71-73. In some embodiments, variants of LGP2019 have genomic DNA comprising one or more polynucleotide marker fragments having at least 50, 60, 100, 120, 180, 200, 240, or 300 nucleotides having SEQ ID NO: 25-27.

[0012] In some embodiments of the compositions and methods provided herein, 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 embodiments of any of the methods described above, the composition comprises a titer greater than 1 × 10⁻⁶ for liquid compositions. 3 CFU / gm or a titer of approximately 1 × 10⁻⁶ 6 CFU / gm to approximately 1 × 10 14 CFU / gm, or for liquid compositions with a titer greater than 1 × 10⁻⁶. 3 CFU / ml or a titer of approximately 1 × 10⁻⁶6 CFU / mL to approximately 1 × 10⁻⁶ 11 CFU / mL of Methylobacterium.

[0013] This document also provides a method for improving the growth and yield of rice plants by treating rice plants, plant parts, or seeds with one or more Methylobacterium spp. isolates. In some embodiments, the harvested seed yield and / or nutrient content of the rice plants are improved. In some embodiments, rice seeds are treated and such treatment provides increased rice seed yield. In some embodiments, the Methylobacterium spp. isolates are selected from the group consisting of LGP2016 (ISO 117), LGP2017 (ISO 118), LGP2019 (ISO 120), and variants of these isolates. This document also provides rice plants, plant parts, or seeds coated with Methylobacterium spp. isolates and / or compositions. In some embodiments, the chromosomal genomic DNA of Methylobacterium spp. has at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity with the chromosomal genomic DNA of LGP2016, LGP2017, or LGP2019. In some embodiments, the genus *Methylobacterium* has genomic DNA comprising one or more polynucleotide marker fragments having at least 50, 60, 100, 120, 180, 200, 240, or 300 nucleotides having SEQ ID NO: 37-39 or SEQ ID NO: 25-27.

[0014] This document also provides a method for improving the growth and production of cannabis plants by treating cannabis plants, plant parts, or seeds with one or more Methylobacterium spp. isolates. In some embodiments, the nutrient content of the treated plants is improved. In some embodiments, cannabis cuttings from mature plants are treated. In some embodiments, such treatment improves plant growth and rooting of such cuttings. In some embodiments, such treatment provides a reduced cycle time for cannabis plant production due to the increased plant growth and rooting. In some embodiments, the Methylobacterium spp. isolates are selected from the group consisting of LGP2002, LGP2009, LGP2019, and variants of these isolates. This document also provides cannabis plants, plant parts, or seeds coated with Methylobacterium spp. isolates and / or compositions. In some embodiments, the chromosomal genomic DNA of Methylobacterium spp. has at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity with the chromosomal genomic DNA of LGP2002, LGP2009, or LGP2019. In some embodiments, the genus *Methylobacterium* has genomic DNA comprising one or more polynucleotide marker fragments having at least 50, 60, 100, 120, 180, 200, 240, or 300 nucleotides of SEQ ID NO: 13-15, SEQ ID NO: 71-73, or SEQ ID NO: 25-27.

[0015] This document also provides a method for identifying Methylobacterium isolates that increase the content of at least one mineral nutrient and / or at least one vitamin in leafy plants or plant parts, the method comprising: (i) treating leafy plant seeds and / or leafy plants with at least a first Methylobacterium strain to obtain treated seeds and / or treated plants; (ii) harvesting plant parts from cultivated plants, wherein the cultivated plants are grown from the treated seeds or treated plants of step (i); (ii) harvesting plant parts from cultivated control plants, wherein the cultivated control plants are grown from untreated control seeds or untreated control plants; (iii) determining the content of at least one mineral nutrient and / or vitamin in the plant parts from the cultivated plants and cultivated control plants; and (iv) selecting Methylobacterium strains that increase the content of at least one mineral nutrient or vitamin in the cultivated plants or plant parts compared with the content of at least one mineral nutrient or vitamin in the cultivated control plants or plant parts.

[0016] This document also provides a method for identifying Methylbacterium isolates that increase the content of one or more mineral nutrients and / or vitamins by treating and analyzing plants separately with two or more Methylbacterium isolates. Such methods include: (i) treating a first leafy plant seed or plant with at least a first Methylbacterium strain and treating a second leafy plant seed or plant portion with a second Methylbacterium strain; (ii) harvesting plant portions from plants grown from the first seed or plant, plants grown from the second seed or plant, and optionally plants grown from untreated control seeds or untreated control plants; (iii) analyzing the plant portions harvested from the plants grown from the first seed or plant, the plants grown from the second seed or plant, and optionally plants grown from control seeds or plants to determine the content of at least one mineral nutrient and / or vitamin; and (iv) selecting the Methylbacterium strain that provides the largest increase in the content of at least one mineral nutrient and / or vitamin. Such methods can also be used to test three or more Methylbacterium isolates. In such methods, seeds and / or plants are treated with three or more different Methylobacterium isolates or combinations of different Methylobacterium isolates, respectively, to determine the mineral nutrient and / or vitamin content in the plants, shoots, or one or more plant leaves, compared with other different Methylobacterium treatments and optionally with untreated control plants. The treated seeds or seedlings are then cultivated, harvested, and analyzed to determine the mineral nutrient and / or vitamin content in the plants, shoots, or one or more plant leaves.

[0017] A method for producing leafy green food products with increased levels of one or more mineral nutrients and / or vitamins is also provided, the method comprising harvesting leafy green vegetables from one or more cultivated plants grown from seeds, plants, or plant parts treated with *Methylobacterium*, thereby obtaining leafy green vegetables with increased levels of one or more mineral nutrients and / or vitamins. In some embodiments of such methods, the *Methylobacterium* strain is LGP2009 (ISO110 (NRRL B-50938)) or a variant thereof. In some embodiments, the chromosomal genomic DNA of the *Methylobacterium* strain has at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity with the chromosomal genomic DNA of ISO110 (NRRL B-50938). In some embodiments, the *Methylobacterium* strain has genomic DNA comprising one or more polynucleotide marker fragments having at least 50, 60, 100, 120, 180, 200, 240, or 300 nucleotides having SEQ ID NO: 71-73. In some embodiments of the above methods, the composition comprises (i) *Methylobacterium*, wherein the average nucleotide identity (ANI) score of the assembled genomic DNA sequence of *Methylobacterium* is at least 99.00 when compared with an assembled genomic DNA sequence of ISO 110 (NRRL B-50938). A method is also provided for producing a harvested leafy green food product with increased levels of one or more mineral nutrients and / or vitamins, the method comprising harvesting leafy green vegetables from leafy plants grown from seeds and / or seedlings treated with an effective amount of *Methylobacterium* strain, wherein the leafy green plants are mature plants, immature plants with two to four true leaves, or immature plants with fewer than two true leaves, thereby obtaining a leafy, tender, or miniature green food product with increased levels of one or more mineral nutrients and / or vitamins. In some embodiments of such methods, *Methylobacterium* is ISO 110 (NRRL B-50938). In some embodiments, the chromosomal genomic DNA of *Methylobacterium* has at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity with the chromosomal genomic DNA of ISO 110 (NRRL B-50938). In some embodiments, *Methylobacterium* has genomic DNA comprising one or more polynucleotide marker fragments having at least 50, 60, 100, 120, 180, 200, 240, or 300 nucleotides having SEQ ID NO: 71-73. In some embodiments, the leafy plant is cultivated in a hydroponic system. In some embodiments, the leafy plant is a spinach plant.

[0018] This document also provides leafy green plants or plant parts with increased levels of one or more mineral nutrients and / or vitamins. The leafy green plants or plant parts are harvested from cultivated plants grown from seeds, plants, or plant parts treated with *Methylobacterium*, wherein *Methylobacterium* provides increased levels of one or more mineral nutrients and / or vitamins. In some embodiments, *Methylobacterium* is ISO 110 (NRRL B-50938). In some embodiments, the chromosomal genomic DNA of *Methylobacterium* has at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity with the chromosomal genomic DNA of ISO 110 (NRRL B-50938). In some embodiments, *Methylobacterium* has genomic DNA comprising one or more polynucleotide marker fragments having at least 50, 60, 100, 120, 180, 200, 240, or 300 nucleotides having SEQ ID NO: 71-73. In some embodiments, the *Methylobacterium* genus includes *Methylobacterium*, wherein the average nucleotide identity (ANI) score of the assembled genomic DNA sequence of *Methylobacterium* is at least 99.00 when compared with the assembled genomic DNA sequence of ISO 110 (NRRL B-50938). In some embodiments, the leafy plant is a spinach plant. Detailed Implementation

[0019] definition

[0020] The term “and / or” as used herein is to be considered a specific disclosure of each of two or more particular features or components having or not having the other. Therefore, the term “and / or” as used herein in phrases such as “A and / or B” is intended to encompass “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).

[0021] 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.

[0022] As used herein, the term "biologic agent" refers to a component of a composition for treating a plant or plant part comprising or derived from a microorganism. Biologic agents 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 biological agent may include a single culture or co-culture of *Methylobacterium*, or a combination of individually cultured *Methylobacterium* strains or isolates.

[0023] As used herein, “leafy greens” means vegetable crops with edible leaves and includes, but is not limited to, spinach, kale, lettuce (including, but not limited to, longleaf endive, iceberg lettuce, head lettuce, and loose-leaf lettuce), kale, cabbage, beetroot, watercress, Swiss chard, arugula, endive, chicory, bok choy, and turnip leaves. As used herein, leafy greens also refer to plants cultivated for the harvest of miniature leafy greens and / or herbs, including, but not limited to: lettuce, cauliflower, broccoli, cabbage, watercress, arugula, garlic, onion, leeks, amaranth, Swiss chard, beets, spinach, melon, cucumber, squash, basil, celery, coriander, radish, red chicory, sow thistle, dill, rosemary, tarragon, basil, pennisetum, carrot, fennel, beans, peas, chickpeas, and lentils. Leafy greens also refer to mixtures of various leafy greens, such as mixed lettuce (mesclun) or other mixed salad leafy greens or mixed miniature leafy greens. As used in this article, “leafy greens” also includes other Brassica or Brassicaceae field leafy greens not specifically named herein.

[0024] As used in this article, “fruit” or “fruiting plant” refers to a succulent fruiting plant, including but not limited to melons (including watermelons and cantaloupes), berries (including strawberries, blueberries, blackberries, and raspberries), grapes, kiwifruit, mangoes, papayas, pineapples, bananas, peppers, tomatoes, squash, and cucumbers.

[0025] As used herein, the term “Methylbacterium” refers to the genera and species within the family Methylbacteriaceae, including bacterial species within the genus Methylbacterium and proposed Methylbacterium species (Green and Ardley (2018)). The genus Methylbacterium includes pink facultative methyltrophs (PPFM) and also encompasses non-pink nodular Methylbacterium, as well as colorless mutants of Methylbacterium isolates. For example, but not limited to, "Methylobacterium" refers to the following species of bacteria, as well as any new methylobacteria that have not yet been reported or described, which can be characterized as Methylobacterium / Methylorubrum based on phylogenetic analysis: Methylobacterium adhaesivum; Methylobacterium oryzae; Methylobacterium aerolatum; Methylobacterium oxalidis; Methylobacterium aquaticum; Methylobacterium persicinum; Methylobacterium braachiatum; 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 aminovorans) 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 nodulans; Methylobacterium zatmanii. *Zatmanii* or *Methylobacterium organophilum*.

[0026] As used herein, “colonization efficiency” refers to the relative ability of a given microbial strain to colonize plant host cells or tissues compared to a non-colonization control sample or other microbial strains. Colonization efficiency can be assessed, for example but not limited to, by determining colonization density, reported as colony-forming units (CFU) per mg of plant tissue, or by quantifying strain-specific nucleic acids in a colonization sieve, for example using qPCR.

[0027] As used in this article, “mineral nutrients” (sometimes simply referred to as “nutrients”) are micronutrients or macronutrients that are required or useful for plants or plant parts, including, but not limited to, nitrogen (N), potassium (K), calcium (Ca), magnesium (Mg), phosphorus (P) and sulfur (S), as well as the micronutrients chlorine (Cl), iron (Fe), boron (B), manganese (Mn), zinc (Z), copper (Cu), molybdenum (Mo) and nickel (Ni).

[0028] As used in this article, “vitamin” refers to small amounts of organic compounds required for normal growth and metabolism. Vitamins are important for the growth of humans and / or animals, and some vitamins have been reported to be beneficial to plants. Vitamins include, but are not limited to, vitamin A (including, but not limited to, all-trans retinol, all-trans retinyl esters, all-trans beta-carotene, and other provitamin A carotenoids), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B7 (biotin), vitamin B9 (folic acid or folate), vitamin B12 (cobalamin), vitamin C (ascorbic acid), vitamin D (calciferol), vitamin E (tocopherols and tocotrienols), and vitamin K (quinones).

[0029] As used herein, the term “strain” should include all isolates of such strains.

[0030] As used herein, when applied in the context of *Methylobacterium* isolates, "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 a reference *Methylobacterium* isolate, such as 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 preserved isolates. Sequence analysis tools such as BLAST (as taught by Altschul et al. (1990)) or clustalw (www.ebi.ac.uk / Tools / msa / clustalw2 / ) can be used to sequence Methylobacterium isolates or strains (as taught by Sanger et al. (1977), Bentley et al. (2008), or Caporaso et al. (2012)) and to perform genome-scale comparisons of the sequences (Konstantinidis et al. (2005)).

[0031] As used herein, when applied in the context of *Methylobacterium* isolates, “derivative” 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 selected by mutagenesis and selection, and genetically transformed *Methylobacterium* isolates obtained from *Methylobacterium* isolates. A “derivative” can be identified, for example, based on genetic identity with the strain or isolate from which it was obtained, and will generally be 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.

[0032] 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 for fast genome comparison within multiple bacterial strains"; *Database*, 2016, 1–5) to determine the average nucleotide identity (ANI) score.

[0033] As used herein, the term "cultivated" means the growing of plants. Cultivated plants can be plants grown and raised on a large agricultural scale or a smaller scale, and may include, for example, a single plant.

[0034] As used in this article, the terms “hydroponics,” “hydroponics method,” or “hydroponic land” refer to the method of cultivating plants in the absence of soil.

[0035] Where the terminology is provided in singular form, other embodiments described in plural form of the terminology are also provided.

[0036] 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.

[0037] Other descriptions

[0038] This document provides isolated *Methylobacterium* strains that enhance early plant growth, improve propagation / transplanting vigor, increase nutrient uptake, improve seedling establishment, and / or improve stress tolerance, and compositions that can be used to treat plants with such strains. In some embodiments, the *Methylobacterium* in the composition is selected from the group consisting of LGP2022, LGP2023, and LGP2021. In some embodiments, the *Methylobacterium* in the composition is a variant of LGP2022, LGP2023, or LGP2021. In some embodiments, early plant development is enhanced, for example, before the plant reaches the two true leaf stage. In some embodiments, the plant is a fruit or vegetable plant. In some embodiments, the plant is a leafy plant. In some embodiments, the plant is grown in a greenhouse. In some embodiments, the plant is grown hydroponically or in an aeroponic plant cultivation system. Isolated *Methylobacterium* strains selected from LGP2022, LGP2023, and LGP2021 are also provided.

[0039] Further, methods are provided for improving plant production by treating plants with the *Methylobacterium* strains provided herein, said plants including leafy plants, fruit and vegetable plants, row crops such as corn, soybeans, wheat, barley, etc., and specialty crops including hemp. In some embodiments, production is improved by enhanced early growth of the treated plants or plants grown from treated seeds compared to untreated control plants or control plants grown from untreated seeds. Such enhanced early growth is measured, for example, by an increase in biomass in the treated plants, comprising increased biomass in shoots, leaves, roots, or whole seedlings. Increased early growth can cause various improvements in plant production compared to untreated control plants or control plants grown from untreated seeds, including, for example, increased biomass production or yield of harvested plants, increased and / or more uniform fruit production, faster seed setting, earlier maturity, increased leaf growth rate, increased root growth rate, increased seed yield, and reduced cycle time. In some embodiments, the application of *Methylobacterium* strains, as provided herein, provides an increase of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 30%, or 40% of any of the aforementioned traits compared to untreated control plants or control plants grown from untreated seeds. In some embodiments, production is increased, for example, by increased rooting of plant cuttings, wherein such increased rooting can lead to a reduction in cycle time and / or an increase in biomass or yield of the treated plants.

[0040] This document also provides various methods for identifying Methylobacterium strains that increase the content of at least one mineral nutrient and / or at least one vitamin in leafy plants or plant parts. In such methods, leafy plant seeds and / or leafy plant seedlings are treated with at least a first Methylobacterium strain to obtain treated seeds and / or treated plants or plant parts, such as plant cuttings. After cultivation of the treated seeds, plants, or plant parts, plant parts are harvested from the cultivated plants and control plants grown from untreated control seeds or untreated control plants, or from plants treated with a second Methylobacterium strain. The levels of at least one mineral nutrient and / or vitamin in the harvested plant parts of the treated and control plants are determined compared to the content of at least one mineral nutrient or vitamin in the cultivated control plants or plant parts, or compared to plants treated with other Methylobacterium strains, and Methylobacterium strains that increase the content of at least one mineral nutrient or vitamin in the cultivated plants or plant parts are selected. In some embodiments, leafy plant seeds are treated. In other embodiments, leafy plant seedlings or parts thereof are treated. In some embodiments, leafy plant seeds or seedlings are treated with two, three, four, or more strains of *Methylobacterium*, and the levels of one or more mineral nutrients and vitamins in the plants or plant parts treated with different strains are compared. One or more *Methylobacterium* strains that exhibit increased levels of one or more mineral nutrients and vitamins are selected. In some embodiments, plants are treated with a combination of *Methylobacterium* strains, and combinations that can be used to treat leafy plants to increase vitamins and / or nutrients are identified.

[0041] In some embodiments, leafy plant seeds are treated. In some other embodiments, plant seedlings or portions thereof are treated. In some embodiments, leafy plant shoots or seedlings are treated. In some embodiments, leafy plant seedlings are treated before the first true leaf appears. In some embodiments, treated leafy plants are cultivated to the second true leaf stage and harvested to determine the levels of at least one mineral nutrient and / or vitamin. In some embodiments, treated leafy plants are cultivated to the third or fourth true leaf stage. In some embodiments, treated leafy plants are cultivated for 10 to 14 days. In some embodiments, treated leafy plants are cultivated for 14 to 28 days. In some embodiments, treated leafy plants are cultivated for 28 days or longer before harvesting and analyzing tissue samples to determine the levels of mineral nutrients and vitamins. In some embodiments, treated leafy plant seeds or seedlings are cultivated in a hydroponic or aeroponic plant growth system. The hydroponic system may be a hydroponic system, nutrient film technology, fluctuation system, drip irrigation system, or wick system. In an aeroponic system, plants are grown in air or a mist environment without the use of soil. In some embodiments, the hydroponic or aeroponic system can be a variant of any of these types or a combination of one or more systems. In some embodiments, hydroponic or aeroponic systems are superior to soil-based cultivation systems in determining the role of *Methylobacterium* strains due to the presence of fewer background microorganisms. Various inert substrates can be used to support plants, seedlings, and root systems in hydroponic or aeroponic growth, including, but not limited to, perlite, rock wool, clay granules, foam cubes, rock, peat moss, or vermiculite.

[0042] In some embodiments, Methylobacterium strains tested in the disclosed methods for identifying strains that increase the content of at least one mineral nutrient and / or at least one vitamin in leafy plants or plant parts colonize plant host cells or tissues more effectively than other Methylobacterium strains. A method for identifying microbial strains with enhanced colonization efficiency is described in WO2020163027 (PCT / US2020 / 012041), which is incorporated herein by reference in its entirety.

[0043] In some embodiments, Methylobacterium strains that increase the content of at least one mineral nutrient and / or at least one vitamin in leafy plants or plant parts also confer on the leafy plants improved traits selected from: increased biomass production, reduced cycle time, increased leaf growth rate, reduced time to develop two true leaves, increased root growth rate, and increased seed yield.

[0044] This document provides various methods for using *Methylobacterium* strains to enhance early growth or rooting, increase mineral nutrient and / or vitamin content, improve propagation / transplanting viability, improve seedling emergence, and / or improve the stress tolerance of plants such as leafy plants, row crops, hemp, and other specialty crops. In some embodiments, treatment with *Methylobacterium* strains in row crops including, but not limited to, corn, soybean, rice, rapeseed, and wheat resulted in increased plant growth and yield. In some embodiments, the crop is rice, and the *Methylobacterium* strain is selected from the group consisting of: LGP2016 (ISO 117), LGP2017 (ISO 118), LGP2019 (ISO 120), and variants thereof. In some embodiments, *Methylobacterium* strains selected from combinations of LGP2001, LGP2002, LGP2009, LGP2015, LGP2002, and LGP2015, and variants thereof, are applied to rosemary, tarragon, basil, pennisetum, and other herbs to improve growth and root development. In some embodiments, treatment of soil, seeds, leaves, stems, roots, or shoots with *Methylobacterium* can enhance early growth, propagation / transplanting vigor, seedling establishment, and / or stress tolerance, and may alternatively increase the content of one or more mineral nutrients or vitamins in the leafy greens or plant parts harvested from plants grown from *Methylobacterium*-treated plant parts or seeds as described herein. In some embodiments, *Methylobacterium* LGP2022, *Methylobacterium* LGP2023, *Methylobacterium* LGP2021, or variants thereof are applied to plants, plant parts, or seeds to enhance early plant growth and improve plant production.

[0045] Alternatively, such Methylobacterium species can be applied to soil or other growth media for plant growth. Soil treatment or application of Methylobacterium species may include, but is not limited to, application in furrows (e.g., before, during, and / or after seed deposition), soil irrigation, and the distribution of granules or other dry formulations to the soil (e.g., before, during, and / or after seed deposition or plant growth). Methylobacterium treatment for plants grown in hydroponic systems may include pre-germination seed treatment, foliar application to germinating plants or portions thereof, and application in liquid solutions used in hydroponic systems. In some embodiments, Methylobacterium treatment for leafy plants may include application to seeds, plants, and / or portions of plants, and thus may include any Methylobacterium treatment or application that leads to colonization of leafy plants by Methylobacterium species. In some embodiments, application of Methylobacterium species to crops propagated by cuttings may enhance the growth and / or rooting of such plants. Field transplantation of such treated and rooted cuttings due to such treatment may show reduced cycle times and / or increased biomass and / or yield. In some embodiments, Methylobacterium species selected from LGP2002, LGP2009, LGP2019 and their variants are applied to cannabis cuttings to improve growth and root development.

[0046] Treatment or application of the plants described herein may include, but is not limited to, spraying, coating, partially coating, impregnating, and / or absorbing seeds, plants, or plant parts with the Methylobacterium strains provided herein and compositions comprising therein. In some embodiments, soil, seeds, leaves, stems, roots, tubers, or shoots may be sprayed, impregnated, and / or absorbed with liquids, semi-liquids, emulsions, or slurries of the compositions provided herein. Compared to untreated plants or plants grown from untreated seeds, such treatments, applications, seed impregnation, or absorption may be sufficient to provide enhanced early growth and / or increased levels of one or more mineral nutrients and / or vitamins in harvestable tissues from treated plants or plants grown from treated seeds. Enhanced early growth may result in further improvements in plant production, including increased biomass in treated plants, such as shoots, roots, or whole seedlings. Enhanced early growth can lead to various additional improvements in plant production, including, for example, increased yield of harvested plants or harvested plant parts, increased and / or more uniform fruit production, faster seed setting, earlier maturity, increased leaf growth rate, increased root growth rate, increased seed yield, and reduced cycle time. In some embodiments, plant seeds or cuttings may be immersed and / or absorbed for at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, such immersion and / or absorption may be carried out at temperatures harmless to plant seeds or Methylobacterium. In some embodiments, seeds may be treated at about 15°C to about 30°C or about 20°C to about 25°C. In some embodiments, seed percolation and / or immersion may be carried out with gentle agitation. Seed treatment may be carried out using continuous and / or intermittent seed treatment machines. In some embodiments, coated seeds may be prepared by pulping the seeds with a coating composition comprising a Methylobacterium strain that increases the levels of one or more mineral nutrients and / or vitamins. Air drying can be performed at any temperature that is harmless to the seeds or *Methylobacterium*, but generally will not exceed 30 degrees Celsius. The coating proportions including *Methylobacterium* strains include, but are not limited to, the following ranges: 0.1 to 25% by weight of the seeds or other plant parts, 0.5 to 5% by weight of the seeds or other plant parts, and 0.5 to 2.5% by weight of the seeds or other plant parts. In some embodiments, due to growth in a biphasic medium comprising *Methylobacterium* strains, solid material, and liquid culture medium, the solid material used for seed coating or treatment will contain *Methylobacterium* strains, which increase the mineral nutrient and / or vitamin content adhering to the solid material. Methods for culturing *Methylobacterium* in a biphasic medium include those described in U.S. Patent No. 9,181,541, which is incorporated herein by reference in its entirety.In some embodiments, compositions suitable for treating seeds or plant parts can be obtained by the method provided in U.S. Patent No. 10,287,544, which is specifically and entirely incorporated herein by reference. Various seed treatment compositions and methods for seed treatment 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 are applicable to the treatment of seeds with compositions comprising strains of the genus *Methylobacterium*.

[0047] In some embodiments of treating plant seeds with the Methylobacterium compositions provided herein, the compositions further include one or more lubricants to ensure smooth flow and separation (single-cut) of the seeds in a seeding apparatus such as a seeding cylinder. Lubricants used in such compositions include talc, graphite, polyethylene wax-based powders (such as fluidity agents), protein powders such as soy protein powder, or combinations of protein powders and lipids such as lecithin or vegetable oils. The lubricant may be applied to the seeds simultaneously with the Methylobacterium or may be mixed with the Methylobacterium prior to applying the composition to the seeds.

[0048] In some embodiments, the treated plants are cultivated in a hydroponic system. In some embodiments, plant seeds are treated and plants are continuously grown from the treated seeds in the same cultivation system. In some embodiments, plant seeds are treated and cultivated in a hydroponic nursery to produce seedlings. The seedlings are transferred to different hydroponic systems for the commercial production of leafy green vegetables. In some embodiments, Methylobacterium strains that enhance early growth or increase the levels of one or more mineral nutrients and / or vitamins persist in the seedlings transferred to the greenhouse production system and continue to provide advantages, such as increased micronutrient and / or vitamin content and / or biomass production, through further growth of the leafy plants. In some embodiments, the plant seedlings transferred to the greenhouse production system may be further treated with LGP2009, LGP2022, LGP2023, LGP2021 or variants thereof, or with one or more other Methylobacterium strains that increase the levels of one or more mineral nutrients and / or vitamins before, during or after transfer to the production system.

[0049] In some embodiments, the composition for treating seeds or plant parts may contain a strain of *Methylobacterium* and agriculturally acceptable excipients. Agriculturally acceptable excipients include, but are not limited to, wood flour, clay, activated carbon, diatomaceous earth, fine-grained inorganic solids, calcium carbonate, etc. Clays and inorganic solids that can be used with these include, but are not limited to, calcium bentonite, kaolin, porcelain clay, talc, perlite, mica, vermiculite, silica, quartz powder, montmorillonite, and mixtures thereof. Agriculturally acceptable excipients also include various lubricants such as talc, graphite, polyethylene wax-based powders (such as lubricants), protein powders such as soy protein powder, or combinations of protein powders and lipids such as lecithin or vegetable oils.

[0050] Agriculturally acceptable adjuvants that can be used to promote adhesion to seeds include, but are 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. Further, agriculturally acceptable adjuvants also include various lubricants (which can provide smooth flow and separation (single-cut) of seeds) such as talc, graphite, polyethylene wax-based powders (such as flow agents), protein powders such as soy protein powder, or combinations of protein powders and lipids such as lecithin or vegetable oils. Various surfactants, dispersants, anti-caking agents, defoaming agents, and dyes disclosed herein and in U.S. Patent No. 8,181,388 can be adapted to compositions including suitable *Methylobacterium* strains. In some embodiments, seeds and / or seedlings are exposed to the composition by providing *Methylobacterium* strains in soil in which plants or seed-produced plants are grown, or in other plant growth media in which plants or seed-produced plants are grown. Examples of methods for providing *Methylobacterium* strains in soil include furrow application, soil irrigation, etc.

[0051] Non-limiting examples of treatments with *Methylobacterium* to plant seeds, seedlings, or other plant parts that provide enhanced early growth and / or increase the content of one or more mineral nutrients and / or vitamins in the harvested plant parts include treatments on vegetable crops with edible leaves, including but not limited to spinach, kale, lettuce (including but not limited to longleaf endive, iceberg lettuce, cabbage endive, and loose-leaf lettuce), and field green leafy vegetables including *Brassica* var. *brassica*. Specific leafy green vegetables that can be treated with *Methylobacterium* as described herein include kale, cabbage, beetroot, watercress, Swiss chard, arugula, endive, chicory, bok choy, and turnip leaves. Other leafy greens grown for the production and harvesting of miniature leafy vegetables and / or herbs may also be treated in the methods described herein to provide increased content of one or more mineral nutrients and / or vitamins in the harvested miniature leafy greens and herbs, including but not limited to lettuce, cauliflower, broccoli, cabbage, watercress, arugula, garlic, onion, leek, amaranth, Swiss chard, beet, spinach, melon, cucumber, squash, basil, celery, coriander, radish, red chicory, sow thistle, dill, rosemary, tarragon, basil, foxtail grass, carrot, fennel, beans, peas, chickpeas, and lentils. Treatment of plants grown for the harvesting of fleshy fruits is also provided herein. Such plants include, for example, melons (including watermelon and cantaloupe), berries (including strawberries, blueberries, blackberries, and raspberries), grapes, kiwifruit, mango, papaya, pineapple, banana, pepper, tomato, squash, and cucumber plants.

[0052] In some embodiments, LGP2022, LGP2023, LGP2021, or variants thereof will also be used to treat other plant species to enhance early growth, including, for example, field crops, ornamental plants, turfgrasses, and trees grown in commercial production such as conifers. Unrestrictedly, such additional plant species include corn, soybeans, cruciferous plants or Brassica vegetables (e.g., canola, turnip, Indian mustard), alfalfa, rice, rye, wheat, barley, oats, sorghum, millet (e.g., pearl millet, sorghum sorghum, foxtail millet, and finger millet), sunflower, safflower, tobacco, potato, peanut, cotton, cannabis species (including but not limited to hemp and industrial hemp varieties), sweet potato, cassava, coffee, coconut, and ornamental plants (including but not limited to azaleas, hydrangeas, etc.). Hibiscus, rose, tulip, daffodil, morning glory, carnation, poinsettia and chrysanthemum), conifers (including but not limited to pine such as loblolly pine, slash pine, American yellow pine, black pine and Montreal pine, Douglas fir, hemlock, western spruce, redwood, fir such as silver fir and balsam fir; and cedar such as western red cedar and Alaskan yellow cedar) and turfgrass (including but not limited to annual Kentucky bluegrass, annual ryegrass, Canada bluegrass, fescue, creeping bentgrass, wheatgrass, Kentucky bluegrass, orchard grass, ryegrass, redtop grass, Bermuda grass, St. Augustine grass and Zoysia grass).

[0053] In some embodiments, the *Methylobacterium* strain used to treat the seeds, plants, or plant parts of a given cultivar or variety may be a *Methylobacterium* strain isolated from a different plant species or a different cultivar or variety of the plant species being treated, and is therefore heterologous or non-resident in the treated plant or plant part. Plant parts that have increased levels of one or more mineral nutrients and / or vitamins due to treatment with *Methylobacterium* as provided herein include, but are not limited to, leaves, stems, flowers, roots, seeds, fruits, tubers, coleoptiles, etc. In some embodiments, plants that have enhanced early growth due to treatment with LGP2022, LGP2023, LGP2021, or variants thereof, or plants that have enhanced levels of one or more mineral nutrients due to treatment with the *Methylobacterium* compositions provided herein, are leafy plants. In some embodiments, leaves contain increased levels of one or more mineral nutrients and / or vitamins. In some embodiments, harvested leafy green vegetables comprising leaves and shoots contain increased levels of one or more mineral nutrients and / or vitamins.

[0054] In some embodiments, a composite composition comprising two or more strains of the genus *Methylobacterium* can be used to treat seeds or plant parts in any of the methods provided herein. Such a composite composition can be prepared by a method comprising harvesting a single culture of each *Methylobacterium* strain and mixing the harvested single cultures to obtain the composite composition. In some embodiments, the composite composition may comprise *Methylobacterium* strains isolated from different plant species or from different cultivars or varieties of a given plant.

[0055] In some embodiments, the effective amount of one or more Methylobacterium strains used for treating plants, seeds, or plant parts is a composition having the following Methylobacterium titers: at least about 1 × 10⁻⁶ per milliliter. 6 Each colony-forming unit, at least approximately 5 × 10⁶ per milliliter 6 Each colony-forming unit, at least approximately 1 × 10⁻⁶ per milliliter 7 Each colony-forming unit, at least approximately 5 × 10⁶ per milliliter 8 Each colony-forming unit, at least approximately 1 × 10⁻⁶ per milliliter 9 Each colony-forming unit, at least approximately 1 × 10⁻⁶ per milliliter 10 Each colony-forming unit or at least approximately 3 × 10⁶ units per milliliter 10 Each colony-forming unit. In some embodiments, the effective amount of one or more Methylobacterium strains is a composition having the following Methylobacterium titers: at least about 1 × 10⁻⁶ per milliliter of liquid or emulsion. 6 Each colony-forming unit, at least approximately 5 × 10⁶ per milliliter 6 Each colony-forming unit, at least approximately 1 × 10⁻⁶ per milliliter 7 Each colony-forming unit or at least approximately 5 × 10⁶ units per milliliter 8 The number of colony-forming units is at least approximately 6 × 10⁶ per milliliter. 10 Each colony-forming unit. In some embodiments, the effective amount of one or more Methylobacterium strains is a composition having at least about 1 × 10⁻⁶ Methylobacterium species per gram of composition. 6 Each colony-forming unit, at least approximately 5 × 10⁻⁶ per gram 6 Each colony-forming unit, at least approximately 1 × 10⁻⁶ per gram 7 Each colony-forming unit or at least approximately 5 × 10⁶ units per gram 8 The number of colony-forming units is at least approximately 6 × 10⁶ per gram. 10 Methylobacterium spp. colony-forming units. In some embodiments, an effective amount of the composition provided herein may be a composition having the following Methylobacterium spp. titer: at least about 1 × 10⁻⁶ per gram of particles in the composition. 6Each colony-forming unit, at least approximately 5 × 10⁻⁶ per gram 6 Each colony-forming unit, at least approximately 1 × 10⁻⁶ per gram 7 Each colony-forming unit or at least approximately 5 × 10⁶ units per gram 8 The number of colony-forming units is at least approximately 6 × 10⁶ per gram. 10 The composition comprises colony-forming units of *Methylobacterium*, wherein particles including solid matter are to which single or co-cultures of one or more *Methylobacterium* strains adhere. In some embodiments, the compositions provided herein that are effective in a plant or plant part may have the following *Methylobacterium* titers: at least about 1 × 10⁻⁶ per mL of composition. 6 Each colony-forming unit, at least approximately 5 × 10⁻⁶ per mL 6 Each colony-forming unit, at least approximately 1 × 10⁻⁶ per mL 7 One colony-forming unit or at least about 5 × 10⁶ per mL 8 The number of colony-forming units is at least approximately 6 × 10⁻⁶ per mL. 10 The composition comprises a colony-forming unit of *Methylobacterium*, wherein a single culture or co-culture of one or more *Methylobacterium* strains adhering to a solid material is provided therein or in which such a culture is grown. In some embodiments, an effective amount of the composition provided herein may be a composition having the following *Methylobacterium* titer: at least about 1 × 10⁻⁶ per mL of the composition. 6 Each colony-forming unit, at least approximately 5 × 10⁻⁶ per mL 6 Each colony-forming unit, at least approximately 1 × 10⁻⁶ per mL 7 One colony-forming unit or at least about 5 × 10⁶ per mL 8 The number of colony-forming units is at least approximately 6 × 10⁻⁶ per mL. 10 The composition comprises a colony-forming unit of *Methylobacterium*, wherein the composition provides or grows a single culture or co-culture of one or more *Methylobacterium* strains therein. In some embodiments, any of the aforementioned compositions comprising a single culture or co-culture of one or more *Methylobacterium* strains may further comprise a single culture or co-culture of *Rhizobium* and / or *Bradyrhizobium*.

[0056] In some embodiments, the effective amount of one or more *Methylobacterium* strains provided in the treatment of seeds or plant parts to provide increased early growth and / or increased mineral nutrients and / or vitamin content is at least about 10 per seed or treated plant part. 3 10 4 10 5 Or 106 CFU. In some embodiments, the effective amount of Methylobacterium provided in the treatment of seeds or plant parts is at least about 10 CFU per seed or treated plant part. 3 10 4 10 5 Or 10 6 CFU to approximately 10 7 10 8 10 9 Or 10 10 CFU. In some embodiments, the effective amount of Methylobacterium provided in the treatment of the seed or plant part is an amount in which the CFU per seed or treated plant part will exceed the CFU number of any resident naturally occurring Methylobacterium strain by at least 5, 10, 100, or 1000 times. In some embodiments, the effective amount of Methylobacterium provided in the treatment of the seed or plant part is an amount in which the CFU per seed or treated plant part will exceed the CFU number of any resident naturally occurring Methylobacterium strain by at least 2, 3, 5, 8, 10, 20, 50, 100, or 1000 times. In some embodiments where the treated plants are cultivated in a hydroponic system, the population of naturally occurring Methylobacterium or other soil microorganisms will be minimal.

[0057] Non-limiting examples of *Methylobacterium* strains that can be used in the methods provided herein are disclosed in Table 1. Other *Methylobacterium* strains that can be used in some of the methods provided herein include variants of the *Methylobacterium* strains disclosed in Table 1. Various combinations of two or more strains or variants of *Methylobacterium* strains disclosed in Table 1 can also be used for the treatment of plants or parts thereof.

[0058]

[0059] 1The accession number of the strain deposited in the Agricultural Research Service Culture Collection (NRRL) of the National Center for Agricultural Utilization Research, Agricultural Research Service, US Department of Agriculture, at 1815 North University Street, Peoria, Illinois 61604 USA, in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, is hereby granted. Pursuant to 37 CFR §1.808(b), all restrictions imposed by the depositor on public access to the deposited material will be irrevocably lifted upon the granting of any patent derived from this patent application.

[0060] The variants of the *Methylobacterium* isolates listed in Table 1 comprise isolates obtained through genetic transformation, mutagenesis, and / or insertion of heterologous sequences. In some embodiments, such variants 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 they are derived. In some embodiments, such variants are characterized by the presence of one or more unique DNA sequences comprising: (i) SEQ ID NO: 1 to 3, SEQ ID NO: 13 to 15, SEQ ID NO: 25 to 27, SEQ ID NO: 37 to 39, SEQ ID NO: 49 to 51 and SEQ ID NO: 61 to 73; or (ii) sequences having at least 98% or 99% sequence identity in the full length of SEQ ID NO: 1 to 3, SEQ ID NO: 13 to 15, SEQ ID NO: 25 to 27, SEQ ID NO: 37 to 39, SEQ ID NO: 49 to 51, SEQ ID NO: 61 to 73 and SEQ ID NO: 74 to 76.

[0061] In some embodiments of the methods provided herein, one or more strains of the genus *Methylobacterium* used for treating leafy plant seeds and / or plant parts are selected from the group consisting of: ISO 101 (NRRL B-50929), ISO 102 (NRRL B-50930), ISO 103 (NRRL B-50931), ISO 104 (NRRL B-50932), ISO 105 (NRRL B-50933), ISO 106 (NRRL B-50934), ISO 107 (NRRL B-50935), ISO 108 (NRRL B-50936), ISO 109 (NRRL B-50937), ISO 110 (NRRL B-50938), ISO 111 (NRRL B-50939), ISO 112 (NRRL B-50940), ISO 113 (NRRL B-50941), ISO 114 ...2 (NRRL B-50939), ISO 113 (NRRL B-50941), ISO 114 (NRRL B-50939), ISO 112 (NRRL B-50939), ISO 115 (NRRL B-67339), ISO 116 (NRRL B-67340), ISO 117 (NRRL B-67341), ISO 118 (NRRL B-67741), ISO 119 (NRRL B-67742), ISO 120 (NRRL B-67743), ISO 121 (NRRL-B-67892), its variants or any combination thereof.In some embodiments, one or more Methylobacterium strains used in the method may include total genomic DNA (chromosomal and plasmid DNA) or average nucleotide identity (ANI), and may be associated with ISO 101 (NRRL B-50929), ISO 102 (NRRL B-50930), ISO 103 (NRRL B-50931), ISO 104 (NRRL B-50932), ISO 105 (NRRL B-50933), ISO 106 (NRRL B-50934), ISO 107 (NRRL B-50935), ISO 108 (NRRL B-50936), ISO 109 (NRRL B-50937), ISO 110 (NRRL B-50938), ISO 111 (NRRL B-50939), ISO 112 (NRRL B-50940), ISO 113 (NRRL B-50941), and ISO 114 (NRRL B-50939). The total genomic DNA of the samples (NRRL B-50942), (NRRL B-67339), (NRRL B-67340), (NRRL B-67341), (NRRL B-67741), (NRRL B-67742), (NRRL B-67743), or (NRRL B-67892) has at least 99%, 99.9%, 99.8%, 99.7%, 99.6%, or 99.5% sequence identity or ANI. In some embodiments, the ANI percentage can be determined, as disclosed by Konstantinidis et al. 2006. In some embodiments of the methods provided herein, one or more Methylobacterium strains used for treating seeds and / or plant parts are ISO 110 or LGP 2009, deposited with NRRL accession number NRRL B-50938. In some embodiments, strains identified as ISO 110 or LGP 2009 and deposited with NRRL accession number NRRL B-50938 are used as controls or reference standards for comparison with one or more new test or candidate Methylbacterium isolates in methods for identifying new Methylbacterium species that can enhance the levels of one or more mineral nutrients and / or vitamins in leafy green vegetables harvested from treated plants.

[0062] In some embodiments of the methods provided herein, plants, plant seeds, and / or plant parts are treated with a strain of *Methylobacterium* and at least one additional component. In some embodiments, the additional component may be another active ingredient, such as a pesticide or a second biological agent. In some embodiments, the pesticide may be an insecticide, fungicide, herbicide, nematicide, or other biocidal agent. The second biological agent may be a strain that increases yield or controls insects, pests, fungi, weeds, or nematodes. In some embodiments, the second biological agent is a strain of *Methylobacterium*.

[0063] 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, the insecticides and nematicides comprise 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.

[0064] 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.Other non-limiting examples of biocides include isothiazolinones such as 1,2-benzothiazolin-3-one (BIT), 5-chloro-2-methyl-4-isothiazolin-3-one (CIT), 2-methyl-4-isothiazolin-3-one (MIT), octylisothiazolinone (OIT), dichlorooctylisothiazolinone (DCOIT), and butylbenzisothiazolinone (BBIT); 2-bromo-2-nitropropane-1,3-diol (Bronopol), 5-bromo-5-nitro-1,3-dioxane (Bronidox), tris(hydroxymethyl)nitromethane, 2,2-dibromo-3-nitropropamide (DBNPA), and alkyl dimethyl benzyl ammonium chloride.

[0065] Non-limiting examples of herbicides include ACCase 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, flumioxazin, fomesafen, glyphosate, glufosinate, mesotrione, quizalofop, saflufenacil, sulcotrione, and 2,4-D.

[0066] In some embodiments, the compositions or methods disclosed herein may include strains of the genus *Methylobacterium* and additional active ingredients selected from the group consisting of: clothianidin, ipconazole, imidacloprid, metalaxyl, mefenoxam, thiazoxafen, azoxystrobin, thiomethoxam, fluopyram, prothioconazole, pyraclostrobin, and sedaxane.

[0067] In some embodiments, the compositions or methods disclosed herein may include additional active ingredients, which may be a second biological agent. The second biological agent may be a biocontrol agent, other beneficial microorganisms, microbial extracts, natural products, plant growth activators, or plant defense agents. Non-limiting examples of the second biological agent may include bacteria, fungi, beneficial nematodes, and viruses. In some embodiments, the second biological agent may be a *Methylobacterium* species. In some embodiments, the second biological agent is a *Methylobacterium* species listed in Table 1. In some embodiments, the second biological agent may be a *Methylobacterium* species selected from: *Methylobacterium gravidii*, *Methylobacterium radiation-resistant*, *Methylobacterium truncatum*, *Methylobacterium quorum*, *Methylobacterium oilfieldii*, *Methylobacterium armatibrum*, and *Methylobacterium foetida*.

[0068] In some embodiments, the second biological agent may be one of the following bacterial genera: *Actinomycetes*, *Agrobacterium*, *Arthrobacter*, *Alcaligenes*, *Aureobacterium*, *Azobacter*, *Azorhizobium*, *Azospirillum*, *Azotobacter*, *Beijerinckia*, *Bacillus*, and *Brevibacillus*. cillus, Burkholderia, Chromobacterium, Clostridium, Clavibacter, Comomonas, Corynebacterium, Curtobacterium, Enterobacter, Flavobacterium, Gluconacetobacter, Herbaspirella *Irillum*, *Hydrogenophage*, *Klebsiella*, *Luteibacter*, *Lysinibacillus*, *Mesorhizobium*, *Methylobacterium*, *Microbacterium*, *Ochrobactrum*, *Paenibacillus*, *Pantoea*, *Pasteuria*, *Phingob...* The genera *Acterium*, *Photorhabdus*, *Phyllobacterium*, *Pseudomonas*, *Rhizobium*, *Rhodococcus*, *Bradyrhizobium*, *Serratia*, *Sinorhizobium*, *Sphingomonas*, *Streptomyces*, *Stenotrophomonas*, and *Variovorax* are mentioned.Xanthomonas and Xenorhadbus genera. In specific embodiments, 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 stabulina, Pasteuria usage, and Pseudomonas fluorescens.

[0069] In some embodiments, the second biological agent may be one of the following fungal genera: *Acremonium*, *Alternaria*, *Ampelomyces*, *Aspergillus*, *Aureobasidium*, *Beauveria*, *Botryosphaeria*, *Cladosporium*, *Cochliobolus*, *Colletotrichum*, *Coniothyrium*, *Embellisia*, *Epicoccum*, *Fusarium*, and *Gigaspora*. The genera *Gliocladium*, *Glomus*, *Laccaria*, *Metarhisium*, *Muscodor*, *Nigrospora*, *Paecilonyces*, *Paraglomus*, *Penicillium*, *Phoma*, *Pisolithus*, *Podospora*, *Rhizopogon*, *Scleroderma*, *Trichoderma*, *Typhula*, *Ulocladium*, and *Verticilium*. In specific embodiments, the fungus is Beauveria bassiana, Coniothyrium minitans, Gliocladium vixens, Muscodor albus, Paecilomyces lilacinus, or Trichoderma polysporum.

[0070] In another embodiment, the second biological agent may be a plant growth activator or a plant defense agent, including but not limited to allergenic proteins, Polygonum cuspidatum (Reynoutria sachalinensis), jasmonate, lipochitooligosaccharides, and isoflavones.

[0071] In another embodiment, the second biological agent may comprise, but is not limited to, various Bacillus sp., Pseudomonas sp., Coniothyrium sp., Pantoea sp., Streptomyces sp., and Trichoderma sp. The microbial biopesticide may be bacteria, fungi, viruses, or protozoa. Particularly useful biopesticide microorganisms include various Bacillus subtilis, Bacillus thuringiensis, Bacillus pumilis, Pseudomonas syringae, Trichoderma harzianum, Trichoderma virens, and Streptomyces lydicus strains. 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 second biological agent may be provided in the composition in the form of spores.

[0072] Provided are leafy green plants or harvested plant parts having increased levels of at least one mineral nutrient and / or at least one vitamin compared to control plants or plant parts, and methods for obtaining and using such plants and plant parts. In some embodiments, the content of at least one mineral nutrient and / or at least one vitamin in the plant or harvested plant part is increased by at least about 1%, or 2%, to about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% per gram of dry or wet weight compared to the content of at least one mineral nutrient and / or at least one vitamin in the control plant or plant part. In other embodiments, the content of at least one mineral nutrient and / or at least one vitamin in the plant, plant parts, food ingredients, and feed ingredients is increased by more than 30%, including 35%, 40%, 45%, 50%, or more than 50%, compared to the content of at least one mineral nutrient and / or at least one vitamin in the control plant or plant parts. In some embodiments, the content of more than one mineral nutrient and / or more than one vitamin is increased in leafy plants or harvested plant parts, and the percentage increase of each of the mineral nutrient and / or vitamin may vary, wherein each increased mineral nutrient and vitamin increases by at least about 1%, or 2%, to about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% or more per gram of dry or wet weight. The control comprises a plant or plant part harvested from a control plant, which is grown from untreated control seeds or untreated controls.

[0073] Mineral nutrients and / or their content in leafy green plants or their harvested parts grown from seeds or seedlings treated with effective amounts of one or more strains of *Methylobacterium* can be determined using a variety of different techniques or combinations thereof. Methods for determining nitrate and nitrite nitrogen content include cadmium reduction and colorimetric analysis via a flow injection system (Lachat, Inc.); AOAC 968.07. Mineral digestion can be performed using an open-vessel microwave SW846-3051A (AOAC 991-10D(e)). Mineral analysis can be performed using inductively coupled argon plasma (ICAP); AOAC 985.01. The mineral and vitamin content of seeds and various food products can also be determined using the standard methods described in AOAC as described in AACC, Official Methods of Analysis of AOAC INTERNATIONAL, 21st edition (2019), and Codex Alimentarius of International Food Standards as described by the Food and Agriculture Organization of the United Nations (FAO) or WHO (CXS 234-19991, adopted in 1999).

[0074] Preservation Information

[0075] In accordance with the provisions of the Budapest Treaty on the International Recognition of Microbial Deposits Used in Patent Proceedings, samples of the following Methylbacillus strains are deposited at the Agricultural Research Service Culture Collection (NRRL) of the National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604, USA. Methylbacillus NRRL B-50929, NRRL B-50930, NRRL B-50931, NRRL B-50932, NRRL B-50933, NRRL B-50934, NRRL B-50935, NRRL B-50936, NRRL B-50937, NRRL B-50938, NRRL B-50939, NRRL B-50940, NRRL B-50941, and NRRL B-50942 were deposited at the NRRL on March 12, 2014. Methylbacterium NRRL B-67339 was deposited in the NRRL on November 18, 2016. Methylbacterium NRRL B-67340 was deposited in the NRRL on November 18, 2016. Methylbacterium NRRL B-67341 was deposited in the NRRL on November 18, 2016. Methylbacterium NRRL B-67741 was deposited in the NRRL on December 20, 2018. Methylbacterium NRRL B-67742 was deposited in the NRRL on December 20, 2018. Methylbacterium NRRL B-67743 was deposited in the NRRL on December 20, 2018. Methylbacterium NRRL-B-67892 was deposited in the NRRL on November 26, 2019. Methylbacterium NRRL-B-68032, NRRL-B-68033 and NRRL-B-68034 were deposited in NRRL on May 20, 2021.

[0076] According 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 shall be irrevocably lifted.

[0077] Example

[0078] The following examples are for illustrative purposes only and not for limiting purposes.

[0079] Example 1. Effects of treatment with *Methylobacterium* strain ISO110 (NRRL B-50938) on the mineral nutrient content of harvested leaves in spinach.

[0080] Spinach seeds were treated with Methylobacterium strain ISO 110 at 10 mg / seed. 6CFU treatment was performed and paralleled with 15 plots of untreated spinach seeds in 15 plots (26 seeds per plot) of mixed soil (Fick Garden Mix) in a greenhouse. The plots were thinned to contain at least 20 plants. Twenty-eight days after planting (approximately 7 true leaves), 15 or more plants were randomly selected from each plot, and shoots were collected by cutting one inch above the soil line. The shoots were incubated in sample bags at 45°C for 4 days to dry and for macronutrient and micronutrient content analysis. One-tailed unequal variance (Welch) t-tests were used to analyze the data to determine whether treatment with ISO 110 resulted in a significant increase in nutrient content. ISO 110 significantly increased the foliar content of three nutrients: nitrogen (N), magnesium (Mg), and iron (Fe). Other nutrients that showed higher UTC levels after ISO 110 treatment were copper, calcium, potassium, and sulfur. Compared with the untreated control plants, the ISO 110-treated plants had lower levels of zinc, boron, phosphorus, and manganese.

[0081] The percentage differences in macronutrients and micronutrients measured in this experiment between the ISO110 treatment and the UTC treatment are shown in Table 2. P-values ​​were estimated using Student's t-test. Results showing differences of p < 0.1 are indicated in italics.

[0082]

[0083] Example 2. Determination of the effects of *Methylobacterium* on micronutrient content and early growth enhancement in hydroponic systems.

[0084] The experiments were conducted using a randomized, fully block design. Experiments with three treatment levels were performed to compare the growth-enhancing effects of Methylobacterium isolates on plant biomass treated with two Methylobacterium strains and water with a control treated with water alone. The experiments had n = 10 and were arranged in 10 completely randomized blocks. Each experimental unit consisted of 24 individual plants grown on a quarter (3 × 8 cube) horticube sheet, with the biomass in bulk. Ten horticube sheets (104-cell Oasis HorticubeXL™, single-spot; Smithers-Oasis NorthAmerica, Kent, OH, USA) were each divided into four 3 × 8 cube blocks, and 30 blocks were placed in their respective clean 1020-mesh trays. Completely saturate the horticube block with UV-filtered RO water and place a seed (lettuce or spinach) into each of the horticube's holes (pre-formed seed pores). By using 10... 6 The CFU-tested Methylobacterium strain was applied directly to each seed for inoculation.

[0085] The seeds were allowed to grow undisturbed at 23–25°C for 14 hours a day. Plants were watered and fertilized on Mondays, Wednesdays, and Fridays (15–16–17). On all other days, the plants were watered with UV-filtered RO water. Fourteen days after planting (approximately two true leaf stages), the shoots from each plant were harvested by cutting directly below the cotyledons, and all shoots from the same tray were bundled together. The shoots were dried in an oven at 45°C for at least three days, and the bundled shoots per piece / tray were weighed to identify the *Methylobacterium* strain that increased the biomass of lettuce or spinach shoots after seed treatment. The shoots could be from the same sample measured for the biomass determination or from separate experiments as described above.

[0086] Tables 3 and 4 below present the results of the analysis of the effects of treatments with different strains of *Methylobacterium* on enhanced early growth in two-true-leaf stage lettuce and spinach plants as described above. The lettuce results in Table 3 are from biomass data only. Data are combined results from at least three independent replicates of a given isolate. Comparative p-values ​​are taken from a post-hoc Studden's t-test of a linear mixture model. The lettuce results in Table 3 show that using LGP2002, LGP2001, LGP2010, LGP2012, LGP2000, LGP2009, LGP2006, LGP2011, LGP2007, LGP2004, LGP2025, LGP2026, LGP2021, LGP2020, LGP2017, LGP2028, LGP2029, LGP2030, LGP2019, LGP2031, LGP2016, LGP2033, LGP2034, LGP2022, LGP2023, and the combination of LGP2002 and LGP2015 resulted in a percentage increase in positive growth relative to the control.

[0087]

[0088] The spinach results in Table 4 are based on image data as representative of aboveground biomass. Data are combined results from two independent replicates of the experiment. Comparative p-values ​​are taken from a post-hoc Studden's t-test of a linear mixture model. The spinach results in Table 4 show the percentage of positive growth enhancement relative to the control caused by the combination of LGP2001, LGP2010, LGP2009, LGP2021, LGP2022, LGP2023, and LGP2002 and LGP2015.

[0089]

[0090] Example 3. Detection or identification of Methylobacterium strains, variants, and derivatives.

[0091] Assays for detecting or identifying specific *Methylobacterium* strains and closely related derivatives have been disclosed. Genomic DNA fragments specific to *Methylobacterium* strains have been identified, and a qPCR-based locked nucleic acid (LNA) assay has been developed.

[0092] Genomic DNA sequences of *Methylobacterium* strains were compared with whole genome sequences of over 1000 common and proprietary *Methylobacterium* isolates using BLAST analysis with a sliding window of approximately 300 bp having a length of 1–25 nucleotides. Genomic DNA fragments with weak BLAST alignments were identified, indicating approximately 60–95% identity to their corresponding fragments in the *Methylobacterium* genus of interest. Fragments from the LGP2015 genome corresponding to the identified weakly aligned regions were selected for assay development and are provided as SEQ ID NO: 1–3.

[0093]

[0094] Regions in SEQ ID NO:1-3 were selected, with corresponding regions in other *Methylobacterium* strains identified as having one or more nucleotide mismatches from the LGP2015 sequence. qPCR primers were designed using Primer 3 software (Untergasser et al. (2012), Koressaar et al. (2007)) to side-join 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 with 5' FAM reporter dye, 3' Iowa Black FQ quencher, and one to six 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 raise the Tm. The target Tm for the probe sequence was 10 degrees higher than the primer Tm.

[0095] Primer and probe sequences used for the specific detection of LGP2015 are provided as SEQ ID NO:4-12 in Table 6. Each probe contains a 5' FAM reporter dye and a 3' Iowa Black FQ quencher.

[0096]

[0097] LGP2015 was detected using a primer / probe set on the isolated DNA and differentiated from related Methylobacterium isolates.

[0098] Each 10 µl qPCR reaction contains 5 µl Quantabio PerfeCTa qPCR ToughMix 2x master mix, low ROX from VWR, 0.5 µl 10 µM forward primer, 0.5 µl 10 µM reverse primer, 1 µl 2.5 µM probe, 1 µl nuclease-free water, and 2 µl DNA template. Approximately 1 ng of DNA template is used per reaction. Reactions are performed using the following procedure on a ThermoFisher QuantStudio. TM The 6-cycle Flex real-time PCR was performed: 95°C for 3 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The analysis software on the PCR instrument calculated the threshold and Ct value for each sample. Each sample was run in triplicate on the same qPCR plate. A positive result was considered achieved when the ΔCt between the positive and negative controls was at least 5.

[0099] The differentiation of LGP2015 from closely related isolates using three primer / probe sets by analyzing the isolated DNA is shown in Table 7 below. The similarity scores shown for related isolates consider average nucleotide identity and the alignment score between the isolate and LGP2015. One of the test strains, LGP2035, was used as an additional positive control. LGP2035 is a clonal isolate of LGP2015 obtained from a culture of LGP2015, which was confirmed to be identical to LGP2015 by whole-genome sequencing and scored positive in all three reactions. The similarity score of this strain greater than 1.000 compared to LGP2015 may be a result of slightly different genome assembly in this isolate. A ΔCt of approximately 15 or higher between the LGP2015 and LGP2035 isolates and the water-only control is consistent with sequence confirmation of identity with these isolates. Analysis of other isolates less closely related to LGP2015 resulted in ΔCt values ​​similar to those of the water-only control.

[0100]

[0101] LGP2015 was detected on treated plant material using primers / probes.

[0102] To test the effectiveness of LGP2015 foliar spray treatment on maize: Untreated maize seeds were planted in field soil in the grow room and watered with unfertilized RO water. After germination and growth for approximately 3 weeks, the plants were transferred to the greenhouse. At stage V5, the plants were divided into three treatment groups: LGP2015 foliar spray, simulated foliar spray, and untreated. Plants receiving LGP2015 foliar spray were treated with 10x glycerol stock solution at a rate of 71.4 µl per plant using a Solo sprayer. This translates to a rate of 10 L / acre in the field. Simulated-treatment plants were sprayed with 71.4 µl water / plant. Untreated plants did not receive foliar spray treatment. Two weeks after foliar spray treatment, leaves were harvested into sterile tubes, and DNA was isolated from bacteria on the harvested leaves as described above. Each experiment was grown at least twice. As shown in Table 8, LGP2015 was detected on leaves harvested from maize plants treated with foliar spray application of Methylobacterium strains using all three primer and probe sets, as evidenced by a ΔCt value of approximately 10 between the samples and the negative control.

[0103]

[0104] The results demonstrate the applicability of genome-specific primers and probes for detecting *Methylobacterium* strain LGP2015 in various plant tissues after treatment with the strain, and provide a method for distinguishing LGP2015 from closely related isolates. Similar methods were developed for other *Methylobacterium* strains LGP2002, LGP2019, LGP2018, and LGP2017 using the target sequence fragments and primer / probe pairs shown in the table below.

[0105]

[0106]

[0107]

[0108]

[0109] The differentiation of LGP2019 from closely related isolates by analyzing isolated DNA using primer / probe sets is shown in Table 13 below. The similarity scores shown for related isolates consider average nucleotide identity and alignment scores between the isolate and LGP2019. Two test strains, LGP2043 and LGP2014, were used as additional positive controls because a similarity score of 1.00 indicates they are nearly identical to LGP2019. qPCR using LGP2019 as a DNA template consistently maintained low Ct values, and no sequence confirmation of identity with these isolates was detected in the water-only control. Analysis of other isolates less closely related to LGP2019 did not yield similar results to the water-only control.

[0110]

[0111]

[0112]

[0113]

[0114]

[0115] LGP2019 on treated plant material was detected using primers / probes.

[0116] LGP2019 was detected in maize roots treated within furrows.

[0117] At planting, maize seeds in the soil were soaked with LGP2019 and a control strain derived from frozen glycerol stock solution to simulate furrow treatment. To obtain 10 7 The final concentration of CFU / seed is 10. 8 At CFU / ml, 100 µl of each strain was inoculated onto each seed in a pit placed in the soil. A 1 / 10 dilution series was used for lower concentrations of the target. For the control treatment, 100 µl of Milli-Q water was applied to each corn seed in a pit placed in the soil. Pots containing the treated seeds were placed in a growth chamber for approximately two weeks, and the soil was kept moist by watering with unfertilized RO water every 1–2 days. After 2 weeks of growth, roots from approximately 9 plants per replicate were harvested into sterile tubes. Each treatment had at least 2 replicates per experiment, and each experiment was performed at least 3 times.

[0118] DNA from bacteria on harvested maize roots was isolated as follows. A single root was immersed in 20 mL of phosphate-buffered saline (PBS) (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4) in a 50 mL conical tube. The tube was vortexed for 10 minutes, followed by sonication for 10 minutes. Root tissue was removed, and the remaining supernatants from multiple roots of the same sample were combined and centrifuged at 7500 x g for 10 minutes. This process was repeated until each sample was in one tube. Moistened soil particles were vortexed until they uniformly coated the tube walls. The tubes were placed in a laminar flow hood with the caps removed and the open end of the tube facing the blower. After drying, the samples were stored at room temperature. 250 mg of dried soil was used as input for DNA extraction using the Qiagen DNeasyPowerSoil HTP 96 kit (catalog number 12955-4) following the manufacturer's protocol.

[0119] The LGP2019 primers and probes disclosed in Table 12 above were used for qPCR reactions to detect the presence of the LGP2019-specific fragments provided in Table 11. Each 10 µl qPCR reaction contained 5 µl Quantabio PerfeCTa qPCR ToughMix2x master mix, low ROX from VWR, 0.5 µl 10 µM forward primer, 0.5 µl 10 µM reverse primer, 1 µl 2.5 µM probe, 1 µl nuclease-free water, and 2 µl DNA template. Approximately 1 ng of DNA template was used per reaction. The reactions were performed using the following procedure in Thermo Fisher QuantStudio. TM The 6-cycle Flex real-time PCR was performed: 95°C for 3 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The analysis software on the PCR instrument calculated the threshold and Ct value for each sample. Each sample was run in triplicate on the same qPCR plate. A positive result was considered achieved when the ΔCt between the positive and negative controls was at least 5.

[0120] Variants of the Methylobacterium spp. isolates in Table 1 were detected using primers / probes.

[0121] The variants of the *Methylobacterium* isolates listed in Table 1 were identified by the presence of DNA fragments as described above. Unique fragments used for this type of method are provided in Table 18.

[0122]

[0123]

[0124]

[0125]

[0126] Example 4. Analysis of the effects of *Methylobacterium* strains on nutrient content in plant vegetative tissues.

[0127] In the summer of 2019, treated soybean seeds, as described in Example 1, were planted in parallel with untreated control soybean plants in multiple field locations in the Midwestern United States. Canola and wheat seeds underwent similar treatment and testing. To analyze field-grown maize plants, *Methylobacterium* strains were applied in the furrows at planting time. The strains and combinations of strains evaluated are shown in Table 19 below.

[0128]

[0129] Preliminary analysis of soybean vegetative tissues showed that treatment with Methylobacterium strains resulted in increased micronutrients, including increased boron in the R1 stage vegetative tissue of soybean plants grown from seeds treated with ISO 103 and ISO 118, and increased iron in the V6 stage vegetative tissue of soybean plants grown from seeds treated with ISO 102.

[0130] ISO 103, ISO 118, ISO 102, ISO 117, ISO 120 and ISO 121 were tested to assess the effects on micronutrient levels and growth enhancement in leafy plants as described in Example 2, as well as on growth and yield enhancement in inter-row crops such as maize, rice, soybean, rapeseed and wheat.

[0131] Example 5. Growth stimulants of *Methylobacterium* on cannabis plants

[0132] The ability of Methylbacterium isolates LGP2002, LGP2009, and LGP2019 to enhance rooting and growth in cannabis plants (cannabis) was evaluated as follows. Cuttings were taken from mature plants and immersed in a solution of approximately 1 × 10⁻⁶ ppm. 6CFU of Methylobacterium was administered in an aqueous suspension for 2 hours. The control solution (water only) contained no Methylobacterium. The injured stem portions of the cuttings from both the control and Methylobacterium treatments were then immersed in 0.3% indole-3-butyric acid (IBA), a synthetic rooting hormone, and inserted stem-down into potting media plugs in multi-slot trays. A total of fifty plants, ten from each of five different CBD hemp varieties, were treated with each Methylobacterium isolate. After two weeks in the potting medium, the plugs were non-destructively harvested, and the roots were scored using a visual rating scale of 1–5: 1 = visible roots between 0 and 20%; 2 = visible roots between 21 and 40%; 3 = visible roots between 41 and 60%; 4 = visible roots between 61 and 80%; 5 = visible roots between 81 and 100%.

[0133] Compared to the untreated control plants (2.6 score), the rooting scores of plants treated with the tested Methylobacterium isolates ranged from 3 to 3.4. At p < 0.05, the increases induced by treatments with LGP2002 and LGP2019 were significantly different from those in the control, and at p < 0.001, the increase induced by treatment with LGP2009 was significantly different from that in the control.

[0134] Rooted plantlets were transplanted to the field. Aboveground biomass was harvested approximately thirteen weeks after transplanting, dried, and measured. Treatment with three *Methylobacterium* isolates, LGP2002, LGP2009, and LGP2019, resulted in increased aboveground dry biomass compared to the untreated control. Treatment with LGP2009 increased aboveground dry biomass by 18%, LGP2002 by 27%, and LGP2019 by 38% (significantly different from the control at p < 0.05). Treatment with *Methylobacterium* isolates enhances rooting, allowing for earlier transplanting of plantlets without negatively impacting yield, thus reducing cycle time.

[0135] Example 6. Growth stimulants of Methylobacterium spp. on cannabis plants

[0136] The ability of *Methylobacterium* isolates LGP2000, LGP2001, LGP2002, LGP2003, LGP2004, LGP2005, LGP2006, LGP2007, LGP2008, LGP2009, LGP2010, LGP2011, LGP2012, LGP2013, LGP2014, LGP2015, LGP2016, LGP2017, LGP2018, LGP2019, LGP2020, LGP2021, LGP2022, and LGP2023 to enhance the rooting and growth of cannabis plants (cannabis) was evaluated as follows. Cuttings were taken from mature plants and immersed in a solution of approximately 1 × 10⁻⁶ ppm. 6 CFU of Methylobacterium was administered in an aqueous suspension for 2 hours. The control solution (water only) contained no Methylobacterium. The injured stem portions of the cuttings from both the control and Methylobacterium treatments were then immersed in 0.3% indole-3-butyric acid (IBA), a synthetic rooting hormone, and inserted stem-down into potting media plugs in multi-slot trays. A total of fifty plants, ten from each of five different CBD hemp varieties, were treated with each Methylobacterium isolate. After two weeks in the potting medium, the plugs were nondestructively harvested, and the roots were scored using a 1-5 visual rating scale: 1 = 0-20% visible roots; 2 = 21-40% visible roots; 3 = 41-60% visible roots; 4 = 61-80% visible roots; 5 = 81-100% visible roots.

[0137] The rooting score of plants treated with the tested Methylobacterium isolates was determined compared with the untreated control plants.

[0138] The rooted plantlets were transplanted to the field. The aboveground biomass was harvested approximately thirteen weeks after transplanting, dried, and the aboveground dry biomass was measured.

[0139] Example 7. Increasing rice yield through the application of Methylobacterium spp.

[0140] The paddy field trials were conducted at three locations, all near Humphrey, Arkansas, to evaluate the effects of applying three *Methylobacterium* isolates as seed treatments. Treatments included each *Methylobacterium* isolate and an untreated control applied to rice seeds, with basal treatments of insecticide (active ingredient thiamethoxam) alone or without. The trials were conducted using a randomized whole-block design (RCBD) with four replicates at each location. ISO 117 (NRRL B-67341), ISO 120 (NRRL B-67743), and ISO 118 (NRRL B-67741) were used in 10... 6The target concentration of CFU / seed is applied to rice seeds.

[0141] Compared with the untreated control in both pesticide and non-pesticide treatments, the Methylobacterium spp. isolates increased yield in paddy field trials.

[0142] Table 20. Increase in average yield (Bu / A) relative to control and the percentage increase shown.

[0143]

[0144] When describing elements of the invention or preferred embodiments thereof, the articles “a,” “an,” “the,” and “said” are intended to mean the presence of one or more of the elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that additional elements besides those listed may be present.

[0145] Based on the above, it can be seen that several objectives of the present invention have been achieved and other advantageous results have been obtained.

[0146] Since various changes can be made to the above compositions, methods and processes without departing from the scope of the invention, all subject matter contained in the above description and shown in the drawings should be interpreted in an illustrative rather than restrictive sense.

[0147] The invention has been described in detail, and it will be apparent that modifications and variations may be made without departing from the scope of the invention as defined in the appended claims.

[0148] References

[0149] Green, PN 2005. *Methylobacteria*. In Brenner, DJ, NR Krieg, and JTStaley (eds.). "Bergey's Manual of Systematic Bacteriology, Volume two, The Proteobacteria. Part C, The alpha-, beta-, delta-, andepsilonproteobacteria". Second ed. Springer, New York. pp. 567-571.

[0150] Green, PN 2006. *Methylobacteria*. In Dworkin, M., S. Falkow, E. Rosenberg, K.-H. Schleifer, and E. Stackebrandt (eds.). "The Prokaryotes. A Handbook on the Biology of Bacteria. Volume 5. Proteobacteria: Alpha and Beta Subclasses". 3rd ed. Springer, New York, pp. 257-265.

[0151] Green, PN and Ardley, JK 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 (Int J Syst Evol Microbiol.) Sep 2018; 68(9):2727-2748. doi: 10.1099 / ijsem.0.002856.

[0152] Konstantinidis KT, Ramette A., Tiedje JM (2006;). The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361: 1929–1940.

[0153] Lidstrom, ME 2006. Aerobic Methyltrophic Prokaryotes. In Dworkin, M., S. Falkow, E. Rosenberg, K.-H. Schleifer, and E. Stackebrandt (eds.). "The Prokaryotes. A Handbook on the Biology of Bacteria. Volume 2. Ecophysiology and biochemistry". 3rd ed. Springer, New York, pp. 618-634.

[0154] 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.

Claims

1. A method for enhancing plant growth and / or rooting of cannabis plants, the method comprising: (a) Treating cannabis plants, plant parts or seeds with a composition comprising one or more isolates of the genus Methylobacterium; as well as (b) To cause the treated plant to grow or to cause the plant from the treated plant part or seed to grow to produce rooted plant, wherein the cannabis plant growth and / or rooting is increased compared with untreated control plant that has not been treated with the composition or with control plant grown from untreated plant part or seed that has not been treated with the composition.

2. The method according to claim 1, wherein the composition comprises a *Methylobacterium* isolate selected from the group consisting of: LGP2002 (NRRL B-50931), LGP2009 (NRRL B-50938), and LGP2019 (NRRL B-67743).

3. The method according to claim 1 or 2, wherein the plant portion is derived from cuttings of a cannabis plant.

4. The method of claim 3, wherein the cuttings are treated by immersion in a suspension of Methylobacterium.

5. The method of claim 4, wherein the Methylobacterium species are present at a concentration greater than 1 × 10⁻⁶ per milliliter. 3 The concentration of CFU is present in the suspension.

6. The method according to claim 1 or 2, wherein the rooted plants are transplanted to the field, and wherein the production cycle time of the mature cannabis plants is reduced compared to control cannabis plants grown from untreated cuttings.

7. The method according to claim 1 or 2, wherein the enhanced growth causes an improvement in plant traits selected from: increased biomass production, reduced cycle time, increased leaf growth rate, increased root growth rate, and increased seed yield.

8. The method according to claim 1 or 2, 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.

9. Use of a composition comprising a Methylobacterium isolate in the treatment of cannabis plants, portions, or seeds, said cannabis plants, portions, or seeds being at least partially coated with a composition comprising said Methylobacterium isolate, said Methylobacterium isolate being selected from the group consisting of: LGP2002 (NRRL B-50931), LGP2009 (NRRL B-50938), and LGP2019 (NRRL B-67743), wherein said cannabis plants or cannabis plants grown from said cannabis plant portions or seeds, compared to control cannabis plants not treated with said Methylobacterium or cannabis plants grown from control cannabis plant portions or seeds not treated with said Methylobacterium, exhibit enhanced plant growth or rooting or a reduced cycle time from cutting to mature plant.

10. Use of Methylobacterium in the treatment of leafy plants or plant parts, wherein the leafy plants or plant parts have increased levels of one or more mineral nutrients and / or vitamins, wherein the plants or plant parts are harvested from cultivated plants grown from seeds or seedlings treated with Methylobacterium, and wherein the Methylobacterium includes LGP2009 (NRRL B-50938) and provides increased levels of one or more mineral nutrients and / or vitamins.

11. The leafy plant or plant part according to claim 10, wherein the cultivated plant is selected from the group consisting of: spinach, lettuce, beet, Swiss chard, watercress, kale, broccoli, endive, arugula, chicory, bok choy, and turnip, spinach plant or plant part.

12. The use according to claim 10 or 11, wherein the plant or plant part has increased levels of one or more mineral nutrients selected from the group consisting of nitrogen, magnesium and iron.

13. A method for identifying a *Methylobacterium* isolate, said *Methylobacterium* isolate increasing the content of one or more mineral nutrients and / or vitamins in a green leafy plant, said method comprising: (i) Treat green leafy plant seeds or green leafy plant seedlings with at least a *Methylobacterium* isolate; (ii) Cultivate the seeds or seedlings to obtain a treated plant with at least two true leaves; (iii) Harvesting the plant, plant shoots, or one or more true leaves from the treated plant; (iv) Analyze the harvested plants, shoots, or leaves to determine the content of mineral nutrients and / or vitamins; and (v) Select a Methylbacterium isolate that increases the content of at least one mineral nutrient or vitamin in the treated plant, twig or leaf compared to the plant, twig or leaf harvested from an untreated control plant, or the plant, twig or leaf harvested from a plant treated with a Methylbacterium isolate.

14. The method of claim 13, wherein the seeds and / or plants are treated with two or more different Methylobacterium isolates in step (i) compared with other Methylobacterium isolates and optionally with untreated control plants, and are cultivated, harvested and analyzed in steps (ii) to (iv) respectively to determine the mineral nutrient and / or vitamin content in the plants, shoots or one or more plant leaves.

15. The method of claim 13, wherein, in step (i), the seeds and / or plants are treated with three or more different strains of Methylobacterium or combinations of different strains of Methylobacterium, respectively, compared with other different Methylobacterium treatments and optionally with untreated control plants, and the treated seeds or seedlings are cultivated, harvested, and analyzed in steps (ii) to (iv) respectively to determine the mineral nutrient and / or vitamin content in the plants, shoots, or one or more plant leaves.

16. The method according to any one of claims 13 to 15, wherein one or more different Methylobacterium strains of the different Methylobacterium genus have enhanced colonization efficiency.

17. The method according to any one of claims 13 to 15, wherein the leafy plant is cultivated in a hydroponic or aeroponic system.

18. The method according to any one of claims 13 to 15, wherein, due to the increased content of at least one mineral nutrient or vitamin, the selected *Methylobacterium* strain in (v) also confers on the green leaf plant improved traits selected from: increased biomass production, reduced cycle time, increased leaf growth rate, reduced time to develop two true leaves, increased root growth rate, and increased seed yield.

19. The method according to any one of claims 13 to 15, wherein the leafy plant is selected from the group consisting of: spinach, lettuce, beet, Swiss chard, watercress, kale, broccoli, endive, arugula, chicory, bok choy, and turnip.

20. The method according to any one of claims 13 to 15, wherein the leafy plant is cultivated for the production of miniature leafy vegetables and / or herbaceous plants.

21. The method according to any one of claims 13 to 15, wherein the leafy green plant is selected from the group consisting of: lettuce, cauliflower, broccoli, cabbage, watercress, arugula, garlic, onion, leek, amaranth, Swiss chard, beet, spinach, melon, cucumber, squash, basil, celery, coriander, radish, red chicory, sow thistle, dill, rosemary, tarragon, basil, pennisetum, carrot, fennel, beans, peas, chickpeas, and lentils.

22. The method according to any one of claims 13 to 15, wherein the mineral nutrient is selected from nitrogen, potassium, iron, magnesium, copper, calcium and sulfur.

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