methods and compositions to improve corn yield

专利摘要:
METHODS AND COMPOSITIONS TO IMPROVE MAIZE YIELD. The present invention relates to yield enhancing and early vigor compositions comprising Methylobacterium sp., Methods for improving yield and early vigor of corn and methods for producing the compositions. Methylobacterium sp. isolated income increases.
公开号:BR112016012883B1
申请号:R112016012883-4
申请日:2014-12-04
公开日:2021-01-05
发明作者:Marcus Jones；Gregg Bogosian
申请人:Newleaf Symbiotics, Inc.；
IPC主号:

专利说明:

[0001] This international patent application claims the benefit of U.S. Patent Application No. 61 / 911,780, filed on December 4, 2013. BACKGROUND
[0002] Organic carbon compounds such as methane and methanol are found extensively in nature and are used as carbon sources by bacteria classified as metanotrophic and methylotrophic. Metanotrophic bacteria include species in the genera Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, Methylosinus, Methylocystis, Methylosphaera, Methylocaldum and Methylocella (Lidstrom, 2006). Methanotrophs have the enzyme methane monooxygenase which incorporates an oxygen atom of O2 in methane, forming methanol. All metanotrophs are mandatory carbon users who are unable to use compounds that contain carbon-carbon bonds. Methylotrophs, on the other hand, can also use more complex organic compounds, such as organic acids, higher alcohols, sugars and the like. Thus, methylotrophic bacteria are optional methylotrophs. Methylotrophic bacteria include species in the genera Methylobacterium, Hyphomicrobium, Methylophilus, Methylobacillus, Methylophaga, Aminobacter, Methylorhabdus, Methylopila, Methylosulfonomonas, Marinosulfonomonas, Paracoccus, Xanthobacter, Ancylobacter, Thromcobacter, Rhodes, Microbacteria , Arthobacter and Nocardia (Lidstrom, 2006).
[0003] Most methylotrophic bacteria of the genus Methylobacterium are pigmented with rose. They are conventionally referred to as PPFM bacteria, being facultative rose-pigmented methylotrophs. Green (2005, 2006) identified twelve species validated in the genus Methylobacterium, specifically M. aminovorans, M. chloromethanicum, M. dichloromethanicum, M. extorquens, M. fujisawaense, M. mesophilicum, M. organophilum, M. radiotolerans, M. rhodesianum , M. rhodinum, M. thiocyanatum and zatmanii. However, M. nidulans is a nitrogen fixing Methylobacterium that is not a PPFM (Sy et al., 2001). Methylobacterium are ubiquitous in nature, being found in soil, dust, fresh water, sediment and leaf surfaces, as well as in industrial and clinical environments (Green, 2006). SHORT DESCRIPTION
[0004] Methylobacterium sp. for increased yield alone, compositions comprising Methylobacterium sp. for increasing yield, methods for using the compositions to increase the yield of corn plants, plant parts and corn plants derived therefrom and methods for producing the compositions. Such Methylobacterium sp. to increase yield are, in certain cases, referred to in this document simply as “Methylobacterium”. In certain embodiments, Methylobacterium sp. to increase yield, they can be distinguished from other neutral-yielding or negative-yielding Methylobacterium by evaluating Methylobacterium sp. for improved yield in a controlled environment (ie a culture chamber or greenhouse) or in a field test compared to untreated control plants or compared to control plants treated with neutral yield or negative yield Methylobacterium and combinations thereof. In certain embodiments, Methylobacterium sp. to increase yield is a Methylobacterium isolate selected from the group consisting of ISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO04 (NRRL B-50932), ISO11 (NRRL B-50939) and derivatives of the same.
[0005] Methods are provided herein to improve the yield of a corn plant comprising applying a composition comprising a Methylobacterium sp. to a corn plant in the development stage about V6 to about R3. In certain embodiments, the methods comprise (a) applying a composition comprising a Methylobacterium sp. to a corn plant in the development stage from about V6 to about R3, where the composition comprises: (i) a solid substance with Methylobacterium grown on it and adhered to it; (ii) an emulsion that has Methylobacterium grown in it; or (iii) an isolate of Methylobacterium ISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO04 (NRRL B-50932), ISO11 (NRRL B-50939) or a derivative thereof and an agricultural adjuvant or excipient acceptable or combinations thereof; and, (b) cultivating the corn plant until maturity, thus improving the yield of the corn plant. In certain embodiments, the solid substance with Methylobacterium grown on it and adhered to it is supplied in a liquid or in an emulsion. In certain method embodiments, the composition comprises a solid substance with Methylobacterium grown on it and adhered to it or an emulsion that has Methylobacterium grown on it. In certain method embodiments, the composition comprises the solid substance or the emulsion and in which Methylobacterium sp. it is selected from the group consisting of ISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO04 (NRRL B-50932) and derivatives thereof. In certain method modalities, the methods additionally comprise the corn plant until maturity. In certain method modalities, the composition is applied in the development stage of about V6 to about R2, in the development stage of about R1 to R2, in the development stage of about R1 to R3 or in the development stage of about R1. In certain method embodiments, a composition comprises: (i) a solid substance with Methylobacterium grown therein and adhered to it. In certain methods of the methods, the composition is a solid comprising Methylobacterium sp. at a titre of about 1 x 106 CFU / g to about 1 x 1014 CFU / g. In certain method embodiments, the composition is a liquid that contains the solid substance or an emulsion and has a title of Methylobacterium sp. from about 1 x 106 CFU / ml to about 1 x 1011 CFU / ml. In certain methods, Methylobacterium sp. it is selected from the group consisting of ISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO04 (NRRL B-50932) and derivatives thereof. In certain embodiments, Methylobacterium sp. is a glyphosate-resistant or glufosinate-resistant derivative of ISO02 (NRRL B-50930), ISO03 (NRRL B-50931) or ISO04 (NRRL B-50932). In certain modalities of any of the aforementioned methods, the applied composition covers or partially covers the corn plant or a part of it. In certain embodiments of any of the aforementioned methods, the composition is applied to the foliage of the corn plant. In certain embodiments of any of the methods mentioned above, the composition further comprises a fungicidal agent. In certain embodiments of any of the aforementioned methods, the methods additionally comprise the seed harvesting stage of the mature corn plant. In certain embodiments of any of the aforementioned methods, the yield of the harvested seed is increased compared to the yield of the harvested seed obtained from a control corn plant that has not received an application of Methylobacterium sp.
[0006] Also provided herein is a corn plant or part of a corn plant that is coated or partially coated with a composition comprising Methylobacterium sp. In certain embodiments, Methylobacterium sp. is selected from the group consisting of ISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO04 (NRRL B-50932), ISO11 (NRRL B-50939) and derivatives thereof. In certain embodiments, Methylobacterium sp. is ISO11 (NRRL B-50939) or a derivative thereof. In certain embodiments, the composition comprises: (i) a solid substance with Methylobacterium grown on it and adhered to it; or (ii) an emulsion that has Methylobacterium grown in it. In certain embodiments, the composition comprises Methylobacterium sp. at a titre of about 1x106 CFU / g to about 1x1014 CFU / g for a solid composition or at a titre of about 1x106 CFU / ml to about 1x1011 CFU / ml for a liquid composition containing the solid substance or for the emulsion. In certain embodiments, Methylobacterium sp. is the ISO11 isolate from Methylobacterium or a derivative thereof. In certain of any of the aforementioned modalities, the part of the corn plant is selected from the group consisting of a seed, a leaf, an ear or a corut.
[0007] Methods are also provided in this document to improve the yield of a corn plant comprising: (i) applying a composition comprising a Methylobacterium sp. to a corn seed or a corn plant at the stage of about VE to about V5 of the development of the corn plant. In certain embodiments, methods for improving the yield of a corn plant include: (a) applying a composition comprising a Methylobacterium sp. to a corn seed or a corn plant in the stage of about VE to about V5 of the development of the corn plant, in which the composition comprises: (i) a solid substance with Methylobacterium grown on it and adhered to it ; (ii) an emulsion that has Methylobacterium grown in it; or (iii) an isolate of Methylobacterium ISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO04 (NRRL B-50932), ISO11 (NRRL B- 50939) or a derivative thereof and an agricultural adjuvant or excipient acceptable or combinations thereof; and, (b) cultivating a corn plant from seed or the corn plant to maturity, thereby improving the yield of the corn plant. In certain method embodiments, the composition comprises a solid substance with Methylobacterium grown on it and adhered to it or an emulsion that has Methylobacterium grown on it. In certain embodiments, the solid substance with Methylobacterium grown on it and adhered to it is supplied in a liquid or in an emulsion. In certain method modalities, the methods additionally comprise growing a corn plant from seed or the corn plant until maturity. In certain method modalities, the composition is applied in the developmental stage from about VE to about V3, in the developmental stage from about VE to about V5 in the developmental stage from about V1 to V4 or V3. In certain method embodiments, the composition comprises Methylobacterium sp. at a titre of about 1x106 CFU / g to about 1x1014 CFU / g for a solid composition or at a titre of about 1x106 CFU / ml to about 1x1011 CFU / ml for a liquid composition containing the solid substance or for the emulsion. In certain methods, Methylobacterium sp. is the isolate of Methylobacterium ISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO04 (NRRL B-50932), ISO11 (NRRL B-50939) or a derivative thereof. In certain embodiments, their derivative is a strain selected for resistance to a bactericidal agent. In certain modalities of any of the aforementioned methods, the derivative of the Methylobacterium isolate is selected for resistance to glyphosate or resistance to glufosionate. In certain embodiments of any of the aforementioned methods, the corn plant is a glyphosate-tolerant corn plant, and a formulation containing glyphosate is also applied in the about V2 to about V4 stage of corn plant development. In certain methods, Methylobacterium sp. ISO11 isolate from Methylobacterium or a derivative thereof. In certain embodiments of any of the aforementioned methods, the method further comprises harvesting the seed from the mature corn plant. In certain embodiments of any of the methods mentioned above, the yield of harvested seed is increased compared to the yield of harvested seed obtained from a control corn plant that has not received an application of Methylobacterium sp. In certain embodiments of any of the aforementioned methods, the composition applied covers or partially covers the corn seed or the corn plant or a part thereof.
[0008] Methods of improving the early vigor of a corn plant are also provided in this document which comprise: (a) applying a composition comprising a Methylobacterium sp. to a corn seed or a corn plant in the stage of about VE to about V3 of the development of the corn plant, in which the composition comprises: (i) a solid substance with Methylobacterium grown on it and adhered to it ; (ii) an emulsion that has Methylobacterium grown in it; or (iii) an isolate of Methylobacterium ISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO04 (NRRL B-50932), ISO11 (NRRL B-50939) or a derivative thereof and an agricultural adjuvant or excipient acceptable or combinations thereof; and, (b) cultivating a corn plant from seed or corn plant to the development stage V3 to V6, thus improving the early vigor of the corn plant. In certain method modalities, the composition is applied in the developmental stage about VE to about V2, in the developmental stage about VE to about V1 or developmental stage VE. In certain embodiments, the solid substance with Methylobacterium grown on it and adhered to it is supplied in a liquid or in an emulsion. In certain method embodiments, the composition comprises Methylobacterium sp. at a titre of about 1x106 CFU / g to about 1x1014 CFU / g for a solid composition or at a titre of about 1x106 CFU / ml to about 1x1011 CFU / ml for a liquid composition containing the solid substance or for the emulsion. In certain method embodiments, the composition comprises the solid or the emulsion, and Methylobacterium sp. is the isolate of Methylobacterium ISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO04 (NRRL B-50932, ISO11 (NRRL B-50939) or a derivative of the same. is selected for resistance to a bactericidal agent. In certain method modalities, the Methylobacterium isolate is selected for glyphosate resistance or glufosinate resistance. In certain method modalities, the corn plant is a corn plant glyphosate-tolerant, and a formulation containing glyphosate is also applied in the about V2 to about V4 stage of corn plant development. ) is increased compared to the vigor of a control maize plant that has not received an application of Methylobacterium sp. In certain modalities of any of the methods mentioned above, the vigor increases Entente comprises increased height, increased leaf area, increased chlorophyll content, increased stem diameter, an advanced vegetative stage on a scale from V1 to V6, root volume, root length, number of root tips and combinations thereof. In certain embodiments of the aforementioned methods, the applied composition covers or partially covers the corn seed or the corn plant or a part thereof. DESCRIPTION DEFINITIONS
[0009] As used in this document, the phrases “adhered to it” and “adherent” refer to Methylobacterium that are associated with a solid substance by culture or that have been grown on a solid substance.
[0010] As used herein, the phrase "agriculturally acceptable adjuvant" refers to a substance that increases the performance of an active agent in a composition for the treatment of plants and / or plant parts. In certain compositions, an active agent may comprise a monoculture or co-culture of Methylobacterium.
[0011] As used herein, the phrase “agriculturally acceptable excipient” refers to an essentially inert substance that can be used as a diluent and / or carrier for an active agent in a composition for the treatment of plants and / or plant parts. In certain compositions, an active agent may comprise a monoculture or co-culture of Methylobacterium.
[0012] As used herein, the term "Methylobacterium" refers to bacteria that are optional methylotrophs of the genus Methylobacterium. The term Methylobacterium, as used herein, does not therefore cover species in the genera Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, Methylosinus, Methylocystis, Methylosphaera, Methylocaldum and Methylocella which are mandatory methanotrophs.
[0013] As used in this document, the phrase "control plant" refers to a plant that has not been treated with a Methylobacterium to increase yield or early vigor or composition that comprises the same in the seed or at any subsequent stage development of the control plant. In certain embodiments, a control plant may be a plant that has been treated with Methylobacterium sp. neutral income.
[0014] As used herein, the phrase "Methylobacterium coculture" refers to a culture of Methylobacterium comprising at least two strains of Methylobacterium or at least two species of Methylobacterium.
[0015] As used in this document, the phrase “contaminating microorganism” refers to microorganisms in a culture, fermentation broth, fermentation broth product or composition that were not identified prior to introduction into the culture, broth fermentation product, fermentation broth product or composition.
[0016] As used herein, the phrase "derivatives thereof", when used in the context of a Methylobacterium isolate, refers to any strain that is obtained from the Methylobacterium isolate. Derived from a Methylobacterium isolate include however, without limitation, strain variants obtained by selection, strain variants selected by mutagenesis and selection and a genetically transformed strain obtained from the Methylobacterium isolate.
[0017] As used herein, the phrase “early corn vigor” or “early vigor”, when used in the context of applying compositions containing Methylobacterium to corn seeds, plants or parts of plants, refers to any characteristic growth of a corn plant in the development stage V3 to V6 which is indicative of improved growth compared to an untreated corn plant. Such growth characteristics may, however, include, without limitation, increased height, increased leaf area, increased chlorophyll content, increased stem diameter, an advanced vegetative stage on a scale from V1 to V6, increased root volume, root length increased, increased number of root tips and combinations thereof.
[0018] As used herein, the term "emulsion" refers to a colloidal mixture of two immiscible liquids in which one liquid is the continuous phase and the other liquid is the dispersed phase. In certain embodiments, the continuous phase is an aqueous liquid, and the dispersed phase is a liquid that is not miscible, or partially miscible, in the aqueous liquid.
[0019] As used in this document, the phrase “essentially free and contaminating microorganisms” refers to a culture, fermentation broth, fermentation product or composition in which at least about 95% of the microorganisms present by amount or type in the culture, fermentation broth, fermentation product or composition are the desired Methylobacterium or other desired microorganisms of predetermined identity.
[0020] As used in this document, the phrase “solid inanimate substance” refers to a substance that is insoluble or partially soluble in water or aqueous solutions and that is either not alive or not a part of a still living organism. which it was derived.
[0021] As used herein, the phrase “Methylobacterium monoculture” refers to a culture of Methylobacterium that consists of a single strain of Methylobacterium.
[0022] As used herein, the term "peptide" refers to any polypeptide of 50 amino acid residues or less.
[0023] As used herein, the term "protein" refers to any polypeptide that has 51 or more amino acid residues.
[0024] As used herein, a "pesticide" refers to an agent that is an insecticide, fungicide, nematocide, bactericide or any combination thereof.
[0025] As used in this document, the phrase "bacteriostatic agent" refers to agents that inhibit the culture of bacteria, but do not kill bacteria.
[0026] As used in this document, the phrase "the pesticide does not substantially inhibit the culture of said Methylobacterium" refers to any pesticide that, when supplied in a composition that comprises a fermentation product that comprises a solid substance which a Monoculture or co-culture of Methylobacterium is adhered to, results in an inhibition not greater than 50% of the culture of Methylobacterium when the composition is applied to a plant or plant part in comparison to a composition that does not have the pesticide. In certain embodiments, the pesticide results in an inhibition of no more than 40%, 20%, 10%, 5% or 1% of the Methylobacterium culture when the composition is applied to a plant or part of a plant compared to a composition that don't have the pesticide.
[0027] As used herein, the term "PPFM bacteria" refers, without limitation, to bacterial species in the genus Methylobacterium instead of M. nodularis.
[0028] As used herein, the phrase "solid substance" refers to a substance that is insoluble or partially soluble in water or aqueous solutions.
[0029] As used in this document, the phrase "solid phase that can be suspended in it" refers to a solid substance that can be distributed throughout an entire liquid by stirring.
[0030] As used in this document, the term "non-regenerable" refers to either a plant part or a processed plant product that cannot be regenerated in an entire plant.
[0031] As used herein, the phrase "substantially the entire solid phase is suspended in the liquid phase" refers to media in which at least 95%, 98% or 99% of the solid substance (s) ( s) comprising the solid phase is (are) distributed (s) throughout the liquid by stirring.
[0032] As used herein, the phrase "substantially all of the solid phase is not suspended in the liquid phase" refers to media in which less than 5%, 2% or 1% of the solid is in a particulate form that it is distributed throughout the medium by agitation.
[0033] To the extent that any of the above definitions is inconsistent with the definitions provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference cited therein or in any reference to patent or non-patent found elsewhere, it is understood that the previous definition will be used in this document. METHYLOBACTERIUM FOR INCREASED INCOME AND EFFECTIVE INCOME, COMPOSITIONS THAT UNDERSTAND METHYLOBACTERIUM FOR INCREASED INCOME AND INCREASED, METHODS OF USE AND PRODUCTION METHODS
[0034] Various Methylobacterium isolates for yield enhancement, compositions comprising these Methylobacterium, methods for using the compositions to improve corn plant yield and methods for producing the compositions are provided herein. Amounts of the compositions comprising Methylobacterium sp. for sufficient yield increase to provide the increased corn plant yield can be determined by measuring any or all changes in yield in relation to untreated plants or plant parts. In certain modalities, yield can be assessed by measuring seed output on a per unit area basis (ie bushels per acre, kilograms per hectare and the like), in which plants treated with Methylobacterium sp to increase yield or plants grown from seeds treated with Methylobacterium sp are grown at approximately the same density as the control plants. In certain modalities, yield can be assessed by measuring output on a per plant or per plant basis basis (grams of seed per plant, grams of seed per ear, seeds per plant, seeds per ear and the like) of treated plants with Methylobacterium sp to increase yield compared to untreated control plants.
[0035] Methylobacterium sp. for increased yields alone are provided in this document. In certain modalities, Methylobacterium is selected from the group consisting of M. aminovorans, M. extorquens, M. fujisawaense, M. mesophilicum, M. radiotolerans, M. rhodesianum, M. nodularis, M. phyllosphaerae, M. thiocyanatum and M. oryzae. In certain embodiments, Methylobacterium is not M. radiotolerans or M. oryzae. In certain modalities, the Methylobacterium isolate for increased yield or early vigor is selected from the group consisting of ISO02, ISO03, ISO04, ISO11 and derivatives thereof. In certain embodiments, the Methylobacterium isolate for increased yield can increase the yield when applied before or during the reproductive stages of corn development and is a Methylobacterium sp. selected from the group consisting of SO02, ISO03 and ISO04. In certain embodiments, the Methylobacterium isolate to increase yield may increase yield when applied to a corn seed or in vegetative stages of corn development. In certain modalities in which the Methylobacterium isolate for increased yield is applied to a corn seed or in vegetative stages of islet development, Methylobacterium sp. is ISO11. In certain embodiments, Methylobacterium for increased yield provides increases of at least about 2%, at least about 5%, at least about 10% or at least about 15% in the yield of a treated plant or a plant that arises from a treated seed compared to untreated control plants or plants grown from untreated seeds. In certain embodiments, Methylobacterium to increase yield provides increases of at least about 2% or at least about 5% to at least about 10% or at least about 20% in the yield of a treated plant or a cultivated plant from a treated seed compared to untreated control plants or plants that arise from untreated seeds.
[0036] In certain embodiments, Methylobacterium is not M. radiotolerans or M. oryzae. In certain embodiments, Methylobacterium to increase yield or early vigor provides increases in yield and / or early vigor when applied to a seed. In certain embodiments, Methylobacterium for increased yield provides increases in yield when applied just before or during the reproductive stages of corn development. In certain embodiments of any of the aforementioned compositions, the composition comprises a solid substance in which a monoculture or co-culture of Methylobacterium is added to it. In certain embodiments in which Methylobacterium is adhered to a solid substance, the composition comprises a colloid formed by the solid substance in which a monoculture or co-culture of Methylobacterium is adhered to it and a liquid. In certain embodiments, the colloid is a gel. In certain embodiments of certain compositions mentioned above, the composition is an emulsion that does not contain a solid substance. In certain modalities of any of the aforementioned compositions, Methylobacterium for increasing yield or early vigor is selected from the group consisting of ISO02, ISO03, ISO04, ISO11 and derivatives thereof. In certain embodiments of any of the aforementioned compositions, Methylobacterium for increasing yield is selected from the group consisting of ISO02, ISO03, ISO04 and derivatives thereof.
[0037] In certain embodiments, Methylobacterium sp. for increased yield or isolated early vigor can be identified by treating a plant, a seed, the soil on which the plant or a plant that emerges from a seed is grown, or another plant growth medium in which the plant or a plant that emerges from the seed is cultivated and evaluating the increased yield or improved early vigor.
[0038] In certain modalities, seed or corn plants in the vegetative stages of development are treated with Methylobacterium sp. for increased yield or early vigor. The vegetative stages of maize are as follows: VE (emergence of coleoptile until just before the first formation of leaf paste), V1 (first leaf in paste), V2 (first and second leaf in paste), V3 (first to third leaf) in necklace), V4 (first to fourth leaves in necklace), V5 (first to fifth leaves in necklace), V6 (first to sixth leaves in necklace) and so on up to V18 (plants with 18th leaf in necklace). A description of the vegetative stages of maize can be found on the world wide web (internet) at agronext.iastate.edu/ corn / production / management / growth / CornGrowthandDevelopment. html and in "Corn Growth and Development", Abendroth et al. Iowa State University Extension and Outreach publication PMR 1009, March 2011). In certain embodiments, Methylobacterium sp. to increase yield, about VE to about V4, V5, or V6 are applied in the development stage. In certain modalities, Methylobacterium sp. to increase yield, about VE, V1, V2 or V3 are applied in the development stage to about V4, V5, or V6 In certain modalities, Methylobacterium to increase yield or early vigor that is applied to the seed or during the stage vegetative is ISO11.
[0039] In certain modalities, Methylobacterium to increase yield or early vigor are applied before, during or after the application of glyphosate to a transgenic soybean plant that is tolerant to glyphosate. Commercially available glyphosate formulations that can be used include, but are not limited to, Roundup Original MAX®, Roundup PowerMAX®, Roundup UltraMax® or RoundUp WeatherMAX® (Monsanto Co., St. Louis, MO., USA); Touchdown IQ® or Touchdown Total® (Syngenta, Wilmington, Delaware, USA); Glyphomax®, Glyphomax Plus®, or Glyphomax XRT® (Dow Agrosciences LLC, Indianapolis, IN, USA). Corn plants are typically sprayed with glyphosate at the vegetative development stage of about V3 and / or about V6. In certain embodiments, Methylobacterium for increased yield that is applied before, during or after glyphosate application is a Methylobacterium that is selected for glyphosate resistance. The selections for glyphosate resistant bacteria that have been described (Comai et al., Science 221 (4608): 370 and 371) can be adapted for the selection of Methylobacterium to increase yield. The selection and use of Methylobacterium to increase yield or early glyphosate resistant vigor of mutagenized populations or other Methylobacterium populations such as ISO02, ISO03, ISO04, ISO11 and derivatives thereof are provided in this document.
[0040] In certain modalities, the seeds or plants of corn in the late vegetative stages to reproductive stages of development are treated with Methylobacterium sp. to increase yield. As used in this document, the late vegetative stages of corn are the development stages V6 to V (n) (n-th leaf, where the final number of leaves depends on the corn variety and environmental conditions) or VT (formation of coruto). A description of the vegetative and reproductive stages of maize can be found on the world wide web (internet) at agronext.iastate.edu/ corn / production / management / growth / CornGrowthandDevelopment. html and in "Corn Growth and Development", Abendroth et al. Iowa State University Extension and Outreach Publication PMR 1009, March 2011). In certain embodiments, Methylobacterium sp. to increase yield, the stage about V5, V6 to about Vn or the development stage VT are applied to the development stage of about R2, R3, R4, R5 or R6. In certain embodiments, Methylobacterium sp. to increase yield, about V12, V16, V18, Vn or VT are applied in the development stage to the development stage, about R2, R3 or R4. In certain embodiments, Methylobacterium sp. for increase of yield, about R1 are applied in the development stage In certain modalities, the Methylobacterium for increase of yield that is applied to the final vegetative or reproductive stage corn plant is selected from the group consisting of SO02, ISO03, ISO04 and derivatives thereof.
[0041] Several isolates of Methylobacterium sp. provided in this document are revealed in Table 1. TABLE 1. ISOLATES OF METHYLOBACTERIUM sp.

1 Deposit number for the strain deposited with AGRICULTURAL RESEARCH SERVICE CULTURE COLLECTION (NRRL) from the National Center for Agricultural Use Research, Agricultural Research Service, US Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604 USA under the terms of Budapest Treaty on International Recognition of the Deposit of Microorganisms for the Purposes of the Patent Procedure. Subject to 37 CFR § 1,808 (b), all restrictions imposed by the depositor on the availability of the deposited material to the public will be irrevocably removed by granting any patent from this patent application.
[0042] Co-assigned patent applications that reveal additional specific uses of the Methylobacterium strains in Table 1 such as: (1) increase soybean yield (US 61/911698, filed on 12/4/2013; and International Application that claims the benefit of it deposited on 12/4/2014); (2) improving lettuce cultivation (PCT / US 14/68558 International Patent Application filed on 4/12/2014); (3) improve tomato growth (International Patent Application PCT / US14 / 68611 filed on 12/4/2014) and are incorporated by reference in this document. The genomic nucleic acid and amino acid sequences of NLS017, NLS020, NLS037, NLS042, NLS065 and NLS066, which are disclosed in International Application PCT / US 14/68558, deposited on 12/4, are specifically incorporated in this document for reference in their entirety. / 2014. The genomic nucleic acid sequences of NLS017 and NLS066 disclosed in International Patent Application PCT / US 14/68611, filed on 2/4/2014, are also specifically incorporated in this document for reference in their entirety. Such sequences of amino acid and genomic nucleic acids can be used to identify compositions, plant parts, plant seeds or processed plant products that comprise Methylobacterium sp. NLS017, NLS020, NLS037, NLS042, NLS065 and NLS066.
[0043] Methods of improving corn yield are also provided in this document which comprise applying any of the aforementioned compositions provided herein to a plant or part of a plant in an amount that provides increased corn yield in the plant, part of of plant or plant obtained from it in relation to the yield of a control plant, part of plant or plant obtained from it that has not received an application of the composition. In certain embodiments, application of the composition provides at least about 50%, at least about 75%, at least about 85% or at least about 95% of increased corn yield on the plant, plant part or plant derived from it in relation to infection of the control plant, part of a plant or a plant obtained from it. In certain modalities, the plant part is selected from the group consisting of a leaf, a stem, a flower, a root and a seed. In certain embodiments, the method further comprises the step of harvesting at least one part of the plant selected from the group consisting of a leaf, stem, flower, root or seed of the plant or part of the plant. In certain embodiments of any of the aforementioned methods, the methods additionally comprise obtaining a processed food or food composition from the plant or plant part. In certain embodiments, the processed food or food composition is a feed or paste. In certain modalities of any of the methods mentioned above, Methylobacterium to increase yield is selected from the group consisting of ISO02, ISO03, ISO04, ISO11 and derivatives thereof. In certain modalities in which the composition is applied before or during the reproductive stages of corn development, Methylobacterium to increase yield is selected from the group consisting of SO02, ISO03, ISO04 and derivatives thereof. In certain modalities in which the composition is applied to the seed or during the vegetative stages of corn development, Methylobacterium to increase yield is ISO11.
[0044] Methods are also provided to produce compositions useful for improving the yield or early vigor of corn which comprise combining a Methylobacterium for increasing yield or early vigor with an agriculturally acceptable excipient and / or with an agriculturally acceptable adjuvant. In certain methods, Methylobacterium sp. is selected from the group consisting of M. aminovorans, M. extorquens, M. fujisawaense, M. mesophilicum, M. radiotolerans, M. rhodesianum, M. nodularis, M. phyllosphaerae, M thiocyanatum and M. oryzae. In certain methods, Methylobacterium is not M. radiotolerans or M. oryzae. In certain methods, Methylobacterium is adhered to a solid substance. In certain method modalities, Methylobacterium is adhered to the solid substance and combined with a liquid to form a composition that is a colloid. In certain methods, the colloid is a gel. In certain method modalities, Methylobacterium adhered to the solid substance is supplied by growing Methylobacterium in the presence of the solid substance. In certain embodiments of the methods, a composition comprises an emulsion. In certain method modalities, Methylobacterium is provided by growing Methylobacterium in an emulsion. In certain modalities of any of the methods mentioned above, Methylobacterium to increase yield or early vigor is selected from the group consisting of ISO02, ISO03, ISO04, ISO11 and derivatives thereof. In certain modalities in which the composition is applied before or during the reproductive stages of corn development, Methylobacterium to increase yield is selected from the group consisting of SO02, ISO03, ISO04 and derivatives thereof. In certain modalities in which the composition is applied to the seed or during the vegetative stages of corn development, Methylobacterium for increasing yield or early vigor is ISO11.
[0045] Methods in which Methylobacterium are grown in biphasic media comprising a liquid phase and a solid substance have been shown to significantly increase the yield resulting from Methylobacterium compared to methods in which Methylobacterium are grown in liquid media alone. In certain embodiments, the methods may comprise growing the Methylobacterium in liquid media with a particulate solid substance that can be suspended in the liquid by stirring under conditions that provide the culture of Methylobacterium. In certain embodiments in which particulate solid substances are used, at least substantially the entire solid phase can thus be suspended in the liquid phase by stirring. Such solid particulate substances can comprise materials that are about 1 millimeter or less in length or diameter. In certain embodiments, the degree of agitation is sufficient to provide uniform distribution of the particulate solid substance in the liquid phase and / or ideal levels of culture aeration. However, in other embodiments provided herein, at least substantially the entire solid phase is not suspended in the liquid phase or portions of the solid phase are suspended in the liquid phase and portions of the solid phase are not suspended in the liquid phase. Non-particulate solid substances can be used in certain biphasic media in which the solid phase is not suspended in the liquid phase. Such non-particulate solid substances include, but are not limited to, materials that are larger than about 1 millimeter in length or diameter. Such particulate and non-particulate solids also include, but are not limited to, materials that are porous, fibrous or otherwise configured to provide increased surface areas for the Methylobacterium adherent culture. The biphasic media in which portions of the solid phase are suspended in the liquid phase and portions of the solid phase are not suspended in the liquid phase can comprise a mixture of particulate and non-particulate solids. Such particulate and non-particulate solid substances used in any of the two-phase media mentioned above may, however, include, without limitation, materials that are porous, fibrous or otherwise configured to provide increased surface areas for the Methylobacterium adherent culture. In certain embodiments, the medium comprises a colloid formed by a solid and a liquid phase. A colloid comprising a solid and a liquid can be preformed and added to the liquid medium, or it can be formed in the medium containing a solid and a liquid. Colloids comprising a solid and a liquid can be formed by subjecting certain solid substances to chemical and / or thermal change. In certain embodiments, the colloid is a gel. In certain embodiments, the liquid phase of the medium is an emulsion. In certain embodiments, the emulsion comprises an aqueous liquid and a liquid that is not miscible or only partially miscible in the aqueous liquid. Liquids that are not miscible or only partially miscible in water include, without limitation, any of the following: (1) liquids that have a miscibility in water that is equal to or less than that of pentanol, hexanol or heptanol at 25 degrees C; (2) liquids that comprise an alcohol, an aldehyde, a ketone, a fatty acid, a phospholipid or any combination thereof; (3) alcohols selected from the group consisting of aliphatic alcohols containing at least 5 carbons and sterols; (4) an animal oil, microbial oil, synthetic oil, vegetable oil or a combination thereof; and / or (5) a vegetable oil is selected from the group consisting of corn, soy, cotton, peanuts, sunflower, olive, linseed, coconut, palm, rapeseed, sesame seed, saffron and combinations thereof. In certain embodiments, the immiscible or partially immiscible liquid may comprise at least about 0.02% to about 20% of the bulk liquid phase. In certain embodiments, the methods may comprise obtaining a two-phase culture medium comprising liquid, solid and Methylobacterium and incubating the culture under conditions that provide the culture of Methylobacterium. Biphasic culture media comprising liquid, solid and Methylobacterium can be obtained by a variety of methods which include, but are not limited to, any of the following: (a) inoculate a biphasic medium comprising liquid and solid substance with Methylobacterium; (b) inoculate the solid substance with Methylobacterium and then introduce the solid substance comprising Methylobacterium into the liquid medium; (c) inoculate the solid substance with Methylobacterium, incubate Methylobacterium in the solid substance and then introduce the solid substance comprising Methylobacterium into the liquid medium; or (d) any combination of (a), (b) or (c). The methods and compositions for cultivating Methylobacterium in biphasic medium comprising a liquid and a solid are disclosed in US Patent Application No. 13 / 907,161, filed on May 31, 2013, which is incorporated into this document by way of reference in its entirety and in the International Patent Application codigned PCT / US 13/43722, filed on May 31, 2013, which is incorporated in this document as a reference in its entirety.
[0046] The methods in which Methylobacterium are grown in the medium comprising an emulsion have also been shown to significantly increase the yield resulting from Methylobacterium compared to methods in which Methylobacterium are grown in liquid medium only. In certain embodiments, methods for producing the compositions provided herein may comprise culturing the Methylobacterium agent for yield enhancement in an emulsion under conditions that provide the Methylobacterium culture. The means comprising the emulsion and Methylobacterium for increasing yield can be obtained by a variety of methods which include, but are not limited to, any of the following: (a) inoculating a medium comprising the emulsion with Methylobacterium; (b) inoculate the aqueous liquid with Methylobacterium, introduce the non-aqueous liquid and mix to form an emulsion; (c) inoculate the aqueous liquid with Methylobacterium, introduce the non-aqueous liquid and mix to form an emulsion; or (d) any combination of (a), (b) or (c). In certain embodiments, the emulsion comprises an aqueous liquid and a liquid that is not miscible or only partially miscible in the aqueous liquid. Non-aqueous liquids that are not miscible or only partially miscible in water include, without limitation, any of the following: (1) liquids that have a miscibility in water that is equal to or less than that of n-pentanol, n -hexanol or n-heptanol at 25 degrees C; (2) liquids that comprise an alcohol, an aldehyde, a ketone, a fatty acid, a phospholipid or any combination thereof; (3) alcohols selected from the group consisting of aliphatic alcohols containing at least 5, 6 or 7 carbons and sterols; (4) an animal oil, microbial oil, synthetic oil, vegetable oil or a combination thereof; and / or (5) a vegetable oil is selected from the group consisting of corn, soy, cotton, peanuts, sunflower, olive, linseed, coconut, palm, rapeseed, sesame seed, saffron and combinations thereof. In certain embodiments, the immiscible or partially immiscible non-aqueous liquid may comprise at least about 0.02% to about 20% of the mass emulsion. In certain embodiments, the immiscible or partially immiscible non-aqueous liquid may comprise at least about any of about 0.05%, 0.1%, 0.5% or 1% to about 3%, 5%, 10 % or 20% of the mass emulsion. The methods and compositions for cultivating Methylobacterium in an emulsion medium are disclosed in the International Patent Application codigned PCT / US2014 / 040218, filed on May 30, 2014, which is incorporated by reference in its entirety for reference in its entirety .
[0047] In certain embodiments, the fermentation broth, fermentation broth product or compositions comprising Methylobacterium sp. for increased yield or early vigor they may additionally comprise one or more microorganisms of predetermined identity other than Methylobacterium. Other microorganisms that can be added include, but are not limited to, microorganisms that are biopesticides or provide some other benefit when applied to a plant or plant part. Biopesticides beneficial micro-organisms, however, therefore include, without limitation, various biopesticides from Bacillus sp., Pseudomonas sp., Coniothyrium sp., Pantoea sp., Streptomyces sp. and Trichoderma sp. Microbial that can be a bacterium, fungus, virus or protozoan. Particularly useful biopesticidal microorganisms include several strains of Bacillus subtilis, Bacillus thuringiensis, Bacillus pumilis, Pseudomonas syringae, Trichoderma harzianum, Trichoderma virens and Streptomyces lydicus. Other microorganisms that are added can be genetically modified or wild-type isolates that are available as pure cultures. In certain embodiments, it is anticipated that the bacterial or fungal microorganism may be supplied in the fermentation broth, fermentation broth product or composition in the form of a spore.
[0048] In certain embodiments, the liquid culture medium is prepared from inexpensive and readily available components, including, without limitation, inorganic salts such as potassium phosphate, magnesium sulfate and the like, carbon sources such as glycerol, methanol, glutamic acid, aspartic acid, succinic acid and the like and mixtures of amino acids such as peptone, tryptone and the like. Exemplary liquid media that can be used include, but are not limited to, ammonium mineral salts (AMS) medium (Whittenbury et al., 1970), Vogel-Bonner (VB) minimal culture medium (Vogel and Bonner, 1956 ) and LB broth (“Caldo de Luria - Bertani”).
[0049] Generally, the solid substance used in methods and compositions that provide sufficient culture of Methylobacterium can be any suitable solid substance that is insoluble or only partially soluble in water or aqueous solutions. Such suitable solid substances are also non-bacteriocidal or non-bacteriostatic in relation to Methylobacterium sp. to increase yield when solid substances are supplied in the liquid culture medium. In certain embodiments, such suitable solid substances are also solid substances that are readily obtained in sterile form or made sterile. The solid substances used in this document can be sterilized by any method that provides for the removal of contaminating microorganisms, and thus include, without limitation, methods such as autoclaving, irradiation, chemical treatment and any combination thereof. These solid substances include natural substances of animal, vegetable, microbial, fungal or mineral origin, artificial substances or combinations of natural and artificial substances. In certain embodiments, solid substances are inanimate solid substances. Inanimate solid substances of animal, vegetable, microbial or fungal origin can be obtained from animals, plants, microbes or fungi that are unfeasible (that is, no longer alive) or that have been rendered unfeasible. Diatomaceous shells are thus inanimate solid substances when previously associated diatomaceous algae have been removed or otherwise rendered unworkable. As diatom shells are inanimate solid substances, they are not considered to be photosynthetic organisms or photosynthetic micro-organisms. However, in certain embodiments, solid substances include, without limitation, sand, silt, soil, clay, ash, coal, diatomaceous earth and other similar minerals, glass or crushed glass spheres, crushed ceramic materials, ceramic spheres, bentonite, kaolin , talc, perlite, mica, vermiculite, silicas, quartz powder, montmorillonite and combinations thereof. In certain embodiments, the solid substance can be a polymer or polymeric spheres. Polymers that can be used as a solid substance include, without limitation, various polysaccharides such as cellulosic polymers and chitinous polymers that are insoluble or only partially soluble in water or aqueous solutions, agar (ie, galactans) and combinations thereof. . In certain embodiments, the solid substance can be an insoluble salt crystal or only partially soluble. The salt crystals that can be used, however, include, without limitation, insoluble or only partially soluble carbonates, chromates, sulfites, phosphates, hydroxides, oxides and sulfides. In certain embodiments, the solid substance can be a microbial cell, fungal cell, microbial spore or fungal spore. In certain embodiments, the solid substance can be a microbial cell or microbial spore in which the microbial cell or microbial spore is not a photosynthetic microorganism. In certain modalities, the microbial cell or microbial spore is not a photosynthetic microorganism, in which the photosynthetic microorganism is selected from the group consisting of algae, cyanobacteria, diatoms, Botryococcus braunii, Chlorella, Dunaliella tertiolecta, Gracilaria, Pleurochrysis carterae, Sargassum and Ulva. In still other embodiments, the solid substance can be an inactivated (ie, non-viable) microbial cell, fungal cell, microbial spore or fungal spore. In still other embodiments, the solid substance can be a quiescent microbial cell (that is, viable, but not actively dividing), fungal cell, microbial spore or fungal spore. In still other embodiments, the solid substance may be cellular debris of microbial origin. In still other embodiments, the solid substance can be particulate matter from any part of a plant. The plant parts that can be used to obtain the solid substance, however, include, without limitation, ears, barks, hooves, leaves, roots, flowers, stems, corks, seeds and combinations thereof. Products obtained from processed plant parts which include, but are not limited to, bagasse, wheat bran, soybeans, crushed seed cake, straw and the like can also be used. Such plant parts, processed plants and / or processed plant parts can be ground to obtain the solid material in a particulate form that can be used. In certain embodiments, wood or a wood product including, without limitation, wood pulp, sawdust, trim and the like, can be used. In certain embodiments, the solid substance can be a particulate matter from an animal (s), including, without limitation, bone meal, gelatin, crushed or powdered shells, hair, macerated skin and the like.
[0050] In certain modalities, the solid substance is supplied in particulate form that provides the distribution of the solid substance in the culture medium. In certain embodiments, the solid substance is comprised of a particle of about 2 microns to about 1,000 microns in average length or average diameter. In certain embodiments, the solid substance is comprised of a particle of about 1 micron to about 1,000 microns in average length or average diameter. In certain embodiments, the solid substance is a particle of about 1, 2, 4, 10, 20 or 40 microns to any one of about 100, 200, 500, 750 or 1,000 microns of average length or average diameter. The desirable characteristics of the particles used in the methods and compositions provided herein include adequate wettability so that the particles can be suspended throughout the medium by stirring.
[0051] In certain embodiments, the solid substance is supplied in the medium as a colloid in which the continuous phase is a liquid and the dispersed phase is the solid. Suitable solids that can be used to form colloids in the liquid medium used to grow Methylobacterium sp. for increased yield, however, they include, without limitation, various solids which are referred to as hydrocolloids. Such hydrocolloids used in the media, methods and compositions provided herein may be hydrophilic polymers, of vegetable, animal, microbial or synthetic origin. The hydrocolloid polymers used in the methods may contain many hydroxyl groups and / or may be polyelectrolytes. The hydrocolloid polymers used in the compositions and methods provided herein, however, include, without limitation, agar, alginate, arabinoxylan, carrageenan, carboxymethylcellulose, cellulose, curdlan, gelatin, gelan, β-glucan, gorma guar, arabic gum, locust bean gum , pectin, starch, xanthan gum and mixtures thereof. In certain embodiments, the colloid used in the media, methods and compositions provided herein may comprise a hydrocolloid polymer and one or more proteins.
[0052] In certain embodiments, the solid substance can be a solid substance that provides the adherent culture of Methylobacterium sp. to increase yield in the solid substance. As Methylobacterium sp. for performance enhancement that are adhered to a solid substance are Methylobacterium which cannot be substantially removed by simply washing the solid substance with Methylobacterium sp. to increase yields adherent with culture medium while adherent Methylobacterium can be substantially removed by washing the solid substance with liquid culture medium. In this context, "substantially removed" means that at least about 30%, 40%, 50%, 60%, 70% or 80% of the Methylobacterium present is removed when the solid substance is washed with three volumes of liquid culture medium. Such washing can be carried out by a variety of methods including, without limitation, decanting the liquid from a washed solid phase or passing the liquid through a solid phase in a filter that allows the bacteria to flow through the liquid. In certain embodiments, Methylobacterium sp. for increasing yields adherents that are associated with the solid may include Methylobacterium which are directly attached to the solid and / or Methylobacterium which are indirectly attached to the solid substance. Methylobacterium which are indirectly attached to the solid substance include, without limitation, Methylobacterium which are attached to another Methylobacterium or to another microorganism which is attached to the solid substance, Methylobacterium which are attached to the solid substance and are attached to another substance which is fixed to the solid substance and the like. In certain modalities, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5% or 99.9% of the Methylobacterium in the fermentation broth, fermentation broth product or compositions are Methylobacterium which are adhered to the solid substance. In certain embodiments, Methylobacterium sp. to increase adherent yield it can be present on the surface of the solid substance in the fermentation broth, fermentation broth product or composition at a density of at least about 1 Methylobacterium / 20 square micrometers, of at least about 1 Methylobacterium / 10 micrometers square, at least about 1 Methylobacterium / 10 square micrometer, at least about 1 Methylobacterium / 5 square micrometer, at least about 1 Methylobacterium / 2 square micrometer or at least about 1 Methylobacterium / square micrometer. In certain embodiments, Methylobacterium sp. to increase adherent yield it can be present on the surface of the solid substance in the fermentation broth, fermentation broth product or composition at a density of at least about 1 Methylobacterium / 20 square micrometers to about 1 Methylobacterium / square micrometer, at least least about 1 Methylobacterium / 10 square micrometer to about 1 Methylobacterium / square micrometer, at least about 1 Methylobacterium / 10 square micrometer to about 1 Methylobacterium / square micrometer, from at least about 1 Methylobacterium / 5 square micrometer to about 1 Methylobacterium / square micrometer or at least about 1 Methylobacterium / 2 square micrometer to about 1 Methylobacterium / square micrometer. In certain embodiments, Methylobacterium sp. to increase adherent yield it can be present on the surface of the solid substance in the fermentation broth, fermentation broth product or composition at a density of at least about 1 Methylobacterium / 20 square micrometers to about 1 Methylobacterium / 2 square micrometers, from at least about 1 Methylobacterium / 10 square micrometers to about 1 Methylobacterium / 2 square micrometers, at least about 1 Methylobacterium / 10 square micrometers to about 1 Methylobacterium / 2 square micrometers or at least about 1 Methylobacterium / 5 square micrometers to about 1 Methylobacterium / 2 square micrometers. The biphasic fermentation broths provided in this document may comprise a liquid phase containing non-adherent Methylobacterium. In certain embodiments, the titers of Methylobacterium adhering to the liquid phase may be less than about 100,000, 10,000 or 1,000 CFU / ml. In certain modalities of any of the aforementioned compositions, Methylobacterium for increasing yield or early vigor is selected from the group consisting of ISO02, ISO03, ISO04, ISO11 and derivatives thereof. In certain modalities in which the composition is applied before or during the reproductive stages of corn development, Methylobacterium to increase yield is selected from the group consisting of SO02, ISO03, ISO04 and derivatives thereof. In certain modalities in which the composition is applied to the seed or during the vegetative stages of corn development, Methylobacterium to increase yield is ISO11 or a derivative of it.
[0053] The biphasic culture methods provided can yield fermentation broths with Methylobacterium sp. for increased yield or early vigor at a rate greater than about 5 x 108 colony-forming units per milliliter, at a rate greater than about 1 x 109 colony-forming units per milliliter, at a rate greater than about 1 x 1010 colony forming units per milliliter, at a rate of at least about 3 x 1010 colony forming units per milliliter. In certain embodiments, the fermentation broths provided herein may comprise Methylobacterium sp. to increase yield at a rate of at least about 5 x 108 colony-forming units per milliliter to at least about 3 x 1010 colony-forming units per milliliter, at least about 5 x 108 colony-forming units per milliliter at least minus about 4 x 1010 colony forming units per milliliter or at least about 5 x 108 colony forming units per milliliter to at least about 6 x 1010 colony forming units per milliliter. In certain embodiments, the fermentation broths provided herein may comprise Methylobacterium sp. to increase yield at a rate of at least about 1 x 109 colony-forming units per milliliter to at least about 3 x 1010 colony-forming units per milliliter, at least about 1 x 109 colony-forming units per milliliter at least minus about 4 x 1010 colony forming units per milliliter or at least about 1 x 109 colony forming units per milliliter to at least about 6 x 1010 colony forming units per milliliter. In certain embodiments, the fermentation broths provided herein will comprise Methylobacterium sp. to increase yield at a rate of at least about 1 x 1010 colony-forming units per milliliter to at least about 3 x 1010 colony-forming units per milliliter, at least about 1 x 1010 colony-forming units per milliliter at least minus about 4 x 1010 colony forming units per milliliter or at least about 1 x 1010 colony forming units per milliliter to at least about 6 x 1010 colony forming units per milliliter. In certain embodiments, the fermentation broths provided herein will comprise Methylobacterium sp. to increase yield at a rate of at least about 3 x 1010 colony-forming units per milliliter to at least about 4 x 1010 colony-forming units per milliliter or at least about 3 x 1010 colony-forming units per milliliter a at least about 6 x 1010 colony forming units per milliliter. In certain embodiments of any of the aforementioned compositions, Methylobacterium for increasing yield is selected from the group consisting of ISO02, ISO03, ISO04, ISO11 and derivatives thereof. In certain modalities in which the composition is applied before or during the reproductive stages of corn development, Methylobacterium to increase yield is selected from the group consisting of SO02, ISO03 and ISO04. In certain modalities in which the composition is applied to the seed or during the vegetative stages of corn development, Methylobacterium for increasing yield or early vigor is ISO11.
[0054] Solid substances with Methylobacterium sp. for increased yield or adherent early vigor can be obtained as fermentation products that can be used to produce various compositions useful for treating plants or plant parts to improve early corn yield or vigor. Alternatively, the compositions provided herein that comprise Methylobacterium sp. to increase yield or early vigor, solid substances with Methylobacterium sp. to increase yield or early vigor grown in them or that contain emulsions with Methylobacterium sp. for increased yield or early vigor grown on them can be used to treat plants or plant parts. Plants, plant parts and, in particular, plant seeds that have been at least partially coated or coated with the fermentation broth products or compositions comprising Methylobacterium sp. to increase yield are thus provided. The partial coating of a plant, part of a plant or seed includes, however, without limitation, the coating of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70 %, 80%, 90%, 95%, 98%, 99% or about 99.5% of the plant's surface area, plant part or plant seed. Processed plant products containing fermentation broth products or compositions with Methylobacterium sp. to increase yield or Methylobacterium sp. to increase adherent performance. Solid substances with Methylobacterium sp. for increasing adherent yield they can be used to produce various compositions that are particularly useful for treating plant seeds. The seeds that have been at least partially coated with the fermentation broth products or compositions are thus supplied. The partial coating of a seed, however, includes, without limitation, the coating of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or about 99.5% of the seed surface area. Processed seed products are also provided, including, without limitation, bran, flour, feed and flakes, which contain the fermentation broth products or compositions provided in this document. In certain embodiments, the processed plant product will be non-regenerable (that is, it will not be able to grow into a plant). In certain embodiments, the solid substance used in the fermentation product or composition that at least partially covers the plant, part of a plant or plant seed or that is contained in the plant, part of a plant or processed seed product comprises a solid substance and Methylobacterium sp. for associated or adherent yield increase that can be easily identified by comparing a plant, plant part, plant seed or processed product from the same treated and untreated. In certain modalities, Methylobacterium to increase yield or early vigor is selected from the group consisting of ISO02, ISO03, ISO04, ISO11 and derivatives thereof. In certain modalities, Methylobacterium to increase yield or early vigor is selected from the group consisting of ISO02, ISO03 and ISO04.
[0055] Compositions useful for treating plants or parts of plants comprising Methylobacterium sp. to increase yield or early vigor, a solid substance with Methylobacterium sp. to increase yield or early adherent vigor or which comprise emulsions with Methylobacterium sp. for increased yield or early vigor grown in them they may also further comprise an agriculturally acceptable adjuvant or an agriculturally acceptable excipient. An agriculturally acceptable adjuvant or an agriculturally acceptable excipient is typically an ingredient that does not cause undue phytotoxicity or other adverse effects when exposed to a plant or plant part. In certain embodiments, the solid substance itself can be an agriculturally acceptable adjuvant or an agriculturally acceptable excipient as long as it is not bactericidal or bacteriostatic to Methylobacterium. In other embodiments, the composition further comprises at least one of an agriculturally acceptable adjuvant or an agriculturally acceptable excipient. Any of the compositions mentioned above can also further comprise a pesticide. The pesticides used in the composition, however, include, without limitation, an insecticide, a fungicide, a nematicide and a bactericide. In certain embodiments, the pesticide used in the composition is a pesticide that does not substantially inhibit the culture of Methylobacterium. Since Methylobacterium are gram-positive bacteria, suitable bactericides used in the compositions may, however, include, without limitation, bactericides that exhibit activity against gram-positive bacteria, but not gram-negative bacteria. The compositions provided herein can also comprise a bacteriostatic agent that does not substantially inhibit the culture of Methylobacterium. Bacteriostatic agents suitable for use in the compositions provided herein, however, include, without limitation, those that exhibit activity against gram-positive bacteria, but not gram-negative bacteria. Any of the aforementioned compositions can also be an essentially dry product (i.e., having about 5% or less water content), a mixture of the composition with an emulsion or a suspension.
[0056] Agriculturally acceptable adjuvants used in compositions comprising Methylobacterium sp. to increase yield or early vigor include, however, without limitation, components that increase the effectiveness of the product and / or products that increase the ease of application of the product. Adjuvants that increase the effectiveness of the product may include various humidifiers / diffusers that promote adhesion to and diffusion of the composition in plant parts, adhesives that promote adhesion to the plant part, penetrants that can promote the contact of the active agent with internal tissues, extenders that increase the active agent half-life by inhibiting environmental degradation and humectants that increase the density or drying time of sprayed compositions. The humidifiers / diffusers used in the compositions may include, without limitation, nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, organosilicate surfactants and / or acidified surfactants. The adhesives used in the compositions may include, without limitation, latex-based substances, terpene / pinolene and pyrrolidone-based substances. Penetrants may include mineral oil, vegetable oil, esterified vegetable oil, organosilicate surfactants and acidified surfactants. The extenders used in the compositions may, however, include, without limitation, ammonium sulfate, or menthol-based substances. The humectants used in the compositions may, however, include, without limitation, glycerol, propylene glycol and diethyl glycol. Adjuvants that enhance the ease of application of the product, however, include, without limitation, acidifying / buffering agents, defoaming / defoaming agents, compatibility agents, diversion reducing agents, dyes and water conditioners. The defoaming / defoaming agents used in the compositions may, however, include, without limitation, dimetopolysiloxane. The compatibility agents used in the compositions may, however, include, without limitation, ammonium sulfate. The drift reducing agents used in the compositions may, however, include, without limitation, polyacrylamides and polysaccharides. The water conditioners used in the compositions may, however, include, without limitation, ammonium sulfate.
[0057] Methods for treating plants and / or plant parts with fermentation broths, fermentation broth products and compositions comprising Methylobacterium sp. for increased yield or early vigor. The treated plants and the treated plant parts obtained therefrom, however, include, without limitation, corn. The parts of the corn plant that are treated, however, include, without limitation, leaves, stems, primary roots, nodal roots, seeds, fruits, corutes, silks, husks, sheaths, ears, coleoptiles and the like. The seeds and other propagules of any of the corn plants mentioned above can be treated with the fermentation broths, fermentation broth products, fermentation products and / or compositions provided in this document.
[0058] In certain embodiments, plants and / or plant parts are treated by applying fermentation broths, fermentation broth products, fermentation products and compositions comprising Methylobacterium sp. for increased yield or early vigor as a sprinkler. Such spray applications include, but are not limited to, treatments of a single plant part or any combination of plant parts. Sprinkling can be achieved with any device that will distribute the fermentation broths, fermentation broth products, fermentation products and compositions to the plant and / or plant part (s). Useful sprinkler devices include a boom sprinkler, hand or backpack sprinkler, vintage sprayers (i.e., aerial sprinkler) and the like. Sprinkler devices and / or methods that provide the application of fermentation broths, fermentation broth products, fermentation products and compositions to either or both the adaxial surface and / or the abaxial surface can also be used. Plants and / or plant parts that are at least partially coated with any one of a biphasic fermentation broth, a fermentation broth product, a fermentation product or compositions comprising a solid substance with Methylobacterium sp. for increasing yield adhered to it are also provided in this document. The partial coating of a plant, part of a plant or seed includes, however, without limitation, the coating of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70 %, 80%, 90%, 95%, 98%, 99% or about 99.5% of the plant's surface area, plant part or plant seed. Processed plant products that comprise a solid substance with Methylobacterium sp. to increase yield or early vigor adhered to it.
[0059] In certain embodiments, seeds are treated by exposing the seeds to fermentation broths, fermentation broth products, fermentation products and compositions comprising Methylobacterium sp. for increased yield or early vigor. The seeds may be treated with the fermentation broths, fermentation broth products and compositions provided herein by methods that include, but are not limited to, soaking, coating, spraying and the like. Seed treatments can be carried out with both continuous and / or batch seed keepers. In certain embodiments, the coated seeds can be prepared by forming a seed paste with a coating composition containing a fermentation broth, fermentation broth product or compositions comprising the solid substance with Methylobacterium sp. for increased yield and air drying of the resulting product. Air drying can be carried out at any temperature that is not harmful to the seed or Methylobacterium, but will typically not be higher than 30 degrees Centigrade. The coating ratio comprising a solid substance and Methylobacterium sp. However, for increased yield it includes, without limitation, a range of 0.1 to 25% by weight of the seed, 0.5 to 5% by weight of the seed and 0.5 to 2.5% by weight of the seed. In certain embodiments, a solid substance used in the seed coating or treatment will have Methylobacterium sp. to increase early income adhered to it. In certain embodiments, a solid substance used in the seed coating or treatment will be associated with Methylobacterium sp. to increase yield and will be a fermentation broth, fermentation broth product or composition obtained by the methods provided in this document. Various seed treatment compositions and seed treatment methods disclosed in US Patent Nos. 5,106,648, 5,512,069 and 8,181,388 are incorporated herein by reference in their entirety and can be adapted for use with an agent active ingredient comprising fermentation broths, fermentation broth products or compositions provided in this document. In certain embodiments, the composition used to treat the seed may contain agriculturally acceptable excipients which include, but are not limited to, wood flour, clays, activated carbon, diatomaceous earth, fine-grained inorganic solids, calcium carbonate and the like. Inorganic clays and solids that can be used with fermentation broths, fermentation broth products or compositions provided herein include, but are not limited to, calcium bentonite, kaolin, Chinese clay, talc, perlite, mica, vermiculite, silicas , powdered quartz, montmorillonite and mixtures thereof. Agrically acceptable adjuvants that promote adherence to the seed that can be used include, but are not limited to, polyvinyl acetates, polyvinyl acetate copolymers, hydrolyzed polyvinyl acetates, polyvinylpyrrolidone-vinyl acetate copolymer, polyvinyl alcohols, alcohol copolymers , polyvinyl methyl ester, polyvinyl methyl ether copolymer - maleic anhydride, waxes, latex polymers, celluloses including ethylcelluloses and methylcelluloses, hydroxymethylcelluloses, hydroxypropylcellulose, hydroxymethylpropylcelluloses, polyvinyl pyrrolidones, alginates, proteins, dextrins, oils, oxides , karaya gum, jaguar gum, tragacanth gum, polysaccharide gums, mucilage, arabic gum, shellac, vinylidene chloride polymers and copolymers, soy-based protein polymers and copolymers, lignosulfonates, acrylic copolymers, starches, polyvinylacrylates, polyvinylacrylates , gelatin, carboxymethylcellulose, chito sano, polyethylene oxide, acrylamide polymers and copolymers, polyhydroxyethyl acrylate, methylacrylamide monomers, alginate, ethylcellulose, polychloroprene and syrups or mixtures thereof. Other useful agriculturally acceptable adjuvants that can promote the coating include, but are not limited to, vinyl acetate polymers and copolymers, polyvinylpyrrolidone-vinyl acetate copolymer and water soluble waxes. Various surfactants, dispersants, anti-molding agents, foam control agents and dyes disclosed herein in US Patent No. 8,181,388 can be adapted for use with an active agent comprising fermentation broths, fermentation broth products or compositions provided in this document.
[0060] Compositions comprising Methylobacterium sp. for increased yield or early vigor that provide increased yield or early vigor for corn plants in relation to plants, plant parts and plants obtained from the same untreated ones that were exposed to the compositions. In certain embodiments, plant parts, including, without limitation, a seed, leaf, fruit, stem, root or coleoptile, can be treated with the compositions provided in this document to increase plant yield of corn. Treatments or applications may include, without limitation, spraying, coating, partial coating, dipping and / or soaking the plant or plant parts with the compositions provided herein. In certain embodiments, a seed, leaf, fruit, stem, root, tuber or coleoptile can be immersed and / or soaked with a liquid, semi-liquid, emulsion or slurry from a composition provided herein. Such seed immersion or soaking may be sufficient to provide increased yield in a treated corn plant or corn plant grown from a treated seed compared to an untreated corn plant or corn plant grown from an untreated seed. treated. In certain embodiments, plant seeds can be immersed and / or soaked for at least 1, 2, 3, 4, 5 or 6 hours. Such immersion and / or imbibition can, in certain modalities, be carried out at temperatures that are not harmful to the plant seed or Methylobacterium. In certain embodiments, the seeds can be treated at about 15 to about 30 degrees Fahrenheit or about 20 to about 25 degrees Fahrenheit. In certain embodiments, seed soaking and / or immersion can be performed with gentle agitation. In certain embodiments, the seed and / or coleoptile is exposed to the composition by providing the composition in a furrow. Provides the furrow composition represents one of several methods provided herein to apply a composition to a corn seed or corn plant at about the VE stage of corn plant development.
[0061] It is therefore expected that the compositions provided herein that comprise Methylobacterium sp. to increase yield or early vigor and related methods are useful in improving yield and / or early vigor in a wide variety of corn plants, including, without limitation, several hybrids, inbreeding strains, haploids, subspecies and varieties of Zea mays. In certain modalities, the yield and / or early vigor can be improved in toothed corn (Zea mays var. Indentata), collared corn (Zea mays var. Indurata), starch corn (Zea mays var. Amylacea), popcorn corn (Zea mays var. everta), tunicate corn (Zea mays var. Larranaga tunicata ex A. St. Hil.), striped corn (Zea mays var. japonica), sweet corn (Zea mays var. saccharata and Zea mays var. rugosa) and / or waxy corn (Zea mays var. keratin).
[0062] In certain embodiments, an amount of a composition provided herein that is sufficient to provide increased corn yield and / or early vigor may be a composition with Methylobacterium sp. to increase yield or early vigor at a rate of at least about 1 x 106 colony-forming units per milliliter, at least about 5 x 106 colony-forming units per milliliter, at least about 1 x 107 colony-forming units per milliliter, at least about 5 x 108 colony-forming units per milliliter, at least about 1 x 109 colony-forming units per milliliter, at least about 1 x 1010 colony-forming units per milliliter or at least about 3 x 1010 colony forming units per milliliter. In certain embodiments, an amount of a composition provided herein that is sufficient to provide increased corn yield and / or early vigor to a plant or plant part may be a composition with Methylobacterium sp. to increase yield or early vigor at a rate of at least about 1 x 106 colony-forming units per milliliter, at least about 5 x 106 colony-forming units per milliliter, at least about 1 x 107 colony-forming units per milliliter or at least about 5 x 108 colony-forming units per milliliter to at least about 6 x 1010 colony-forming units per milliliter of a liquid or an emulsion. In certain embodiments, an amount of a composition provided herein that is sufficient to provide increased corn yield and / or early vigor may be a fermentation broth product with a Methylobacterium sp. to increase yield or early vigor of a solid phase of this product that is at least about 1 x 106 colony forming units per milliliter, at least about 5 x 106 colony forming units per milliliter, at least about 1 x 107 colony-forming units per milliliter, or at least about 5 x 1010 colony-forming units per gram to at least about 6 x 1010 colony-forming units of Methylobacterium per gram of solid phase. In certain embodiments, an amount of a composition provided herein that is sufficient to provide increased corn yield and / or early vigor may be a composition with a Methylobacterium titer of at least about 1 x 106 colony forming units per gram at least about 5 x 106 colony-forming units per gram, at least about 1 x 107 colony-forming units per gram or at least about 5 x 108 colony-forming units per gram at least about 6 x 1010 colony-forming units of Methylobacterium per gram of particles in the composition containing the particles comprising a solid substance, in which a monoculture or co-culture of Methylobacterium sp. to increase yield or early vigor it is adhered to. In certain embodiments, an amount of a composition provided herein that is sufficient to provide an increased corn yield and / or early vigor to a plant or plant part may be a composition with a Methylobacterium titre of at least about 1 x 106 colony-forming units per ml, at least about 5 x 106 colony-forming units per ml, at least about 1 x 107 colony-forming units per ml or at least about 5 x 108 colony-forming units per ml a at least about 6 x 1010 colony-forming units of Methylobacterium per ml in a composition comprising an emulsion, in which a monoculture or co-culture of a Methylobacterium sp. to increase yield or early vigor adhered to a solid substance it is supplied there or grown therein. In certain embodiments, an amount of a composition provided herein that is sufficient to provide an increased corn yield and / or early vigor to a plant or plant part may be a composition with a Methylobacterium titre of at least about 1 x 106 colony forming units per ml, at least about 5 x 106 colony forming units per ml, at least about 1 x 107 colony forming units per ml or at least about 5 x 108 colony forming units per ml a at least about 6 x 1010 colony-forming units of Methylobacterium per ml in a composition comprising an emulsion, in which a monoculture or co-culture of a Methylobacterium sp. to increase yield or early vigor it is supplied there or grown there.
[0063] In certain embodiments, an amount of a composition provided herein that is sufficient to provide increased corn yield and / or early vigor may be a composition with a Methylobacterium sp. at a rate of at least about 1 x 104 colony-forming units per milliliter, at least about 1 x 105 colony-forming units per milliliter, at least about 1 x 106 colony-forming units per milliliter, at least about 5 x 106 colony-forming units per milliliter, at least about 1 x 107 colony-forming units per milliliter, at least about 5x108 colony-forming units per milliliter, at least about 1 x 09 colony-forming units per milliliter, at least about 1 x 1010 colony forming units per milliliter or at least about 3 x 1010 colony forming units per milliliter. In certain embodiments, an amount of a composition provided herein that is sufficient to provide increased corn yield and / or early vigor may be a composition with Methylobacterium sp. at a rate of at least about 1 x 104 colony forming units per milliliter, at least about 1 x 105 colony forming units per milliliter, at least about 1 x 106 colony forming units per milliliter, at least about 5 x 106 colony forming units per milliliter, at least about 1 x 107 colony forming units per milliliter or at least about 5 x 108 colony forming units per milliliter at least about 6 x 1010 colony forming units per milliliter of a liquid or an emulsion. In certain embodiments, an amount of a composition provided herein that is sufficient to provide increased corn yield and / or early vigor may be a fermentation broth product with a Methylobacterium sp. a solid phase of that product that is at least about 1 x 104 colony forming units per gram, at least about 1 x 105 colony forming units per gram, at least about 1 x 106 colony forming units per gram, at least about 5 x 106 colony-forming units per gram, at least about 1 x 107 colony-forming units per gram or at least about 5 x 108 colony-forming units per gram at least about 6 x 1010 units colony-forming Methylobacterium per gram, at least about 1 x 1011 colony-forming units of Methylobacterium per gram, at least about 1 x 1012 colony-forming units of Methylobacterium per gram, at least about 1 x 1013 colony-forming units Methylobacterium per gram or at least about 5 x 1013 colony-forming units of Methylobacterium per gram of solid phase. In certain embodiments, an amount of a composition provided herein that is sufficient to provide increased corn yield and / or early vigor may be a composition with a Methylobacterium titer of at least about 1 x 106 colony forming units per gram at least about 5 x 106 colony-forming units per gram, at least about 1 x 107 colony-forming units per gram or at least about 5 x 108 colony-forming units per gram at least about 6 x 1010 colony-forming units of Methylobacterium per gram, at least about 1 x 1011 colony-forming units of Methylobacterium per gram, at least about 1 x 1012 colony-forming units of Methylobacterium per gram, at least about 1 x 1013 forming units colony of Methylobacterium per gram or at least about 5 x 1013 colony forming units of Methylobacterium per gram of particles in the composition what contains particles that comprise a solid substance, in which a monoculture or co-culture of Methylobacterium sp. is adhered to. In certain embodiments, an amount of a composition provided herein that is sufficient to provide increased corn yield and / or early vigor may be a composition with a Methylobacterium titre of at least about 1 x 106 colony forming units per ml at least about 5 x 106 colony-forming units per ml, at least about 1 x 107 colony-forming units per ml or at least about 5 x 108 colony-forming units per ml at least about 6 x 1010 colony-forming units of Methylobacterium per ml in a composition comprising an emulsion, in which a monoculture or co-culture of a Methylobacterium sp. adhered to a solid substance is supplied therein or grown in it. In certain embodiments, an amount of a composition provided herein that is sufficient to provide increased corn yield and / or early vigor may be a composition with a Methylobacterium titre of at least about 1 x 106 colony forming units per ml at least about 5 x 106 colony-forming units per ml, at least about 1 x 107 colony-forming units per ml or at least about 5 x 108 colony-forming units per ml at least about 6 x 1010 colony-forming units of Methylobacterium per ml in a composition comprising an emulsion, in which a monoculture or co-culture of a Methylobacterium sp. it is supplied there or grown there. In certain embodiments of any of the aforementioned compositions, Methylobacterium sp. is an isolate of Methylobacterium ISO02 (NRRL B-50930), ISO03 (NRRL B-50931), ISO04 (NRRL B-50932), ISO11 (NRRL B- 50939) or a derivative thereof. Maize plants and maize plant parts (e.g., seeds) which are coated or partially coated with any of the aforementioned compositions are also provided. Methods are also provided to improve corn yield or early corn stamina using any of the aforementioned compositions. EXAMPLES
[0064] The following examples are included to demonstrate illustrative and non-limiting modalities of disclosure. It will be appreciated by those skilled in the art that the sets of procedures disclosed in the following examples represent sets of procedures found by Applicants to work well in the practice of the invention. However, those skilled in the art should, in the light of the present disclosure, appreciate that many changes can be made in the specific modalities from which they are revealed while still obtaining similar or similar results without departing from the scope of the disclosure. EXAMPLE 1. INCREASES IN CORN YIELD BY APPLICATION OF METHYLOBACTERIUM COMPOSITIONS
[0065] Corn field tests were established at six locations in Illinois, USA for the purpose of evaluating 14 isolates of PPFM (optional rose pigmented methylotrophs of the species Methylobacterium) applied as a leaf sprinkling to corn plants in one stage early vegetative (V3) and also in a reproductive stage (R1). The locations were established in Cropsey, Dana, Homer, Farmer City, Farmington and Homer, all in the state of Illinois, USA. The Cropsey test experienced severe damage from maize cow feeding during vegetative cultivation, and the Homer test experienced green breakage due to a straight wind event in early July; these two sites were thus not included in the analysis of the leaf test of corn. EXPERIMENTAL PROJECT
[0066] The test was conducted as a split plot design that consisted of four rows of 76.20 centimeters (30 inches) and 6.10 meters (20 feet) in length. The middle two rows were the treatment rows, the two outer rows were used as untreated edge rows. There were eight replications of each of the 14 treatments with PPFM for application in the V3 and R1 culture stages. The 14 treatments with PPFM plus the control (without PPFM, also referred to as “verification”) comprised the entire plot, and the V3 and R1 culture stage comprised the subdivided plot. There is a V3 and R1 control included with each of the 8 replications. METHODS
[0067] In preparation for field tests, PPFM cultures were grown in AMS + glycerol + peptone + diatomaceous earth at 30 ° C for 6 days. The ammonium mineral salt (AMS) medium contains, per liter, 700 milligrams of anhydrous dibasic potassium phosphate, 540 milligrams of anhydrous monobasic potassium phosphate, one gram of heptahydrate magnesium sulphate, 500 milligrams of anhydrous ammonium chloride and 200 milligrams of calcium chloride dihydrate.
[0068] The basic AMS medium was prepared from three stock solutions, listed below: STOCK SOLUTION I: FOR A LITER THE CONCENTRATION 50X dibasic potassium phosphate, anhydrous 35 grams monobasic potassium phosphate, anhydrous 27 grams STOCK SOLUTION II: FOR ONE LITER THE CONCENTRATION 50X magnesium sulfate heptahydrate 50 grams ammonium chloride, anhydrous 25 grams STOCK SOLUTION III: FOR ONE LITER THE CONCENTRATION 50X calcium chloride dihydrate 10 grams The stock solutions I, II and III were autoclaved separately . To prepare one liter of liquid AMS medium with glycerol, peptone and diatomaceous earth, the following were added to 920 ml of distilled water: 20 ml of stock solution I 20 ml of stock solution II 20 ml of stock solution III 20 ml of a 50% glycerol stock solution 10 grams of peptone 2 grams of diatomaceous earth
[0069] The resulting solution with suspended diatomaceous earth was sterilized by autoclaving. The cultures were collected by centrifugation at 5,000 rpm for 15 minutes and then resuspended in AMS + gicerol + peptone with 20% glycerol as a cryoprotectant at 10X concentration. The cultures were aliquoted and frozen at -80 ° C until thawed for use. Liquid PPFM preparations were applied to corn plants in stages V3 or R1 at a rate of 56.78 l (15 gal) per acre with the use of a chemical costal sprinkler. The titles of PPFMs applied in the various locations for both PPFM applications in R1 and V3 PPFM are provided in Tables 5 and 6, respectively. The tests were established within the existing farmer field sites and were managed with local agronomic methods that the farmer practices throughout the growing season. All hybrids used were Roundup Ready ™ hybrids, and the tests were sprayed with glyphosate in the V4 culture stage. The tests were collected for yield at physiological maturity with a commercial combine harvester. Table 2 indicates the planted hybrid, the date of planting and the date of harvest in the four leaf sites of corn. TABLE 2. PLANTED HYBRID, PLANTING DATE AND HARVEST DATE AT THE FOUR MAIZE FOLIAR SITES
RESULTS
[0070] The analysis of variance (ANOVA) was conducted with the routine Analyze - Fit Model in JMP version 11.0 (SAS Institute). After the parameter, the estimates were obtained from the models, residue plots and studentized residue tables were examined for compliance with the assumptions of normality and constant variance. Comparisons of isolates with verification within a culture stage in the application were performed with two-tailed t-tests applied to the paired differences between minimum squares averages estimated from the ANOVA model, under the null hypothesis that the difference on average was zero.
[0071] The following model was applied to the plot design subdivided into the four individual locations: [1] Yijk = M + I, + Sj + ISij + Rk + IRlk + eljk, where Yijk is the yield of isolate i in stage j in replica k, M represents the general mean, Ii is the fixed effect of isolate i, Sj is the fixed effect of stage j, ISij is the fixed effect of the interaction of isolate ie stage j, Rk is the random effect of replica k, IRik is the random effect of the isolate interaction ie replica k, and eijk is the random error.
[0072] Cross-site analyzes for the four sites were conducted according to the following model: [2] Yhijk = M + Ii + Sj + ISij + Lh + R (L) k (h) + LIhi + IR (L) ik (h) + LShj + LIShij + ehijk, where Yhijk is the yield in site h of isolate i in stage j in replica k, M represents the general mean, Ii is the fixed effect of isolate i, Sj is the fixed effect stage j, ISij is the fixed effect of the isolate interaction ie stage j, L is the random effect of location h, R (L) k (h) is the random effect of replica k housed within location h, LIhi is the random effect of the isolated he-site interaction i, IR (L) ik (h) is the random effect of the isolated iteration ie replica k housed within the h-location, LShj is the random effect of the he-site interaction and stage j, LIShij is the random effect of the three-way interaction of site h with isolate ie stage j, and ehijk is the random error.
[0073] Thirteen of the 14 PPFM isolates showed a significant yield response (p = 0.25) vs the V3 or R1 check which were compared in at least one location (Table 3). Only one site, Pesotum, showed no significant increase. Six isolates showed a significant increase vs verification through the four sites combined in stage R1 (Table 4). TABLE 3. AVERAGE YIELD, YIELD CLASSIFICATION AND P VALUES OF PPFM ISOLATES IN EACH OF THE FOUR LOCATIONS BY CULTURE STAGE




TABLE 4. AVERAGE YIELD, YIELD CLASSIFICATION AND P-VALUES OF PPFM ISOLATES THROUGH THE FOUR LOCATIONS COMBINED BY CULTURE STAGE


TABLE 5. PPFMS TITLES APPLIED TO STAGE R1 IN INDICATED LOCATIONS (IN CFU / ML)
1 ND: Not determined. TABLE 6. PPFMS TITLES APPLIED TO STAGE V3 IN INDICATED LOCATIONS (IN CFU / ML)

1 ND: Not determined. EXAMPLE 2. INCREASES IN CORN YIELD BY APPLICATION OF METHYLOBACTERIUM COMPOSITIONS IN 2014 FIELD TEST EXPERIMENTAL PROJECT
[0074] Corn field tests were established in seven locations for the purpose of evaluating three PPFM (optional pink pigmented methylotrophs of the Methylobacterium species). The isolates were applied as a leaf sprinkler to corn plants in an early vegetative stage (V3) and in furrow at planting. Foliar applications were made at five and 2.5 liters per acre. The furrow application was applied to sowing using 1.25 L and 0.625 L / acre. Field plots were established in Iowa, Illinois, Nebraska, Missouri, Ohio, South Dakota and Wisconsin. The tests were conducted using conventional row spacing (76.20 centimeters (30 inches)) with a minimum plot size of four rows by 6.10 meters (20 feet). Each treatment was conducted using six replications in a Randomized Complete Block Design (unless otherwise indicated). All observations were made from the two central rows of the plot. All destructive sampling was taken from the two outer rows. The treatments were applied in a furrow at planting with a nozzle over the seed furrow opened before covering. Linear current nozzles or flat fan nozzles were adjusted so that the fan pattern was parallel to the seed groove. Leaf sprays were applied with a conventional bar using a flat fan or cone jet nozzles. A minimum of 18.93 liters (five gallons) / acre of total volume was used for furrow applications and 56.78 liters (15 gallons) / acre for foliar applications. Early plant vigor was rated 20 and 60 days after emergence. The visual assessment of plant vigor was based on a scale of 1 to 5, with 1 being insufficient and 5 being excellent. Visual vigor assessments considered factors such as height, leaf area, leaf color and / or percentage canopy closure. The plants were harvested by standard producer practices using a conventional combine harvester. METHODS
[0075] In preparation for field tests, PPFM cultures were grown in AMS + glycerol + peptone + diatomaceous earth at 30 ° C for 6 days. The ammonium mineral salt (AMS) medium contains, per liter, 700 milligrams of anhydrous dibasic potassium phosphate, 540 milligrams of anhydrous monobasic potassium phosphate, one gram of heptahydrate magnesium sulphate, 500 milligrams of anhydrous ammonium chloride and 200 milligrams of calcium chloride dihydrate.
[0076] The basic AMS medium was prepared from three stock solutions, listed below: STOCK SOLUTION I: FOR A LITER THE CONCENTRATION 50X dibasic potassium phosphate, anhydrous 35 grams monobasic potassium phosphate, anhydrous 27 grams STOCK II SOLUTION: FOR ONE LITER THE CONCENTRATION 50X magnesium sulfate heptahydrate 50 grams ammonium chloride, anhydrous 25 grams STOCK SOLUTION III: FOR ONE LITER THE CONCENTRATION 50X calcium chloride dihydrate 10 grams The stock solutions I, II and III were autoclaved separately . To prepare one liter of liquid AMS medium with glycerol, peptone and diatomaceous earth, the following were added to 920 ml of distilled water: 20 ml of stock solution I 20 ml of stock solution II 20 ml of stock solution III 20 ml of a 50% glycerol stock solution 10 grams of peptone 2 grams of diatomaceous earth
[0077] The resulting solution with suspended diatomaceous earth was sterilized by autoclaving.
[0078] The cultures were collected by centrifugation at 5,000 rpm for 15 minutes and then resuspended in AMS + gicerol + peptone with 20% glycerol as a cryoprotectant in 10X concentration. The cultures were aliquoted and frozen at -80 ° C until thawed for use. The tests were established within the existing farmer field sites and were managed with local agronomic methods that the farmer practices throughout the growing season. The title ranges for the different strains of NLS used at the field sites were as follows: NLS0017 = 4.7 x 108 to 2.2 x 109 CFU / ml; NLS0020 = 3.0 x 108 to 3.1 x 109 CFU / ml; and NLS021 = 2.3 x 108 to 3.7 x 108 CFU / ml. RESULTS
[0079] Analysis of variance (ANOVA), Tukey HSD and average separations using LSD were conducted with Statistix software, version 9.0. The initial Tukey HSD analysis was performed at 90% confidence intervals that evaluate tests of comparisons entirely in pairs of vigor and yield for the purposes of treatment. At alpha equal to 0.1, there was no significant pairing difference between the means. However, when the tests of comparisons entirely in LSD pairs of vigor and yield were analyzed in alpha equal to 0.20, (80% CI) several treatments demonstrated significantly better vigor and yield higher than the verification across all sites. per treatment. TABLE 7: TEST OF COMPARISONS FULLY IN LSD PAIRS IN FORCE IN 60 DAYS AFTER PLANTING FOR TRT


[0080] The means followed by the same letter are not significantly different at alpha = 0.20. "H" = 1.25 L / acres furrow treatment (Fur) "L" = 0.625 L / acres furrow treatment (Fur) "H" = 5.0 L / acres foliar treatment (Fol) "L" = 2 , 5 L / acres leaf treatment (Fol) TABLE 8: TEST OF COMPARISONS IN LSD PAIRS OF INCOME IN BUSHELS PER ACRE FOR TRT


[0081] The means followed by the same letter are not significantly different at alpha = 0.20. "H" = 1.25 L / acres furrow treatment (Fur) "L" = 0.625 L / acres furrow treatment (Fur) "H" = 5.0 L / acres foliar treatment (Fol) "L" = 2 , 5 L / acres foliar treatment (Fol) When evaluated by treatment with the use of forced classification, trends that supported the evaluations of vigor and yield data were visible. TABLE 9. CORN YIELD ANALYSIS: RENTERS BY AVERAGE ACRE THROUGH ALL LOCATIONS.
Column values in bold and underlined are Average Treatment where Yields> Average Check
[0082] Of the three PPFM isolates tested (NLS017, NLS020 and NLS021), two showed a significant yield response (alpha = 0.20) when they were compared across all seven sites by these analyzes. The isolate NLS021 generated the greatest increase in yield when applied at 625 ml / acre as a furrow treatment. The isolate NLS017 delivered significant improvements in yield when applied at high rates of application in furrow (1,250 ml / acre) and leaf (5 L / acre).
[0083] Yield data were also analyzed using the statistical analysis software package JMP (Version 9.0). The complete model with all random effects was adjusted first and then reduced to the best fit model based on Akaike information criteria (AIC) values. Across the sites, mean comparisons were conducted using Fisher's LSD test with α = 0.05, 0.10 and 0.20 (Table 10). The gross averages reported in Table 9 differ from the adjusted averages calculated by the mixed effects model used to analyze the data presented in Table 10. This model was adjusted for random location and replication effects. As a result, statistical differences do not necessarily reflect numerical differences in gross income values. In addition, the approach to statistical analysis described in paragraph [93] differs slightly from that approach, resulting in marginally different results between the two analyzes. The analysis and results presented in table 10 do not include a second check, "Treatment 14", which was included in only four of the seven sites. TABLE 10. INCREASE IN INCOME OVER VERIFICATION THROUGH SITES
1 least square means
[0084] Treatment yield relative to verification for individual sites was analyzed in the same way as data across sites (Table 11). In the table below, yields significantly higher than the verification at α = 0.05, 0.10 and 0.20 are represented by "XXX", "XX" and "X", respectively. TABLE 11. NUMERIC INCREASES IN YIELD ON VERIFICATION THROUGH SITES

RESULTS
[0085] Of the three PPFM isolates tested (NLS017, NLS020 and NLS021), two showed a significant yield response (alpha = 0.20) when they were compared across all seven sites. The isolate NLS021 generated the greatest increase in yield when applied at 625 ml / acre as a furrow treatment. The isolate NLS017 delivered significant improvements in yield when applied at high rates of application in furrow (1,250 ml / acre) and leaf (5 L / acre).
[0086] Individual site data indicates that PPFMs, in general, have a beneficial effect on corn yield. Farmers City, a site where PPFMs did not positively influence maize yield, had particularly high overall yields. This could indicate that Farmers City had an "ideal" yield environment and suggests that the PPFM treatments used in these experiments offered increased yield protection and yield in the presence of various biotic and / or abiotic stress factors elsewhere, but did not affect performance under ideal growing conditions in Farmers City. REFERENCES 1. Abanda-Nkpwatt, D., M. Musch, J. Tschiersch, M. Boettner and W. Schwab. 2006. Molecular interaction between Methylobacterium extorquens and seedlings: growth promotion, methanol consumption, and localization of the methanol emission site. J. Exp. Bot. 57, 4,025 to 4,032. 2. Broekaert WF, Terras FR, Cammue BP, Vanderleyden J (1990) An automated quantitative assay for fungal growth inhibition. FEMS Microbiology Letters 69: 55 to 60. 3. Cao, Y-R, Wang, Q., Jin, R-X., Tang, S-K., He, W-X., Lai, H-X, Xu, L-H. and C-L Jiang. 2011. Methylobacterium soli sp. nov. a methanol-utilizing bacterium isolated from the forest soil. Antonie van Leeuwenhoek (2011) 99: 629 to 634. 4. Corpe, W.A. and D.V. Basile. 1982. Methanol-utilizing bacteria associated with green plants. Devel. Industr. Microbiol. 23: 483 to 493. 5. Corpe, W.A. and S. Rheem. 1989. Ecology of the methylotrophic bacteria on living leaf surfaces. FEMS Microbiol. Ecol. 62, 243 to 250. 6. Green, P.N. 2005. Methylobacterium. In Brenner, D.J., N.R. Krieg and J.T. Staley (eds.). “Bergey’s Manual of Systematic Bacteriology. Volume two, The Proteobacteria. Part C, The alpha-, beta-, delta-, and epsilonproteobacteria. ” Second edition. Springer, New York. Pages 567 to 571. 7. Green, P.N. 2006. Methylobacterium. In Dworkin, M., S. Falkow, E. Rosenberg, K.-H. Schleifer e. Stackebrandt (eds.). "The Prokaryotes. A Handbook on the Biology of Bacteria. Volume 5. Proteobacteria: Alpha and Beta Subclasses.” Third edition, Springer, New York, Pages 257 to 265. 8. Holland, MA 1997. Methylobacterium and plants, Recent, Res. Devel. In Plant Physiol. 1. 207 to 1,213, 9. Holland, MA and JC Polacco, 1994 PPFMs and other covert contaminants: Is there more to plant physiology than just plant Annu. Rev. Plant Physiol. Plant Mol. Biol. 45: 197 to 209. 10. Kutschera, U. 2007. Plant-associated methylobacteria as co- evolved phytosymbionts. A hypothesis. Plant Signal Behav. 2, 74 to 78. 11. Lidstrom, ME 2006. Aerobic methylotrophic 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. Ecophysio logy and biochemistry. "Third edition. Springer, New York. Pages 618 to 634. 12. Madhaiyan, M., S. Poonguzhali, HS Lee, K. Hari, SP Sundaram and TM Sa. 2005. Pink- pigmented facultative methylotrophic bacteria accelerate germination, growth and yield of sugarcane clone Co86032 (Saccharum officinarum L.) Biol. Fertil. Soils 41: 350 to 358. 13. Madhaiyan, M., S. Poonguzhali, M. Senthilkumar, S. Seshadri, H. Chung, J. Yang, S. Sundaram and T. Sa. 2004. Growth promotion and induction of systemic resistance in rice cultivar CO-47 (Oryza sativa L.) by Methylobacterium spp. Bot. Bull. Acad. Sin. 45 , 315 to 324. 14. Madhaiyan, M., S. Poonguzhali and T. Sa. 2007. Influence of plant species and environmental conditions on epiphytic and endophytic pink-pigmented facultative methylotrophic bacterial populations associated with field- grown rice cultivars. Biotechnol. October 2007; 17 (10): 1,645 to 1,654. 15. Stanier, RY, NJ Palleroni and M. Doudoroff. 1966. The a erobic pseudomonads: A taxonomic study. J. Gen. Microbiol. 43: 159 to 271. 16. 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 Vegetables. Jour. Bacteriol. 183; 1: 214 to 220.) 17. Sy, A., A.C.J. Timmers, C. Knief and J. A. Vorholt. 2005. Methylotrophic metabolism is advantageous for Methylobacterium extorquens during colonization of Medicago truncatula under competitive conditions. Environ Request Form. Microbiol. 71. 7245-7252,18. Vogel, H.J. and D.M. Bonner. 1956. Acetylornithinase of Escherichia coli: Partial purification and some properties. J. Biol. Chem., 218, 97 to 106. 18. Vogel, H. J. 1956. A convenient growth medium for Neurospora (Medium N). Microbial Genet Bull 13: 42 to 43. 19. Whittenbury, R., S.L. Davies and J.F. Wilkinson. 1970. Enrichment, isolation and some properties of methane- utilizing bacteria. J. Gen. Microbiol. 61. 205 to 218.
[0087] Having illustrated and described the principles of the present disclosure, it should be understood by those skilled in the art that the invention can be modified in layout and details without departing from such principles.
[0088] Although the materials and methods of this invention have been described in terms of various modalities and illustrative examples, it will be realized by those skilled in the art that variations can be applied to the materials and methods described in this document without departing from the concept, spirit and the scope of the invention. All such substitutes and modifications apparent to those skilled in the art are intended to be within the spirit, scope and concept of the invention, as defined by the appended claims.

权利要求:
Claims (14)
[0001]
1. Method for improving the yield of a corn plant characterized by the fact that it comprises: (a) applying a composition comprising a Methylobacterium sp. to a corn seed, to a corn plant or part of it at the stage of about VE at about V5 of development, or to a corn plant or part of it at the stage of about V6 to about R3 of development , wherein the composition comprises: a strain of Methylobacterium NLS0020 (NRRL B-50930), NLS0017 (NRRL B-50931), NLS0042 (NRRL B-50932), NLS0021 (NRRL B-50939), and an agriculturally acceptable adjuvant, excipient or combination thereof; and, (b) cultivating the corn plant until maturity, thus improving the yield of the corn plant.
[0002]
2. Method according to claim 1, characterized by the fact that the composition is applied in the development stage from about V6 to about R2, in the development stage from about R1 to R2, in the development stage of about from R1 to R3, or at the development stage of about R1.
[0003]
3. Method according to claim 1 or 2, characterized by the fact that the composition is applied in the stage of development from about VE to about V3, in the stage of development from about VE to about V5, in the stage of development from about V1 to V4, or V3.
[0004]
Method according to any one of claims 1 to 3, characterized in that the composition is a solid composition comprising Methylobacterium sp. at a titre of about 1x106 CFU / g to about 1x1014 CFU / g.
[0005]
Method according to any one of claims 1 to 4, characterized in that the composition is provided in a furrow or as a seed treatment.
[0006]
Method according to any one of claims 1 to 5, characterized in that the applied composition partly covers or covers the corn seed, the corn plant or a part of the corn plant.
[0007]
Method according to any one of claims 1 to 5, characterized in that the composition is applied to the foliage of the corn plant.
[0008]
Method according to any one of claims 1 to 7, characterized in that the composition additionally comprises a fungicidal agent.
[0009]
Method according to any one of claims 1 to 8, characterized by the fact that it further comprises the step of harvesting the seed from the mature corn plant.
[0010]
10. Method according to claim 9, characterized in that the yield of harvested seed is increased in comparison to the yield of harvested seed obtained from a control corn plant that has not received an application of Methylobacterium sp.
[0011]
11. Use of a composition comprising strain of Methylobacterium NLS0020 deposited as NRRL B-50930, NLS0017 deposited as NRRL B-50931, NLS0042 deposited as NRRL B-50932 or NLS0021 deposited as NRRL B-50939, and an agriculturally acceptable adjuvant, excipient or combination of these characterized by the fact that it is to increase the yield or improve the initial vigor of a corn plant.
[0012]
12. Use according to claim 11, characterized by the fact that the composition further comprises an insecticide, a fungicide, a nematocide or a bactericide.
[0013]
13. Use according to claim 11, characterized by the fact that the composition further comprises a biopesticide or microorganism beneficial in another way, except Methylobacterium.
[0014]
14. Use of an isolated Methylobacterium selected from the group consisting of NLS0020 deposited as NRRL B-50930, NLS0017 deposited as NRRL B-50931, NLS0042 deposited as NRRL B-50932, NLS0021 deposited as NRRL B-50939 characterized by the fact that it is for the preparation of a composition for the treatment of a corn plant, part of a corn plant or propagule of a corn plant.

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法律状态:
2019-05-28| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2020-04-07| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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