Compositions and methods for improving fruit production

专利摘要:
The present invention provides compositions comprising Methylobacterium and depleted of substances that promote growth of resident microorganisms on a plant or seed, methods for improving fruit production, methods of making the compositions, and methods of treating a plant or seed with a composition comprising Methylobacterium. Also provided are methods and compositions comprising Methylobacterium that provide for improved fruit production.
公开号:ES2602992A2
申请号:ES201690024
申请日:2014-12-04
公开日:2017-02-23
发明作者:Rachel Didonato FLORO；Justin Lee；Gregg Bogosian
申请人:NewLeaf Symbiotics Inc；
IPC主号:

专利说明:

description
Compositions and methods to improve fruit production Cross reference to related applications
This patent application claims the benefit of the provisional US 5 patent application. N0 61 / 911,577, filed on December 4, 2013, which is incorporated herein by reference.
Background
Organic compounds of a carbon such as methane and methanol are widely found in nature, and are used as carbon sources by bacteria classified as methotrophs and methylotrophs. Metanotrophic bacteria include species in the genus Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, Methylosinus, Methylocystis, Methylosphaera, Methylocaldum, and Methylocella (Lidstrom, 2006). Methaneotrophs have the enzyme methane monooxygenase, which incorporates an oxygen atom from 02 to methane, forming methanol. All methanotrophs are bound carbon users who are unable to use compounds containing carbon-carbon bonds. Methylotrophs, on the other hand, can also use more complex organic compounds, such as organic aids, higher alcohols, sugars and the like. Thus, methylotrophic bacteria are facultative methylotrophs. Methylotrophic bacteria include species in the genus Methylobacterium, Hyphomicrobium, 20 Methylophilus, Methylobacillus, Methylophaga, Aminobacter, Methylorhabdus, Methylopila, Methylosulfonomonas, Marinosulfonomonas, Paracoccus, Xanthobacter, Ancylobacteronebacterium, Rhocylbacterus, Rhocylbacterus, Rhocylbacterus, Rhocylobacterus, Rhocylbacterus, Rhocytobacterus, Rhocylbacterus, Rhocylbacterus, Rhocylbacterus, Rhocycology , Mycobacterium, Arthobacter, and Nocardia (Lidstrom, 2006).
Most of the methylotrophic bacteria of the genus Methylobacterium are pigmented 25 pink. They are conventionally called PPFM bacteria, which are pink pigmented facultative 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. 30 thiocyanatum, and M. zatmanii. However, M. nidulans is a Methylobacterium fixative of
nitrogen that is not a PPFM (Sy et al., 2001). Methylobacterium are ubiquitous in nature, found in soil, dust, fresh water, sediments and leaf surfaces, as well as in industrial environments and elmieos (Green, 2006).
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Compendium
Compositions comprising Methylobacterium are provided herein that are free of substances that promote the growth of resident bacteria on the plant or seed, compositions comprising a solid substance with adherent Methylobacterium grown thereon or an emulsion having Methylobacterium grown therein. same, compositions comprising certain strains of Methylobacterium and derivatives thereof and an adjuvant and / or excipient acceptable from the agricultural point of view, methods for using the compositions to improve fruit production, and methods for preparing the compositions. Such compositions are referred to herein in certain cases as simply "compositions containing Methylobacterium". In certain embodiments, the Methylobacterium in the composition or that is used is strain NLS0038, NLS0046, NLS0020, NLS0017, NLS0042, NLS0089, NLS0068, NLS0065, NLS0069, NLS0062, NLS0064, NLS0021, NLS0066, or NLS0037. In certain embodiments, the Methylobacterium in the composition or that is used is strain NLS0037, NLS0042, or NLS0062. In certain embodiments, the Methylobacterium in the composition or that is used is strain NLS0037, NLS0042, or NLS0062. In certain embodiments of any of the above embodiments, the composition further comprises an adjuvant and / or excipient acceptable from the agricultural point of view.
Methods for improving fruit production are provided herein which comprise: (a) applying a composition comprising Methylobacterium to a fruit producing plant or seed, composition comprising: (i) a solid substance with adherent Methylobacterium grown on the same; (ii) an emulsion having Methylobacterium cultured therein, or (iii) compositions comprising certain strains of Methylobacterium and derivatives thereof; where the plant or plant grown from the seed exhibits a faster fruit set, increased fruit set, earlier ripening, and / or more uniform fruit ripening compared to an untreated control plant, thus obtaining an improved fruit production. In certain embodiments, the methods further comprise (b) harvesting the fruit of the plant or a plant grown from the seed. In certain embodiments of the methods, the composition comprises Methylobacterium at a rate of about 1x106 CFU / g at about 1x1014 CFU / g for the solid-containing composition or at a rate of about 1x106 CFU / ml at about 1X1011 CFU / ml for the composition It contains emulsion. In certain embodiments, the composition is free of substances that promote the growth of resident bacteria on a plant or seed. In certain embodiments of the method, the fruit producing plant is a plant of
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Apple, pear, grape, Doric, melon, pepper, tomato, berry, kiwi, mango or banana. In certain embodiments, the berry plant is a blackberry, strawberry or blueberry plant. In certain embodiments of the method, the applied composition partially covers or covers the plant, a part thereof, or the seed. In certain embodiments, the Methylobacterium in the composition or that is used is selected from the group consisting of NLS0017 (NRRL B- 50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL B-50941 ), NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932), NLS0046 (NRRL B-50929),
NLS0062 (NRRL B-50937), NLS0064 (NRRL B-50938), NLS0065 (NRRL B-50935),
NLS0066 (NRRL B-50940), NLS0068 (NRRL B-50934), NLS0069 (NRRL B-50936),
NLS0089 (NRRL B-50933), and derivatives thereof. In certain embodiments, the Methylobacterium in the composition or that is used is selected from the group consisting of NLS0037 (NRRL b-50941), NLS0038 (NRRL b-50942), NLS0042 (NRRL b-50932),
NLS0062 (NRRL b-50937), and derivatives thereof. In certain embodiments, the Methylobacterium in the composition or that is used is selected from the group consisting of NLS0038 (NRRL b-50942), NLS0042 (NRRL b-50932), NLS0062 (NRRL b-50937), and derivatives thereof.
Methods for improving fruit production are also provided herein, the method comprising: (a) applying a composition comprising Methylobacterium to a fruit producing plant or seed, wherein the composition comprises a Methylobacterium sp. selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL b-50930), NLS0021 (NRRL b-50939), NLS0037 (NRRL b-50941), NLS0038 (NRRL b-50942), NLS0042 (NRRL b -50932), NLS0046 (NRRL B- 50929), NLS0062 (NRRL b-50937), NLS0064 (NRRL b-50938), NLS0065 (NRRL b-50935), NLS0066 (NRRL b-50940), NLS0068 (NRRL b-50934 ), NLS0069 (NRRL b-50936), NLS0089 (NRRL b-50933), and derivatives thereof and an adjuvant, excipient or combination thereof agriculturally acceptable. In certain embodiments, the methods further comprise (b) harvesting fruit from the plant or a plant grown from the seed, wherein the plant or plant grown from the seed exhibits a faster fruit set, fruit set increased fruit, earlier ripening, and / or more uniform fruit ripening compared to an untreated control plant, thereby obtaining an improved fruit production. In certain embodiments, the composition comprises Methylobacterium in a range of about 1x106 colony forming units per gram (CFU / g) of solid to about 1x1014 CFU / g of solid for a composition comprising a solid substance with adherent Methylobacterium grown thereon or at a rate of about 1x106 CFU / ml to about 1X1011 CFU / ml for a composition
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which comprises an emulsion that has Methylobacterium grown therein. In certain embodiments, the fruit producing plant is an apple, pear, grape, doric, melon, pepper, tomato, berry, kiwi, mango or banana plant. In certain embodiments, the composition is free of substances that promote the growth of resident microorganisms on the plant or seed. In certain embodiments, the Methylobacterium is selected from the group consisting of NLS0037 (NRRL B-50941), NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932), NLS0062 (NRRL B-50937), and derivatives thereof . In certain embodiments, the Methylobacterium is selected from the group consisting of NLS0038 (NRRL B-50942), NLS0042 (NRRL b-50932), NLS0062 (NRRL b-50937), and derivatives thereof. In certain embodiments, the composition partially covers or partially covers the plant or a part thereof, or the seed.
Methods are also provided herein to improve fruit production comprising: (a) applying to a fruit producing plant or seed a composition comprising Methylobacterium that is free of substances that promote the growth of resident bacteria on said plant or seed, where the plant or plant grown from the seed exhibits a faster fruit set, increased fruit set, earlier ripening, and / or more uniform fruit ripening compared to an untreated control plant, thus obtaining an improved fruit production. In certain embodiments, the methods further comprise (b) harvesting fruit from the plant or a plant grown from the seed. In certain embodiments of the method, the composition comprises a solid substance with adherent Methylobacterium cultured thereon, wherein the Methylobacterium title is about 1x106 CFU / g or about 1x1014 CFU / g, or comprises a liquid, a solid substance with attached Methylobacterium thereto in a liquid, a substance solid with Methylobacterium adhered thereto in an emulsion, or an emulsion having Methylobacterium grown therein in a titer of approximately 1x106 CFU / ml to approximately 1X1011 CFU / ml. In certain embodiments of the method, the fruit producing plant is an apple, pear, grape, doric, melon, pepper, tomato, berry, kiwi, mango or banana plant. In certain embodiments, the berry plant is a blackberry, strawberry or blueberry plant. In certain embodiments of the method, the applied composition partially covers or covers the plant, a part thereof, or the seed. In certain embodiments, the Methylobacterium in the composition or that is used is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939),
NLS0037 (NRRL b-50941), NLS0038 (NRRL b-50942), NLS0042 (NRRL b-50932),
NLS0046 (NRRL B-50929), NLS0062 (NRRL b-50937), NLS0064 (NRRL B-50938),
NLS0065 (NRRL B-50935), NLS0066 (NRRL B-50940), NLS0068 (NRRL B-50934),
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NLS0069 (NRRL B-50936), NLS0089 (NRRL B-50933), and derivatives thereof. In certain embodiments, the Methylobacterium in the composition or that is used is selected from the group consisting of NLS0037 (NRRL B-50941), NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932), NLS0062 (NRRL B-50937 ), and derivatives thereof. In certain embodiments, the Methylobacterium is selected from the group consisting of NLS0038 (NRRL b-50942), NLS0042 (NRRL b-50932), NLS0062 (NRRL b-50937), and derivatives thereof.
Methods for preparing a treatment composition of a plant or plant seed comprising Methylobacterium and free of substances that promote the growth of resident bacteria on a plant or seed are also provided herein. Such methods comprise (a) cultivating a mono-culture or co-culture of Methylobacterium in a medium comprising: (i) an aqueous phase; (ii) a liquid phase and a solid phase; or (iii) an emulsion, thereby obtaining a medium containing Methylobacterium; (b) separating the Methylobacterium from at least one other portion of the medium containing Methylobacterium; and (c) reconstitute Methylobacterium into a matrix that lacks substances that promote the growth of resident bacteria on a plant or seed. In certain embodiments, the separation step is carried out by centrifugation, filtration or sedimentation of the medium containing Methylobacterium and removal of excess liquid or emulsion thereof. In certain embodiments, the substance that promotes the growth of resident bacteria on a plant or seed is selected from the group consisting of a carbon source, a source of nitrogen, a source of phosphorus, a source of sulfur, a source of magnesium, and combinations thereof. In certain embodiments, the matrix is a liquid, an emulsion, or one or more solids, and comprises an adjuvant and / or excipient acceptable from the agricultural point of view. In certain embodiments; The Methylobacterium are grown in media comprising a liquid phase and a solid substance with adherent Methylobacterium grown thereon, the solid substance is separated from the liquid phase of the medium containing Methylobacterium, and the solid substance with Adherent Methylobacterium grown thereon is reconstituted in the matrix mentioned above. In certain embodiments, the solid substance with adherent Methylobacterium is not a substance that promotes the growth of resident microorganisms on a plant, plant part or plant seed. In certain embodiments, the Methylobacterium in the composition or that is used is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL b-50941 ), NLS0038 (NRRL b-50942), NLS0042 (NRRL b-50932), NLS0046 (NRRL B-50929), NLS0062 (NRRL b-50937), NLS0064 (NRRL B-50938), NLS0065 (NRRL B-50935), NLS0066 (NRRL B-50940), NLS0068 (NRRL B-50934),
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NLS0069 (NRRL B-50936), NLS0089 (NRRL B-50933), and derivatives thereof. In certain embodiments, the Methylobacterium in the composition or that is used is selected from the group consisting of NLS0037 (NRRL B-50941), NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932), NLS0062 (NRRL B-50937 ), and derivatives thereof. In certain embodiments, the Methylobacterium is selected from the group consisting of NLS0038 (NRRL b-50942), NLS0042 (NRRL b-50932), NLS0062 (NRRL b-50937), and derivatives thereof.
A method of treating a plant or seed with a composition comprising Methylobacterium and free of substances that promote the growth of resident bacteria on a plant or seed is provided herein. Such method comprises: (a) preparing a composition containing Methylobacterium which is free of substances that promote the growth of resident bacteria on a plant or seed according to any of the methods mentioned above; and, (b) apply the composition to a plant or seed. In certain embodiments of the methods, the composition is applied to the plant or a part thereof or seed as a pulverization, or said composition is applied to said plant, part thereof or seed by immersion. In certain embodiments of the methods, the applied composition partially covers or covers the plant, the part thereof or the seed. In certain embodiments of the methods, the solid substance with adherent Methylobacterium is not a substance that promotes the growth of resident microorganisms on a plant or seed. In certain embodiments of the methods, the composition comprises an adjuvant and / or excipient acceptable from the agricultural point of view. In certain embodiments of any of the methods mentioned above, the composition comprises a solid substance with adherent Methylobacterium cultured thereon, wherein the Methylobacterium rhythm is about 1x106 CFU / g or about 1x1014 CFU / g or comprises a liquid, a solid substance with Methylobacterium adhered thereto in a liquid, a substance solid with Methylobacterium adhered thereto in an emulsion, or an emulsion having Methylobacterium grown therein in a range of about 1x106 CFU / ml to about 1X1011 CFU / ml. Also provided are parts of plants or plant seeds that are coated or partially coated with a composition comprising Methylobacterium that is free of substances that promote the growth of resident microorganisms on a plant or seed, where parts of plants or plant seeds they are obtained by any of the methods mentioned above. In certain embodiments, the Methylobacterium in the composition or that is used is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL b-50941 ), NLS0038 (NRRL b-50942), NLS0042 (NRRL
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B-50932), NLS0046 (NRRL B-50929), NLS0062 (NRRL B-50937), NLS0064 (NRRL B- 50938), NLS0065 (NRRL B-50935), NLS0066 (NRRL B-50940), NLS0068 (NRRL B- 50934), NLS0069 (NRRL B-50936), NLS0089 (NRRL B-50933), and derivatives thereof. In certain embodiments, the Methylobacterium in the composition or that is used is selected from the group consisting of NLS0037 (NRRL B-50941), NLS0038 (NRRL B-50942), NLS0042 (NRRL b-50932), NLS0062 (NRRL b-50937 ), and derivatives thereof.
Methods for treating a plant or seed with Methylobacterium are provided herein. Such methods comprise applying to a plant or seed a composition comprising a solid substance with adherent Methylobacterium grown thereon and free of substances that promote the growth of resident microorganisms on a plant or seed. In certain embodiments, the composition is applied to the plant or a part thereof as a spray, or the composition is applied to the plant, a part thereof or seed by immersion. In certain embodiments, the applied composition partially covers or covers the plant, the part thereof, or the seed. In certain embodiments, the substance solid with Methylobacterium is not a substance that promotes the growth of resident microorganisms on a plant or seed. In certain embodiments, the composition comprises an adjuvant and / or excipient acceptable from the agricultural point of view. In certain embodiments of the methods, the applied composition partially covers or covers the plant, the part thereof, or the seed. In certain embodiments of the methods, the solid substance with adherent Methylobacterium is not a substance that promotes the growth of resident microorganisms on a plant or seed. In certain embodiments of any of the methods mentioned above, the composition comprises a solid substance with adherent Methylobacterium cultured thereon, wherein the Methylobacterium rhythm is about 1x106 CFU / g or about 1x1014 CFU / g or comprises a liquid, a solid substance with Methylobacterium adhered thereto in a liquid, a substance solid with Methylobacterium adhered thereto in an emulsion, or an emulsion having Methylobacterium grown therein in a range of about 1x106 CFU / ml to about 1X1011 CFU / ml. Also provided are parts of plants or plant seeds that are coated or partially coated with a composition comprising Methylobacterium that is free of substances that promote the growth of resident microorganisms on a plant or seed, where the parts of plants or plant seeds they are obtained by any of the methods mentioned above. In certain embodiments, the Methylobacterium in the composition or that is used is selected from the group consisting of NLS0017 (NRRL B- 50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL b-50941 ),
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NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932), NLS0046 (NRRL B-50929),
NLS0062 (NRRL B-50937), NLS0064 (NRRL B-50938), NLS0065 (NRRL B-50935),
NLS0066 (NRRL B-50940), NLS0068 (NRRL B-50934), NLS0069 (NRRL B-50936),
NLS0089 (NRRL B-50933), and derivatives thereof. In certain embodiments, the Methylobacterium in the composition or that is used is selected from the group consisting of NLS0037 (NRRL B-50941), NLS0038 (NRRL b-50942), NLS0042 (NRRL b-50932),
NLS0062 (NRRL b-50937), and derivatives thereof.
A composition comprising a solid substance with adherent Methylobacterium grown thereon is also provided herein, wherein the composition is free of substances that promote the growth of resident microorganisms on a plant or seed. In certain embodiments, the adherent Methylobacterium are in a range of at least about 1 x 106 CFU / g to at least about 1 x 1014 CFU / g. In certain embodiments, the composition is a solid composition and the Methylobacterium are in a range of about 1x106 CFU / g to about 1x1014 CFU / g. In certain embodiments, the adherent Methylobacterium in the solid composition are in a range of at least about 5 x 108 CFU / g to at least about 5 x 1013 CFU / g. In certain embodiments, the composition is a liquid or an emulsion containing the solid substance and the Methylobacterium are in a range of about 1x106 CFU / ml to about 1X1011 CFU / ml. In certain embodiments, the Methylobacterium in the liquid or emulsion is in a range of about 5 x 108 CFU / ml to about 1X1011 CFU / ml. In certain embodiments, the adherent Methylobacterium are in a range of at least about 5 x 108 CFU / g to at least about 1 x 1014 CFU / g and the composition is free of substances that promote the growth of resident microorganisms on a plant or seed. In certain embodiments, the substance that promotes the growth of resident microorganisms on a plant or seed is selected from the group consisting of a carbon source, a source of nitrogen, a source of phosphorus, a source of sulfur, a source of magnesium, and combinations thereof. In certain embodiments, the composition comprises an adjuvant and / or excipient acceptable from the agricultural point of view. In certain embodiments of any of the above-mentioned compositions, Methylobacterium is strain NLS0038, NLS0046, NLS0020, NLS0017, NLS0042, NLS0089, NLS0068, NLS0065, NLS0069, NLS0062, NLS0064, NLS0021, NLS0066, or NLS0037. In certain embodiments, the Methylobacterium in the composition or that is used is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL b-50941 ), NLS0038 (NRRL b-50942), NLS0042 (NRRL b-50932),
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NLS0046 (NRRL B-50929), NLS0062 (NRRL B-50937), NLS0064 (NRRL B-50938),
NLS0065 (NRRL B-50935), NLS0066 (NRRL B-50940), NLS0068 (NRRL B-50934),
NLS0069 (NRRL B-50936), NLS0089 (NRRL B-50933), and derivatives thereof. In certain embodiments, the Methylobacterium in the composition or that is used is selected from the group consisting of NLS0037 (NRRL B-50941), NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932), NLS0062 (NRRL b-50937 ), and derivatives thereof. In certain embodiments of any of the above-mentioned compositions, the solid substance with adherent Methylobacterium grown thereon is not a substance that promotes the growth of resident microorganisms on a plant or seed. In certain embodiments of any of the compositions mentioned above, the composition is adapted for use in the treatment of a plant or seed. Also provided herein is a plant part or plant seed that is partially coated or coated with any of the above-mentioned compositions. Methods for treating plants and / or parts of plants with the compositions are also provided herein. Treated plants, treated plant parts thereof, include, but are not limited to, corn, Brassica sp. (eg, B. napus, B. rapa, B. juncea), alfalfa, rice, rye, sorghum, millet (eg, pearl millet (Pennisetum glaucum), prose millet (Panicum miliaceum), millet foxtail (Setaria italica), finger millet (Eleusine coracana)), sunflower, safflower, soy, tobacco, potato, peanuts, cotton, sweet potato (Ipomoea batatus), cassava, coffee, coconut, pineapple, doric trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, sugar beets, sugar cane, oatmeal, barley, tomatoes, lettuce, green beans, Lima beans, chickpeas, cucurbitaceae such as cucumber, cantaloupe and musk melon, ornamental, and commens. The parts of the plants that are treated include, but are not limited to, leaves, stems, flowers, rates, seeds, fruit, tubers, coleoptils and the like. Ornamental plants and parts of plants that can be treated include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia and chrysanthemum. Common plants and plant parts that can be treated include, but are not limited to, pines such as taeda pine, ellioti pine, ponderosa pine, contorta pine and Monterrey pine; Douglas fir; Paduga Tsuga; Sitka spruce; sequoia true firs such as bianco fir and balsam fir; and cedars such as western red cedar and yellow Alaska cedar. Lawn plants and plant parts that can be treated include, but are not limited to, annual blue grass, annual English grass, Canada blue grass, fescue, agrostis, wheat grass, Kentucky blue grass, ball grass, English grass , agrostis gigantea, Bermuda grass, St. Agustm grass, and zoysia grass. Seeds or other propangles of any of the plants mentioned above may be treated with the compositions provided in the
present memory In certain embodiments, the Methylobacterium in the composition or that is used is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B- 50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL B-50941 ), NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932), NLS0046 (NRRL B-50929), NLS0062 (NRRL B-50937),
5 NLS0064 (NRRL B-50938), NLS0065 (NRRL B-50935), NLS0066 (NRRL B-50940),
NLS0068 (NRRL B-50934), NLS0069 (NRRL B-50936), NLS0089 (NRRL B-50933), and derivatives thereof. In certain embodiments, the Methylobacterium in the composition or that is used is selected from the group consisting of NLS0037 (NRRL b-50941), NLS0038 (NRRL b-50942), NLS0042 (NRRL b-50932), NLS0062 (NRRL b-50937 ), and derivatives thereof.
Description
Definitions
As used herein, the phrases "adhered thereto" and "adherent" refer to Methylobacterium that are associated with a solid substance per culture, or that have been cultivated, on a solid substance.
As used herein, the phrase "agriculturally acceptable adjuvant" refers to a substance that increases the yield of an active agent in a composition for treatment of plants and / or parts of plants. In certain compositions, an active agent may comprise a mono-culture or co-culture of Methylobacterium.
As used herein, the phrase "agriculturally acceptable excipient" refers to an essentially inert substance that can be used as a diluent and / or vehicle for an active agent in a plant treatment composition and / or parts of plants. In certain compositions, an active agent may comprise a mono-culture or co-culture of Methylobacterium.
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 encompass species in the genus Methylobacter, Methylomonas, Methylomicrobium , Methylococcus, 30 Methylosinus, Methylocystis, Methylosphaera, Methylocaldum, and Methylocella, which are obligate methanotrophs.
As used herein, the phrase "co-culture of Methylobacterium" refers to a culture of Methylobacterium comprising at least two strains of
Methylobacterium or at least two species of Methylobacterium.
As used herein, the phrase "contaminating microorganism" refers to microorganisms in a culture, fermentation broth, fermentation broth product or composition that were not identified before introduction into the culture, 5 fermentation broth, product of fermentation broth or composition.
As used herein, the phrase "derivatives thereof", when used in the context of a strain of Methylobacterium, refers to any strain that is obtained from the strain of Methylobacterium. Derivatives of a strain of Methylobacterium include, but are not limited to, variants of the strain obtained by selection, variants of the strain selected by mutagenesis and selection, and genetically transformed isolates obtained from the strain of Methylobacterium.
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.
As used herein, the phrase "essentially free of contaminating microorganisms" refers to a culture, fermentation broth, fermentation product or composition where at least about 95% of the microorganisms present by quantity or type in the culture, fermentation broth, fermentation product 20 or composition are the desired Methylobacterium or other desired microorganisms of predetermined identity.
As used herein, the phrase "inanimate solid 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 an organism still alive. from which it was derived.
As used herein, the phrase "mono-culture of Methylobacterium" refers to a culture of Methylobacterium consisting of a single strain of Methylobacterium.
As used herein, the term "peptide" refers to any polypeptide of 50 amino acid residues or less.
As used herein, the term "pepper" refers to Capsicum sp. Capsicum sp. include, but are not limited to, C. annuum, C. baccatum,
C. chinense, C. frntescens, and C. pubescens.
As used herein, the term "protema" refers to any polypeptide having 51 or more amino acid residues.
As used herein, a "pesticide" refers to an agent that is insecticidal, fungicidal, nematocidal, bactericidal, or any combination thereof.
As used herein, the phrase "bacteriostatic agent" refers to agents that inhibit the growth of bacteria but do not kill bacteria.
As used herein, the phrase "pesticide does not substantially inhibit the growth of said Methylobacterium" refers to any pesticide which, when provided in a composition comprising a fermentation product comprising a solid substance wherein a monkey -cultivation or co-cultivation of Methylobacterium is adhered to it, results in no more than 50% inhibition of growth of Methylobacterium when the composition is applied to a plant or part of plant compared to a composition lacking the pesticide . In certain embodiments, the pesticide results in no more than 40%, 20%, 10%, 5%, or 1% inhibition of growth of Methylobacterium when the composition is applied to a plant or part of the plant compared to a composition that lacks the pesticide.
As used herein, the term "PPFM bacteria" refers without limitation to bacterial species in the genus Methylobacterium other than M. nodulans.
As used herein, the phrase "solid substance" refers to a substance that is insoluble or partially soluble in water or aqueous solutions.
As used herein, the phrase "solid phase that can be suspended therein" refers to a solid substance that can be distributed throughout a liquid by agitation.
25 As used herein, the term "non-regenerable" refers either to a plant part or a processed plant product that cannot be regenerated in an entire plant.
As used herein, the phrase "substantially the entire solid phase is suspended in the liquid phase" refers to media where at least 95%, 98%, or 99% of solid substance (s) which comprise the solid phase are distributed throughout the liquid by stirring.
As used herein, the phrase "substantially the entire solid phase does not
it is suspended in the Kquida phase ”refers to media where less than 5%, 2%, or 1% of the solid is in a form of particles that is distributed throughout the medium by agitation.
As used herein, the phrase "resident microorganism" refers to resident bacteria, fungi or yeasts.
5 As used herein, the phrase "substance that promotes the growth of resident microorganisms on a plant or seed" refers to a source of carbon, a source of nitrogen, a source of phosphorus, and combinations thereof.
To the extent that any of the foregoing definitions is inconsistent with definitions provided in any patent or non-patent reference incorporated herein or by reference, any patent reference or non-patent reference cited herein, or in any reference to patent or non-patent found elsewhere, it is understood that the foregoing definition will be used herein.
Compositions containing Methylobacterium free of substances that promote the growth of resident bacteria on a plant or seed, methods of use, and methods of preparation
Compositions comprising Methylobacterium that are free of substances that promote the growth of resident bacteria on a plant or seed, methods for using the compositions to improve fruit production, and methods for preparing the compositions are provided herein. In certain embodiments of any of the above-mentioned compositions, the composition comprises a solid substance wherein a mono-culture or co-culture of Methylobacterium is adhered thereto. In certain embodiments where the Methylobacterium is adhered to a solid substance, the composition comprises a colloid formed by the solid substance wherein a mono-culture or co-culture of Methylobacterium is adhered thereto 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.
Compositions comprising a solid substance with adherent Methylobacterium grown thereon are provided. In certain embodiments, the adherent Methylobacterium may be in a titer of at least about 5 x 108 CFU / g at least about 5 x 1013 CFU / g or about 1 x 1014 CFU / g and the composition is free of substances that promote the growth of microorganisms
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residents on a plant or seed.
In certain embodiments, compositions containing Methylobacterium provided or used herein are exempt from substances that promote the growth of resident microorganisms when one or more of those substances are absent or essentially absent. In certain embodiments, the composition is free of substances that promote the growth of resident microorganisms when those substances are present in a percentage of no more than about 5%, 2%, 1%, 0.5%, 0.2%, or 0.1% of the total mass, mass / total volume, or total volume of the composition. In certain embodiments, the substance that promotes the growth of resident microorganisms on a plant or seed is selected from the group consisting of a carbon source, a source of nitrogen, a source of phosphorus, a source of sulfur, a source of magnesium, and combinations thereof. Carbon sources include, but are not limited to, alcohols, monosaccharides, disacarids, polysaccharides, Kpids, fatty acids and the like. Alcohols that are exempt include, but are not limited to, methanol, ethanol, glycerol and the like. Sources of nitrogen include, but are not limited to, ammonia and various compounds that contain amino groups that can be metabolized by microorganisms. In certain embodiments, the substance that is exempt is a source of two or more of a carbon source, a source of nitrogen, a source of phosphorus, a source of sulfur and a source of magnesium. For example, the composition that is free of amino acids or peptides and lacks other carbon or nitrogen sources is exempt from both a carbon and nitrogen source. In certain embodiments, the composition comprises an adjuvant and / or excipient acceptable from the agricultural point of view.
Resident microorganisms on the plant or seed include, but are not limited to, bacteria, fungi and yeasts. Substances that promote the growth of such microorganisms can be identified by methods that include, but are not limited to, testing the surface of the plant or seed as to the amount or number of microorganisms present before the exposure of the plant or seed to the substance (or a composition containing the substance), expose the tested plant or seed to the substance or composition in parallel with a control composition lacking the substance, and then re-test the surface of the plant or seed as to the amount or number of microorganisms present after an adequate time interval and under suitable temperature conditions to allow the growth of resident microorganisms. Assays for numbers of microorganisms include, but are not limited to, determinations of colony forming units by an amount of plant or seed
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exposed to substance and control.
Without seeking to be limited by theone, it is believed that compositions containing Methylobacterium provided or used herein that are free of substances that promote the growth of resident microorganisms can result in superior results compared to other compositions containing such substances when applied to plants, parts of plants or seeds. It is believed that such superior results include, but are not limited to, improved plant yield, improved fruit ripening and the like. Although it is not sought to be limited by theory, it is believed that compositions containing Methylobacterium that are free of substances that promote the growth of resident microorganisms allow a more effective and extensive colonization of the plant, part thereof, or seed, since competition for one or more of the space or nutrients by resident microorganisms is reduced.
Methods for improving fruit production are also provided herein which comprise applying any of the aforementioned compositions provided herein to a fruit producing plant, plant part, or seed, and harvesting fruit from the plant or a plant grown from the seed. In certain embodiments, the composition partially covers or covers the fruit producing plant, part of the plant or seed. The plant or plant grown from the seed exhibits a faster fruit set, increased fruit set, earlier ripening, and / or produces more ripe fruit compared to an untreated control plant, thereby obtaining An improved fruit production. In certain embodiments, the fruit that is produced by the methods and compositions provided herein exhibits a more uniform ripening of the fruit in which more fruit matures in a shorter period of time compared to the fruit produced by a control plant. untreated The increased uniformity of fruit ripening provided herein may allow the harvest of a fruit that is more uniform in its ripeness, such that less immature, inflated, and / or overripe fruit is harvested compared to the fruit harvested from an untreated control plant. In certain embodiments, the application of the compositions provides about 5% or 10% to about 15%, 20%, 25%, 30%, 35%, 50%, 60%, 75% or 80% increase in the number of ripened fruit present for a given period of time in the treated plant, part of the plant, or a plant derived therefrom compared to the untreated control plant, part of the plant, or plant obtained from them. In certain embodiments, the application of the compositions provides approximately a
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5% or 10% to approximately 15%, 20%, 25%, 30%, 35%, 50%, 60%, 75% or 80% decrease in the number of unripe, infused, and / or overripe fruit harvested of the treated plant, part of the plant, or a plant derived therefrom compared to the untreated control plant, part of the plant, or plant obtained therefrom. In certain embodiments, the application of the compositions provides at least about 5%, 10%, 15% or 20% decrease in time for fruit set or for ripening of the fruit in the plant, part of the plant , or a plant derived therefrom compared to the untreated control plant, part of the plant, or plant obtained therefrom. In certain embodiments, the application of the composition provides about 5% or 10% to about 15%, 20%, 25%, 30%, or 35% increase in fruit set or ripe fruit production. in the plant, part of the plant, or a plant derived therefrom compared to the untreated control plant, part of the plant, or plant obtained therefrom. In certain embodiments, the application of the composition provides at least about 5%, 10%, 15% or 20% decrease in time for fruit set or ripening of the fruit in the plant, part of the plant, or a plant derived therefrom compared to the untreated control plant, part of the plant, or plant obtained therefrom. In certain embodiments, the application of the composition provides about 5% or 10% to about 15%, 20%, 25%, 30%, or 35% increase in fruit set or ripe fruit production in the plant, part of the plant, or a plant derived therefrom compared to the untreated control plant, part of the plant, or plant obtained therefrom. In certain embodiments, the fruit-producing plant is an apple, pear, grape, doric, melon, pepper, tomato, berry, kiwi, mango or banana plant, and the plant part is a leaf, a stem, a flower, a root, a tuber or a seed. In certain embodiments, the berry plant is a blackberry, strawberry or blueberry plant. In certain embodiments, the plant is a pepper plant. In certain embodiments, the method further comprises the stages of growing the plant and / or the stage of harvesting at least a part of the plant selected from the group consisting of a leaf, a stem, a flower, a root, a tuber or a seed of the plant or part of the plant. In certain embodiments of any of the above methods, the methods further comprise obtaining a processed food or feed composition from the plant or part of the plant. In certain embodiments, the processed food or feed composition is a flour or a paste.
Methods are also provided for preparing a plant or plant treatment composition comprising Methylobacterium and free of substances that promote the growth of resident bacteria on a plant or seed. Such method
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it comprises (i) cultivating a mono-culture or co-culture of Methylobacterium in a medium comprising an aqueous phase, a liquid phase and a solid phase, or an emulsion, thereby obtaining a medium containing Methylobacterium; (ii) separating the Methylobacterium from at least one other portion of the medium containing Methylobacterium; and (iii) reconstitute Methylobacterium into a matrix that lacks substances that promote the growth of bacteria resident in a plant or seed. In certain embodiments, the separation step is carried out by centrifugation, filtration or sedimentation of the medium containing Methylobacterium and removal of excess liquid or emulsion thereof. In certain embodiments, the substance that promotes the growth of resident bacteria on a plant or seed is selected from the group consisting of a carbon source, a source of nitrogen, a source of phosphorus, and combinations thereof. In certain embodiments, the matrix is a liquid, an emulsion, or one or more solids, and comprises an adjuvant and / or excipient acceptable from the agricultural point of view. Even in certain embodiments; Methylobacterium are grown in media comprising a liquid phase and a solid substance with adherent Methylobacterium grown thereon. The solid substance is separated from the liquid phase of the medium containing Methylobacterium, and the solid substance with adherent Methylobacterium grown thereon is reconstituted in the matrix mentioned above. In certain embodiments of the methods, Methylobacterium sp., Is selected from the group consisting of M. aminovorans, M. extorquens, M. fujisawaense, M. mesophilicum, M. radiotolerans, M. rhodesianum, M. nodulans, M. phyllosphaerae , M thiocyanatum, and M. oryzae. In certain embodiments of the methods, Methylobacterium is not M. radiotolerans or M. oryzae. In certain embodiments of the methods, the Methylobacterium is adhered to a solid substance. In certain embodiments of the methods, the Methylobacterium that is adhered to the solid substance is combined with a liquid to form a composition that is a colloid. In certain embodiments of the methods, the colloid is a gel. In certain embodiments of the methods, the Methylobacterium adhered to the solid substance is provided by culturing the Methylobacterium in the presence of the solid substance. In certain embodiments of the methods, the composition comprises an emulsion. In certain embodiments of the methods, the Methylobacterium is provided by culturing the Methylobacterium in an emulsion.
It has been found that methods where Methylobacteriums are grown in biphasic media comprising a liquid phase and a solid substance significantly increase the resulting yield of Methylobacterium relative to methods where Methylobacteriums are grown in liquid media alone. In certain embodiments, the methods may comprise culturing the Methylobacterium in liquid media with a solid substance in particles that can be suspended in the liquid by low agitation.
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conditions that provide the growth of Methylobacterium. In certain embodiments where solid substances are used in particles, at least substantially the entire solid phase can be suspended in the liquid phase after stirring. Such particulate solids may comprise materials that are approximately 1 millimeter or less in length or diameter. In certain embodiments, the degree of agitation is sufficient to provide a uniform distribution of the solid substance in particles in the liquid phase and / or optimal 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 solid phase. liquid phase. Solid substances not in the form of particles can be used in certain two-phase media where the solid phase is not suspended in the liquid phase. Such solid substances not in the form of particles include, but are not limited to, materials that are greater than about 1 millimeter in length or diameter. Such solid substances in the form of particles and not in the form of particles also include, but are not limited to, materials that are porous, fibrous, or otherwise configured to provide increased surface areas for adherent growth of the Methylobacterium. Two-phase means where portions of the solid phase are suspended in the liquid phase and portions of the solid phase are not suspended in the liquid phase may comprise a mixture of solid substances in the form of particles and not in the form of particles. Such solid substances in the form of particles and not in the form of particles used in any of the biphasic media mentioned above also include, but are not limited to, materials that are porous, fibrous, or otherwise configured to provide increased surface areas for Adherent growth Methylobacterium. In certain embodiments, the medium comprises a colloid formed by a solid and a liquid phase. A colloid comprising a solid and a liquid may be preformed and added to a liquid medium or it may be formed in a medium containing a solid and a liquid. Colloids comprising a solid and a liquid can be formed by subjecting certain solid substances to a 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, but are not limited to, 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 comprising an alcohol, an aldehyde, a ketone, a fatty acid, a phospho Kido, or any combination thereof; (3) selected alcohols
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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
combination thereof; and / or, (5) a vegetable oil that is selected from the group that
It consists of corn, soy, cotton, peanut, sunflower, olive, flax, coconut, palm, rapeseed, sesame, safflower, and combinations thereof. In certain embodiments, the immiscible or partially immiscible liquid may comprise at least about 0.02% at
approximately 20% of the mass liquid phase. In certain embodiments, the methods may comprise obtaining a two-phase culture medium comprising the Kido, the solid, and Methylobacterium and incubating the culture under conditions that provide the growth of Methylobacterium. Biphasic culture media comprising Kido, solid, and Methylobacterium can be obtained by various methods that include, but are not limited to, any of: (a) inoculating a biphasic medium comprising the liquid and the substance solid with Methylobacterium; (b) inoculate the solid substance with Methylobacterium and then introduce the solid substance comprising the
Methylobacterium in the liquid medium; (c) inoculate the solid substance with Methylobacterium, incubate the Methylobacterium on the solid substance, and then introduce the solid substance comprising the Methylobacterium into the liquid medium; or (d) any combination of (a), (b), or (c). Methods and compositions for cultivating Methylobacterium in biphasic media comprising a liquid and a solid are described in US patent application. assigned with the present N0 13 / 907,161, filed on May 31, 2013, which is incorporated herein by reference in its entirety, and in the international patent application assigned with this PCT / US13 / 43722, filed on 31 May 2013, which is incorporated herein by reference in its entirety.
It has also been found that methods where Methylobacterium are grown in media comprising an emulsion significantly increase the resulting yield of Methylobacterium in relation to methods where Methylobacterium is grown in liquid media alone. In certain embodiments, the methods for preparing the compositions provided herein may comprise culturing the Methylobacterium agent in an emulsion under conditions that provide the growth of Methylobacterium. Means comprising the emulsion and Methylobacterium can be obtained by various methods that include, but are not limited to, any of:
(a) inoculate a medium comprising the emulsion with Methylobacterium; (b) inoculate the aqueous liquid with the Methylobacterium, introduce the non-aqueous liquid, and mix to form an emulsion; (c) inoculate the aqueous liquid with the 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.
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which is not miscible, or only partially miscible, in the aqueous Kido. Non-aqueous liquids that are not miscible, or only partially miscible, in water include, but are not limited to, 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 comprising an alcohol, an aldeWdo, a ketone, a fatty acid, a phospho Kpido, or any combination thereof; (3) alcohols that are 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 combination thereof; and / or, (5) a vegetable oil that is selected from the group consisting of corn, soy, cotton, peanut, sunflower, olive, flax, coconut, palm, rapeseed, sesame, safflower, 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 bulk 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. Methods and compositions for cultivating Methylobacterium in media comprising an emulsion are described in the international patent application assigned with this PCT / US2014 / 040218, filed May 30, 2014, which is incorporated herein by reference in its entirety .
In certain embodiments, the fermentation broth, fermentation broth product, or compositions comprising Methylobacterium sp. they may also comprise one or more introduced 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 part of the plant. Biopesticidal or otherwise beneficial microorganisms include as ^ but are not limited to, various Bacillus sp., Pseudomonas sp., Coniothyrium sp., Pantoea sp., Streptomyces sp., And Trichoderma sp. Microbial biopesticides can be a bacterium, fungus, virus or protozoan. Particularly useful biopesticidal microorganisms include various strains of Bacillus subtilis, Bacillus thuringiensis, Bacillus pumilis, Pseudomonas syringae, Trichoderma harzianum, Trichoderma virens, and Streptomyces lydicus. Other microorganisms that are added may be isolates treated by genetic or wild-type engineering that are available as pure cultures. In certain embodiments, it is envisioned that the bacterial or fungal microorganism may be provided in the fermentation broth, fermentation broth product, or composition in the form of a spore.
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In certain embodiments, the Kido liquid culture medium is prepared from cheap and readily available components, including, but not limited to, inorganic salts such as potassium phosphate, magnesium sulfate and the like, carbon sources such as glycerol, methanol, glutamic acid, aspartic acid, sucdnic acid and the like, and mixtures of amino acids such as peptone, tryptone and the like. Illustrative liquid media that may be used include, but are not limited to, ammonium mineral salts (AMS) medium (Whittenbury et a /., 1970), minimal Vogel-Bonner (VB) culture medium (Vogel and Bonner, 1956), and LB broth ("Luria-Bertani Broth").
The solid substance used in the methods and compositions that provide effective growth of Methy / obacterium 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-bactericidal or non-bacteriostatic with respect to Methy / obacterium when the solid substances are provided in the liquid culture media. 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 herein can be sterilized by any method that provides for the removal of contaminating microorganisms, and therefore include, but are not limited to, methods such as autoclaving, irradiation, chemical treatment, and any combination of the same. These solid substances include natural substances of animal, plant, microbial, fungic or mineral origin, man-made substances, or combinations of natural and man-made substances. In certain embodiments, solid substances are inanimate solid substances. Inanimate solid substances of animal, plant, microbial or fungal origin can be obtained from animals, plants, microbes or fungi that are unviable (that is, do not live longer) or that have been made unfeasible. Diatomaceous shells are thus inanimate solid substances when associated diatomaceous algae have previously been removed or otherwise made unfeasible. Since diatom shells are inanimate solid substances, they are not considered to be photosynthetic organisms or photosynthetic microorganisms. In certain embodiments, solid substances include, but are not limited to, sand, silt, earth, clay, ash, charcoal, diatomaceous earth and other similar minerals, ground glass or glass beads, ground ceramic materials, ceramic beads, Bentonite, caolm, talc, perlite, mica, vermiculite, sflices, quartz powder, montmorillonite, and combinations thereof. In certain embodiments, the solid substance may be a polymer or polymeric beads. Polymers that can be used as a solid substance include, but are not limited to, various polysaccharides such as cellulosic polymers and chitinous polymers that are insoluble or only partially soluble in water or aqueous solutions, agar (eg.
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galactans), and combinations thereof. In certain embodiments, the solid substance may be an insoluble or only partially soluble salt crystal. Salt crystals that may be used include, but are not limited to, carbonates, chromates, sulphites, phosphates, hydroxides, oxides, and insoluble or only partially soluble sulfides. In certain embodiments, the solid substance may be a microbial cell, fungal cell, microbial spore or fungal spore. In certain embodiments, the solid substance may be a microbial cell or microbial spore where the microbial cell or microbial spore is not a photosynthetic microorganism. In certain embodiments, the microbial cell or microbial spore is not a photosynthetic microorganism, where the photosynthetic microorganism is selected from the group consisting of algae, cyanobacteria, diatoms, Botryococcus braunii, Chlorella, Dunaliella tertiolecta, Gradlaria, Pleumchrysis portfolio, Sargassum, and Ulgassum, and Ulgassum . In still other embodiments, the solid substance may be a microbial cell, fungal cell, microbial spore or inactivated fungal spore (i.e., unviable). In still other embodiments, the solid substance may be a microbial cell, fungal cell, microbial spore or quiescent fungal spore (i.e., viable but not actively divided). In still other embodiments, the solid substance may be cellular debris of microbial origin. In still other embodiments, the solid substance may be particulate matter of any part of a plant. Parts of plants that can be used to obtain the solid substance include, but are not limited to, cobs, husks, pods, leaves, rates, flowers, stems, barks, seeds, and combinations thereof. Products obtained from parts of processed plants that include, but are not limited to, bagasse, wheat fiber, soybean meal, crushed seed paste, stubble and the like can also be used. Such parts of plants, processed plants, and / or parts of processed plants can be ground to obtain the solid material in a particle form that can be used. In certain embodiments, wood or a wood product may be used that includes, but is not limited to, wood pulp, sawdust, chips and the like. In certain embodiments, the solid substance may be a particulate matter of an animal (s), which includes, but is not limited to, bone meal, jelly, ground or powdered shells, hair, macerated skin and the like. .
In certain embodiments, the solid substance is provided in a particulate form that provides the distribution of the solid substance in the culture medium. In certain embodiments, the solid substance is comprised of particles of about 2 micrometers to about 1,000 micrometers of average length or average diameter. In certain embodiments, the solid substance is comprised of partroules of about 1 micrometer to about 1,000 micrometers of average length or average diameter. In certain embodiments, the solid substance is a particle of
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approximately 1, 2, 4, 10, 20, or 40 micrometers to any of about 100, 200, 500, 750, or 1,000 micrometers of medium length or average diameter. Desirable characteristics of the particles used in the methods and compositions provided herein include adequate wettability, such that the particles can be suspended throughout the medium after agitation.
In certain embodiments, the solid substance is provided in the medium as a colloid where the continuous phase is a liquid and the dispersed phase is the solid. Suitable solids that can be used to form colloids in liquid media used to grow Methylobacterium include, but are not limited to, various solids that are called hydrocolloids. Such hydrocolloids used in the media, methods and compositions provided herein may be hydrophilic polymers, of plant, animal, microbial or synthetic origin. The hydrocolloidal polymers used in the methods may contain many hydroxyl groups and / or may be polyelectrolytes. Hydrocolloidal polymers used in the compositions and methods provided herein include, but are not limited to, agar, alginate, arabinoxylan, carrageenan, carboxymethyl cellulose, cellulose, curdlana, gelatin, gelana, p-glucan, guar gum, arabic gum, gum garrofm, pectin, starch, xanthan gum and mixtures thereof. In certain embodiments, the colloid used in the media, methods and compositions provided herein may comprise a hydrocolloidal polymer and one or more proteins.
In certain embodiments, the solid substance may be a solid substance that provides an adherent growth of Methylobacterium on the solid substance. Methylobacterium that are adhered to a solid Methylobacterium substance that cannot be substantially removed by simply washing the solid substance with the adherent Methylobacterium with culture medium, while non-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 are removed when the solid substance is washed with three volumes of culture medium liquid.This washing can be carried out by various methods that include, but are not limited to, decanting liquid from a washed solid phase or passing liquid through a solid phase on a filter that allows the flow through bacteria into the In certain embodiments, the adherent Methylobacterium that are associated with the solid may include both Methylobacterium that are directly bound to the solid and / or Methylobacterium that are indirectly bound to the solid substance. Methylobacterium that are indirectly bound to the solid substance include, but They are not limited to,
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Methylobacterium that are bound to other Methylobacterium or to another microorganism that is bound to the solid substance, Methylobacterium that are bound to the solid substance being bound to another substance that is bound to the solid substance, and the like. In certain embodiments, 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 that are adhered to the solid substance. In certain embodiments, adherent Methylobacterium may 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  square micrometers, of at least about 1
 Methylobacterium / 10  square micrometers, of at least about 1
 Methylobacterium / 5  square micrometers, of at least about 1
 Methylobacterium / 2  square micrometers, or at least about 1
Methylobacterium / square micrometer. In certain embodiments, adherent Methylobacterium may 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 at
about 1 Methylobacterium / square micrometer, at least about
 one  Methylobacterium / 10 square micrometers to approximately
 one  Methylobacterium / square micrometer, at least approximately
 one  Methylobacterium / 10 square micrometers to approximately
 one  Methylobacterium / square micrometer, at least approximately
 one  Methylobacterium / 5 square micrometers to approximately
 one  Methylobacterium / square micrometer, or at least about
 one  Methylobacterium / 2 square micrometers at approximately
 one  Methylobacterium / square micrometer. In certain embodiments, Methylobacterium
Adhesives may be present on the surface of the solid substance in the fermentation broth, fermentation broth product or composition in a density of at least about 1 Methylobacterium / 20 square micrometers to about 1 Methylobacterium / 2 square micrometers, 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 at about 1 Methylobacterium / 2 square micrometers. The broths of
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Biphasic fermentation provided herein may comprise a liquid phase containing non-adherent Methylobacterium. In certain embodiments, the non-adherent Methylobacterium titres in the liquid phase may be less than about 100,000, 10,000, or 1,000 CFU / ml.
The biphasic culture methods provided may yield fermentation broths with Methylobacterium in a titer greater than approximately 5 x 108 colony forming units per milliliter, in a titer greater than approximately 1 x 109 colony forming units per milliliter, in a trtule greater than about 1 x 1010 colony forming units per milliliter, in a title of at least about 3 x 1010 colony forming units per milliliter. In certain embodiments, the fermentation broths provided herein may comprise Methylobacterium in a titer 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 to at least 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 in a titer 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 to at least 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 in a titer 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
approximately 1 x 1010 colony forming units per milliliter to at least
approximately 4 x 1010 colony forming units per milliliter, or at least
approximately 1 x 1010 colony forming units per milliliter to at least
approximately 6 x 1010 colony forming units per milliliter. In certain
embodiments, the fermentation broths provided herein will comprise Methylobacterium in a titer 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 units
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colony forming agents per milliliter to at least about 6 x 1010 colony forming units per milliliter.
Solid substances with adherent Methylobacterium which can be obtained as fermentation products can be used to prepare various compositions useful for treating plants or plant parts to improve plant yield, and / or improve fruit production of the fruit producing plant. In certain embodiments, the composition comprises Methylobacterium and is free of substances that promote the growth of resident bacteria. Compositions provided herein comprising Methylobacterium, substances solid with Methylobacterium grown thereon, or comprising emulsions with Methylobacterium grown therein can be used to treat plants or parts of plants. Thus, plants, parts of plants, and, in particular, plant seeds that have been at least partially coated or coated with fermentation broth products or compositions comprising Methylobacterium are provided. Processed vegetable products containing fermentation broth products or compositions with adherent Methylobacterium or Methylobacterium are also provided. Solid substances with adherent Methylobacterium can be used to prepare various compositions that are particularly useful for treating plant seeds. Seeds are therefore provided that have been at least partially coated with the fermentation broth products or compositions. Processed seed products are also provided, which include, but are not limited to, flour, feed and flakes containing the fermentation broth products or compositions provided herein. In certain embodiments, the processed vegetable product will be non-regenerable (that is, it will be unable to develop to a plant). In certain embodiments, the solid substance used in the fermentation or composition product that at least partially covers the plant, part of the plant or plant seed or that is contained in the plant, part of the processed plant or seed product comprises a solid substance and associated or adherent Methylobacterium that can be easily identified by comparing a plant, plant part, plant seed or processed product thereof, treated and untreated. The partial coating of a plant, a part of a plant or a seed includes, but is not limited to, coating at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% , 80%, 90%, 95%, 98%, 99%, or approximately 99.5% of the surface area of the plant, part of the plant or plant seed.
Methods for preparing a treatment composition of a plant or plant seed comprising Methylobacterium and this are also provided herein.
free of substances that promote the growth of resident bacteria on a plant or seed. Such methods may comprise (i) cultivating a mono-culture or co-culture of Methylobacterium in medium comprising: (a) an aqueous phase; (b) a liquid phase and a solid phase; or (c) an emulsion, thereby obtaining a medium containing 5 methylobacterium; (ii) separate the Methylobacterium from at least one other portion of the medium
which contains Methylobacterium; and (iii) reconstitute the Methylobacterium in a matrix that lacks substances that promote the growth of resident bacteria on a plant or seed. In certain embodiments, the separation step is carried out by centrifugation, filtration or sedimentation of the medium containing Methylobacterium and removal of the excess Kqido or emulsion thereof. In certain embodiments where the Methylobacterium are grown in the presence of a solid substance, the separation will provide a fraction containing Methylobacterium with growth adherent to the solid substance and some non-adherent Methylobacterium that can be reconstituted in the matrix. In certain embodiments, the substance that promotes the growth of bacteria resident on a plant or seed is selected from the group consisting of a carbon source, a source of nitrogen, a source of phosphorus, a source of sulfur, a source of magnesium , and combinations thereof. In certain embodiments, the matrix is a liquid, an emulsion, or one or more solids, and comprises an adjuvant and / or excipient acceptable from the agricultural point of view. In certain embodiments, the Methylobacterium are grown in media comprising a liquid phase and a solid substance with adherent Methylobacterium grown thereon. The solid substance is separated from the liquid phase of the medium containing Methylobacterium, and the solid substance with adherent Methylobacterium grown thereon is reconstituted in the matrix mentioned above. In certain embodiments, the matrix may be a liquid that includes, but is not limited to, water, and aqueous buffer free of substances that promote the growth of resident bacteria on a plant or seed, or an aqueous solution free of substances that promote the growth of resident bacteria on a plant or seed.
Compositions comprising any of the following isolates of Methylobacterium sp. Are also provided herein. provided in the following Table 1, as well as plants, plant parts and plant seeds that are partially coated or coated with the compositions.
Table 1. Isolates of Methylobacterium sp.
 insulation No.  nls No. usda ars NRRL No.1
 isooi  NLS0046 NRRL B-50929
 ISO02  NLS0020 NRRL B-50930
 ISO03  NLS0017 NRRL B-50931
 ISO04  NLS0042 NRRL B-50932
 ISO05  NLS0089 NRRL B-50933
 IS006  NLS0068 NRRL B-50934
 IS007  NLS0065 NRRL B-50935
 IS008  NLS0069 NRRL B-50936
 IS009  NLS0062 NRRL B-50937
 IS010  NLS0064 NRRL B-50938
 isoii  NLS0021 NRRL B-50939
 IS012  NLS0066 NRRL B-50940
 IS013  NLS0037 NRRL B-50941
 IS014  NLS0038 NRRL B-50942
1 Deposit number for strain deposited with the AGRICULTURAL RESEARCH SERVICE CULTURE COLLECTION (NRRL) of the National Center for Agricultural Utilization Research, 5 Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604 U.S.A. under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Subject to 37 CFR §1.808 (b), all restrictions imposed by the depositor on the availability to the public of the deposited material will be irrevocably withdrawn after the granting of any patent from this patent application.
Patent applications assigned herein that describe additional specific uses
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of the strains of Methylobacterium of Table 1 such as: (1) increase the yield of corn (US 61/911780, filed on 12/4/2013; and international application claiming benefit from it presented on 12/4/2014 ); (2) increase soybean yield (US 61/911698, filed on 12/4/2013; and international application claiming benefit from it filed on 12/4/2014); (3) improve lettuce cultivation (international patent application PCT / US14 / 68558, filed on 12/4/2014); (4) improve tomato growth (international patent application PCT / US14 / 68611, filed on 12/4/2014) and each is incorporated by reference herein in its entirety. Specifically incorporated herein by reference in their entirety are the amino acid and genomic nucleic acid sequences of Methylobacterium sp. NLS017, NLS020, NLS037, NLS042, NLS065, and NLS066 which are described in international patent application PCT / US14 / 68558, filed on 12/4/2014. Also specifically incorporated herein by reference in their entirety are the amino acid and genomic nucleic acid sequences of Methylobacterium sp. NLS017 and NLS066 described in international patent application PCT / US14 / 68611 filed on 12/4/2014. Such amino acid and genomic nucleic acid sequences can be used to identify compositions, parts of plants, plant seeds, or processed plant products comprising Methylobacterium sp. NLS017, NLS020, NLS037, NLS042, NLS065, and NLS066.
Compositions provided herein that are useful for treating plants or parts of plants that comprise Methylobacterium, and / or are free of substances that promote the growth of resident bacteria on a plant or seed, contain a solid substance with adherent Methylobacterium grown on the same, or comprising emulsions with Methylobacterium cultured therein may also 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 may itself be an agriculturally acceptable adjuvant or an agriculturally acceptable excipient as long as it is not bactericidal or bacteriostatic for the 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 may also comprise a pesticide. Pesticides used in the composition include, but are not limited to, an insecticide,
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a fungicide, a nematocide and a bactericide. In certain embodiments, the pesticide used in the composition is a pesticide that does not substantially inhibit the growth of Methylobacterium. Since Methylobacterium are gram negative bacteria, suitable bactericides used in the compositions may include, but are not limited to, bactericides that exhibit activity against gram positive bacteria but not gram negative bacteria. The compositions provided herein may also comprise a bacteriostatic agent that does not substantially inhibit the growth of Methylobacterium. Bacteriostatic agents suitable for use in the compositions provided herein include, but are not limited to, those that exhibit activity against gram positive bacteria but not gram negative bacteria. Any of the above-mentioned compositions may also be an essentially dry product (ie, having approximately 5% or less water content), a mixture of the composition with an emulsion, or a suspension. Any of the compositions provided herein may be used to partially coat or coat a plant, plant part or plant seed. The partial coating of a plant, a part of a plant or a seed includes, but is not limited to, coating at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or approximately 99.5% of the surface area of the plant, part of the plant or plant seed.
Agriculturally acceptable adjuvants used in compositions comprising Methylobacterium include, but are not limited to, 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 humectants / spreaders that promote adhesion and extension of the composition on parts of the plant, tackifiers that promote adhesion to the part of the plant, penetrants that can promote agent contact active with inner tissues, extenders that increase the half-life of the active agent by inhibiting environmental degradation, and humectants that increase the density or drying time of the sprayed compositions. Moisturizers / spreaders used in the compositions may include, but are not limited to, non-ionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, organo silicate surfactants, and / or acidified surfactants. Tackifiers used in the compositions may include, but are not limited to, latex-based substances, terpene / pinolene, and pyrrolidone-based substances. Penetrants may include mineral oil, vegetable oil, esterified vegetable oil, organo-silicate surfactants, and acidified surfactants. Extensors used in the compositions may include, but are not limited to, sulfate.
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ammonium, or substances based on menthane. Moisturizers used in the compositions may include, but are not limited to, glycerol, propylene glycol and diethyl glycol. Adjuvants that improve the ease of application of the product include, but are not limited to, acidifying / buffering agents, anti-foaming / defoaming agents, compatibility agents, drift reducing agents, dyes and water conditioners. Defoamers / defoamers used in the compositions may include, but are not limited to, dimetholysiloxane. Compatibility agents used in the compositions may include, but are not limited to, ammonium sulfate. Drift reducing agents used in the compositions may include, but are not limited to, polyacrylamides and polysaccharides. Water conditioners used in the compositions may include, but are not limited to, ammonium sulfate.
Methods for treating plants and / or parts of plants with fermentation broths, fermentation broth products, and compositions comprising Methylobacterium are also provided herein. Treated plants and parts of treated plants obtained therefrom include, but are not limited to, a pepper, tomato, berry or banana plant. Parts of plants that are treated include, but are not limited to, leaves, stems, flowers, roots, seeds, fruit, tubers, coleoptils and the like. Seeds or other propagules of any of the plants mentioned above may be treated with fermentation broths, fermentation broth products, fermentation products, and / or compositions provided herein.
In certain embodiments, plants and / or parts of plants are treated by applying fermentation broths, fermentation broth products, fermentation products and compositions comprising Methylobacterium as a spray. Such spray applications include, but are not limited to, treatments of a single plant part or any combination of plant parts. Spraying can be achieved with any device that distributes fermentation broths, fermentation broth products, fermentation products, and compositions to the plant and / or part (s) of plants. Useful spraying devices include a spear sprayer, a hand or backpack sprayer, crop sprays (eg aerial spraying), and the like. Spray devices and methods that provide the application of fermentation broths, fermentation broth products, fermentation products, and compositions to any one or both of the adaxial surface and / or abaxial surface can also be used. Plants and / or parts of plants that are at least partially coated with any of a two-phase fermentation layer, a fermentation broth product, fermentation product or compositions comprising a
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Solid substance with Methylobacterium adhered thereto are also provided herein. Processed vegetable products comprising a solid substance with Methylobacterium adhered thereto are also provided herein. Any of the compositions provided herein can be used to partially coat or coat a plant, plant part or plant seed. The partial coating of a plant, a part of a plant or a seed includes, but is not limited to, coating at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or approximately 99.5% of the surface area of the plant, part of the plant or plant seed.
In certain embodiments, the seeds are treated by exposing the seeds to the fermentation broths, fermentation broth products, fermentation products and compositions comprising Methylobacterium. Seeds may be treated with fermentation broths, fermentation broth products and compositions provided herein by methods that include, but are not limited to, imbibition, coating, spraying and the like. In certain embodiments, surface-sterilized seeds are treated with a composition comprising Methylobacterium. In certain embodiments, non-sterilized seeds (eg seeds that have not been subjected to surface sterilization) are treated with a composition comprising Methylobacterium that has been devoid of substances that promote the growth of resident microorganisms on the seed. Seed treatments can be carried out with both continuous and discontinuous seed treaters. In certain embodiments, the coated seeds can be prepared by suspending the seeds with a coating composition containing a fermentation broth, fermentation broth product or compositions comprising the substance solid with Methylobacterium and air drying the resulting product. Air drying can be carried out at any temperature that is not harmful to the seed or the Methylobacterium, but will typically be no greater than 30 degrees Celsius. The proportion of coating comprising a solid substance and Methylobacterium includes, but is not limited to, 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. The partial coating of a seed may include, but is not limited to, coating at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95 %, 98%, 99%, or approximately 99.5% of the surface area of the seed. In certain embodiments, a solid substance used in the coating or treatment of the seed will have Methylobacterium adhered thereto. In certain embodiments, a solid substance used in the coating or treatment of the seed will be associated with Methylobacterium and will be a fermentation broth, product of fermentation broth or composition
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obtained by the methods provided herein. Various seed treatment compositions and methods for treating seeds described in US Pat. Nos. 5,106,648, 5,512,069 and 8,181,388 are incorporated herein by reference in their entirety, and may be adapted for use with an active agent comprising fermentation broths, fermentation broth products or compositions provided In the present memory. In certain embodiments, the composition used to treat the seed may contain agriculturally acceptable excipients that include, but are not limited to, wood flour, clays, activated carbon, diatomaceous earth, inorganic solids of fine grain, carbonate of calcium 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, caolm, china clay, talc, perlite, mica, vermiculite, sflices, quartz powder, montmorillonite and mixtures thereof. Agriculturally acceptable adjuvants that promote seed bonding that can be used include, but are not limited to, polyvinyl acetates, polyvinyl acetate copolymer, hydrolyzed polyvinyl acetates, polyvinylpyrrolidone-vinyl acetate co-polymer, poly (vinyl alcohols), poly (vinyl alcohol) co-polymers, poly (vinyl methyl ether), poly (vinyl methyl ether) co-polymer, maleic acid, waxes, latex polymers, celluloses, which they include ethyl celluloses and methyl celluloses, hydroxymethyl celluloses, hydroxypropyl cellulose, hydroxymethylpropyl celluloses, polyvinyl pyrrolidones, alginates, dextrins, maltodextrins, polysaccharides, fats, oils, protemas, karaya gum, jaguar gum, gum tragacanth, gum gums copolymers of vinylidene chloride, pokmeros and copokmeros of soy-based proteins, lignosulfonates, acrylic copolymers, starches, polyvinyl acrylates, zeins, gel atina, carboxymethylcellulose, chitosan, poly (ethylene oxide), pokmeros and copolymers of acrylamide, poly (hydroxyethyl acrylate), monomers of methyl acrylamide, alginate, ethyl cellulose, polychloroprene and syrups or mixtures thereof. Other agriculturally acceptable adjuvants that can promote the coating include, but are not limited to, polymers and copolymers of vinyl acetate, polyvinylpyrrolidone-vinyl acetate copolymer and water-soluble waxes. Various surfactants, dispersants, anti-caking agents, foam control agents and colorants described herein and in US Pat. No. 8,181,388 may be adapted for use with an active agent comprising fermentation broths, fermentation broth products or compositions provided herein.
Compositions comprising Methylobacterium are provided herein.
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which provide improved plant yield, as well as improved fruit production of fruit producing plants in relation to untreated plants that have not been exposed to the compositions. In certain embodiments, parts of plants, which include, but are not limited to, a seed, a leaf, a fruit, a stem, a root, a tuber or a coleoptyl may be treated with the compositions provided herein. to improve plant performance, insect control, fungal control, and / or fruit production. The treatments or applications may include, but are not limited to, pulverization, coating, partial coating, immersion and / or imbibition of the plant or parts of the plant with the compositions provided herein. In certain embodiments, a seed, a leaf, a fruit, a stem, a root, a tuber or a coleoptyl may be submerged and / or embedded with a liquid, semi-liquid, emulsion or suspension of a composition provided herein. Such immersion or imbibition of the seed may be sufficient to provide improved plant yield, insect control, fungal control, and / or fruit production in a treated plant or part of plant compared to an untreated plant or part of plant. . Improved fruit production includes, but is not limited to, faster fruit set, increased fruit set, earlier ripening, and / or more mature fruit production in relation to untreated plants. In certain embodiments, plant seeds may be submerged and / or embedded for at least 1, 2, 3, 4, 5, or 6 hours. Such immersion and / or imbibition may, in certain embodiments, be carried out at temperatures that are not harmful to the plant seed or the Methylobacterium. In certain embodiments, the seeds can be treated at about 15 to about 30 degrees Celsius or at about 20 to about 25 degrees Celsius. In certain embodiments, imbibition 7o immersion of the seed can be performed with gentle agitation.
It is therefore expected that the compositions provided herein that comprise Methylobacterium are useful in improving fruit production in a wide variety of plants, including, but not limited to: an apple, pear, grape, citrus plant , melon, pepper, tomato, berry, kiwi, mango or banana. In certain embodiments, the berry plant is a blackberry, strawberry or blueberry plant. It is therefore expected that the compositions provided herein comprising Methylobacterium are useful in improving plant yield, insect control in plants, and control of plant pathogenic fungi in a wide variety of plants, including, but not are limited to: corn, Brassica sp. (eg, B. napus, B. rapa, B. juncea), alfalfa, rice, rye, sorghum, millet (eg, pearl millet (Pennisetum glaucum), prose millet (Panicum miliaceum), millet foxtail (Setaria italica), finger millet (Eleusine coracana)), sunflower, safflower, soy, tobacco,
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potato, peanuts, cotton, sweet potato (Ipomoea batatus), cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, sugar beets , sugarcane, oats, barley, tomatoes, lettuce, green beans, Lima beans, chickpeas, cucurbitaceae such as cucumber, cantaloupe and musk melon, ornamental, and commens. Plant parts that are treated include, but are not limited to, leaves, stems, flowers, rates, seeds, fruit, tubers, coleoptils and the like. Ornamental plants and parts of plants that can be treated include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia and chrysanthemum. Common plants and plant parts that can be treated include, but are not limited to, pines such as taeda pine, ellioti pine, ponderosa pine, contorta pine and Monterrey pine; Douglas fir; Paduga Tsuga; Sitka spruce; sequoia true firs such as bianco fir and balsam fir; and cedars such as western red cedar and yellow Alaska cedar. Lawn plants and plant parts that can be treated include, but are not limited to, annual blue grass, annual English grass, Canada blue grass, fescue, agrostis, wheat grass, Kentucky blue grass, ball grass, English grass , agrostis gigantea, Bermuda grass, St. Agustm grass, and zoysia grass. Seeds or other propangles of any of the plants mentioned above may be treated with the compositions provided herein.
In certain embodiments, an amount of a composition provided herein that is sufficient to provide improved plant performance, improved plant insect control, improved plant fungal control, as well as improved fruit production may be a composition with Methylobacterium in a titer of at least about 1 x106 colony forming units per milliliter, at least about 5x106 colony forming units per milliliter, at least about 1 x107 colony forming units per milliliter, at least
approximately 5 x 108 colony forming units per milliliter, at least
approximately 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 improved plant yield and / or improved fruit production may be a composition with Methylobacterium in a title of
approximately at least about 1 x106 colony forming units per milliliter, at least about 5x106 colony forming units per milliliter, at least about 1 x107 colony forming units per milliliter, or at least about 5 x 108 colony forming units per milliliter a at least
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approximately 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 improved plant yield and / or improved fruit production may be a fermentation broth product with a Methylobacterium title of a solid phase of that product. it is at least about 5 x 108 colony forming units per milliliter to at least about 5 x 1013 colony forming units per milliliter of Methylobacterium per gram of the solid phase. In certain embodiments, an amount of a composition provided herein that is sufficient to provide improved plant yield and / or improved fruit production may be a composition with a Methylobacterium title of at least about 1 x 106 colony forming units per gram, at least about 5x106 colony forming units per gram, at least about 1 x107 colony forming units per gram, or at least about 5 x 108 colony forming units per gram to 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 mono-culture or co-culture of Methylobacterium is attached to it. In certain embodiments, an amount of a composition provided herein that is sufficient to provide improved plant yield and / or improved fruit production may be a composition with a Methylobacterium title of at least about 1 x 106 colony forming units per ml, at least about 5x106 colony forming units per ml, at least
approximately 1 x107 colony forming units per ml, or at least
approximately 5 x 108 colony forming units per ml to at least
approximately 6 x 1010 colony-forming units of Methylobacterium per ml in a composition comprising an emulsion wherein a mono-culture or co-culture of a Methylobacterium adhered to a solid substance is provided therein or cultivated therein. In certain embodiments, an amount of a composition provided herein that is sufficient to provide improved plant yield and / or improved fruit production may be a composition with a Methylobacterium title of at least about 1 x 106 colony forming units per ml, at least about 5x106 colony forming units per ml, at least
approximately 1 x107 colony forming units per ml, or at least
approximately 5 x 108 colony forming units per ml to at least
approximately 6 x 1010 colony forming units of Methylobacterium per ml in a composition comprising an emulsion wherein a mono-culture or co-culture of a
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Methylobacterium is provided in it or cultivated in it.
In certain embodiments, an amount of a composition provided herein that is sufficient to provide improved plant yield and / or improved fruit production may be a composition with Methylobacterium sp. in a title of at least about 1x104 colony forming units per milliliter, at least about 1 x105 colony forming units per milliliter, at least about 1x106 colony forming units per milliliter, at least
approximately 5x106 colony forming units per milliliter, at least
approximately 1x107 colony forming units per milliliter, at least
approximately 5x108 colony forming units per milliliter, at least
about 1 x 109 colony forming units per milliliter, at least about 1x 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 improved plant yield and / or improved fruit production may be a composition with Methylobacterium sp. in a title of at least about 1x104 colony forming units per milliliter, at least
approximately 1x105 colony forming units per milliliter, approximately at least approximately 1 x106 colony forming units per milliliter, at least approximately 5x106 colony forming units per milliliter, at least
about 1 x107 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 emulsion. In certain embodiments, an amount of a composition provided herein that is sufficient to provide improved plant yield and / or improved fruit production may be a fermentation broth product with a Methylobacterium sp. of a solid phase of that product that is at least about 1 x104 colony forming units per gram, at least about 1x105 colony forming units per gram, at least
about 1x106 colony forming units per gram, at least about 5x106 colony forming units per gram, at least
approximately 1x107 colony forming units per gram, at least
about 5 x 108 colony forming units per gram, at least about 1x109 colony forming units per gram, or at least about 5x109 colony forming units per gram to at least about 6 x 1010 colony forming units of Methylobacterium per
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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 of Methylobacterium per gram, or at least approximately 5 x 1013 colony forming units of Methylobacterium per gram of the solid phase. In certain embodiments, an amount of a composition provided herein that is sufficient to provide improved plant yield and / or improved fruit production may be a composition with a Methylobacterium title of at least about 1 x 106 colony forming units per gram, at least about 5x106 colony forming units per gram, at least
approximately 1x107 colony forming units per gram, at least
about 5 x 108 colony forming units per gram, at least about 1x109 colony forming units per gram, or at least
approximately 5x109 colony forming units per gram to at least
approximately 6 x 1010 colony forming units of Methylobacterium per gram, at least about 1X1011 colony forming units of Methylobacterium per gram, at least about 1x1012 colony forming units of Methylobacterium per gram, at least about 1x1013 colony forming units of Methylobacterium per gram, or at least about 5 x 1013 colony forming units of Methylobacterium per gram of particles in the composition containing the particles comprising a solid substance wherein a mono-culture or co-culture of Methylobacterium sp. is attached to it. In certain embodiments, an amount of a composition provided herein that is sufficient to provide improved plant yield and / or improved fruit production may be a composition with a Methylobacterium title of at least
 approximately  1x106 colony forming units per ml, at least
 approximately  5x106 colony forming units per ml, at least
 approximately  1x107 colony forming units per ml, at least
 approximately  5 x 108 colony forming units per ml, at least
 approximately  1x109 colony forming units per gram, or at least
 approximately  cn X or CD colony forming units per gram to at least
approximately 6 x 1010 colony forming units of Methylobacterium per ml in a composition comprising an emulsion wherein a mono-culture or co-culture of a Methylobacterium sp. adhered to a solid substance is provided in it or cultivated therein. In certain embodiments, an amount of a composition provided herein is sufficient to provide improved plant performance and / or
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Improved fruit production may be a composition with a Methylobacterium titer of at least about 1 x106 colony forming units per ml, at least about 5x106 colony forming units per ml, at least about 1 x107 colony forming units per ml, or at least about 5 x 108 colony forming units per ml to at least about 6 x 1010 colony forming units of Methylobacterium per mL in a composition comprising an emulsion wherein a mono-culture or co-culture of a Methylobacterium sp. is provided in it or cultivated in it.
In certain embodiments, compositions with a Methylobacterium sp. Are provided or used. in a title of at least about 1 x104 colony forming units per milliliter, at least about 1 x105 colony forming units per milliliter, at least about 1 x106 colony forming units per milliliter, at least about 5x106 colony forming units per milliliter , at least about 1 x107 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, compositions with Methylobacterium sp. in a title of at least about 1 x104 colony forming units per milliliter, at least about 1 x105 colony forming units per milliliter, approximately at least about 1 x106 colony forming units per milliliter, at least about 5x106 colony forming units per milliliter at least
about 1 x107 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 emulsion. In certain embodiments, fermentation broth products are provided with a title of Methylobacterium sp. of a solid phase of that product that is at least about 1 x104 colony forming units per gram, at least
approximately 1 x105 colony forming units per gram, at least
approximately 1 x106 colony forming units per gram, at least
approximately 5x106 colony forming units per gram, at least
approximately 1 x107 colony forming units per gram, at least
about 5 x 108 colony forming units per gram, at least about 1x109 colony forming units per gram, or at least
approximately 5x109 colony forming units per gram to at least
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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 1x1012 colony forming units of Methylobacterium per gram, at least about 1x1013 colony forming units of Methylobacterium per gram, or at least about 5x1013 colony forming units of Methylobacterium per gram of the solid phase. In certain embodiments, compositions with a Methylobacterium titer of at least about 1 x106 colony forming units per gram, at least about 5x106 colony forming units per gram, at least about 1 x107 colony forming units per gram are provided at least about 5 x 108 colony forming units per gram, at least about 1x109 colony forming units per gram, or at least about 5x109 colony forming units per gram at least about 6 x 1010 colony forming units of Methylobacterium per gram, at 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 of Methylobacterium per gram, or at least about 5 x 1013 colony forming units of Methylobacterium per gram of particles in the composition containing the particles comprising a solid substance in which a mono-culture or co-culture of Methylobacterium sp. is attached to it. In certain embodiments, compositions with a Methylobacterium titer of at least about 1 x106 colony forming units per ml, at least about 5x106 colony forming units per ml, at least about 1 x107 colony forming units per ml are provided, or at less
approximately 5 x 108 colony forming units per ml to at least
approximately 6 x 1010 colony forming units of Methylobacterium per ml in a composition comprising an emulsion wherein a mono-culture or co-culture of a Methylobacterium sp. adhered to a solid substance is provided in it or cultivated therein. In certain embodiments, compositions with a Methylobacterium titer of at least about 1 x106 colony forming units per mL, at least about 5x106 colony forming units per ml, at least about 1 x107 colony forming units per ml are provided, or at less about 5 x 108 colony forming units per ml to at least
approximately 6 x 1010 colony forming units of Methylobacterium per ml in a composition comprising an emulsion wherein a mono-culture or co-culture of a
Methylobacterium sp. is provided in it or cultivated in it. In certain embodiments of any of the compositions mentioned above, the
Methylobacterium sp. is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL B-50941),
5 NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932), NLS0046 (NRRL B-50929),
NLS0062 (NRRL B-50937), NLS0064 (NRRL B-50938), NLS0065 (NRRL B-50935),
NLS0066 (NRRL B-50940), NLS0068 (NRRL B-50934), NLS0069 (NRRL B-50936),
NLS0089 (NRRL B-50933), and derivatives thereof. In certain embodiments of any of the above-mentioned compositions, the composition may further comprise an agriculturally acceptable adjuvant, an agriculturally acceptable excipient, or combination thereof. In certain embodiments of any of the compositions mentioned above, the
Methylobacterium sp. is selected from the group consisting of NLS0017 (NRRL b-50931), NLS0020 (NRRL b-50930), NLS0021 (NRRL b-50939), NLS0037 (NRRL b-50941),
15 NLS0038 (NRRL b-50942), NLS0042 (NRRL b-50932), NLS0046 (NRRL b-50929),
NLS0062 (NRRL b-50937), NLS0064 (NRRL b-50938), NLS0065 (NRRL b-50935),
NLS0066 (NRRL b-50940), NLS0068 (NRRL b-50934), NLS0069 (NRRL b-50936),
NLS0089 (NRRL b-50933), derivatives thereof; and also comprises an adjuvant acceptable from the agricultural point of view, excipient, or combination thereof. In certain embodiments of any of the compositions mentioned above, the composition is free of substances that promote the growth of resident microorganisms on a plant or seed. Also provided are plants, plant parts, and plant seeds that are coated or at least partially coated with any of the above-mentioned compositions.
25 Examples
The following examples are included to demonstrate certain embodiments of the invention. Those skilled in the art will appreciate that the techniques described in the following examples represent techniques discovered by applicants to function well in the practice of the invention, and therefore can be considered as non-limiting examples for their practice. However, those skilled in the art should, in light of the present description, appreciate that many changes can be made to the specific embodiments described, while still obtaining similar or similar results, without departing from the scope of the invention.
5
10
fifteen
twenty
Example 1. Cultivation of PPFM strains in a Kido culture medium supplemented with a solid substance
The liquid culture medium used to grow the PPFM cultures was a base salt medium supplemented with glycerol, peptone and diatomaceous earth. The base salts medium used was ammonium mineral salts medium (AMS). AMS medium contains, per liter, 700 milligrams of anhydrous dibasic potassium phosphate, 540 milligrams of anhydrous monobasic potassium phosphate, one gram of magnesium sulfate heptahydrate, 500 milligrams of anhydrous ammonium chloride, and 200 milligrams of calcium chloride dihydrate .
The AMS base medium was prepared from three standard solutions, listed below:
Solution pattern I: for a liter at concentration 50X
dibasic potassium phosphate, anhydrous 35 grams
monobasic potassium phosphate, anhydrous 27 grams
Solution pattern II: for a liter at 50X concentration
magnesium sulfate heptahydrate 50 grams
ammonium chloride, anhydrous 25 grams
Solution standard Ill: for a liter at 50X concentration
calcium chloride dihydrate 10 grams
The standard solutions l, ll and Ill were treated in an autoclave separately.
To prepare a liter of liquid AMS medium with glycerol, peptone and diatomaceous earth, the following was added to 920 ml of distilled water:
20 ml of standard solution l
20 ml of solution standard ll
20 ml solution standard Ill
20 ml of a 50% glycerol standard solution
10 grams of peptone
2 grams of diatomaceous earth
The resulting solution with suspended diatomaceous earth was sterilized by treatment in
autoclave.
Two liters of the previous AMS medium were placed in a four-liter flask. Two milliliters of PPFMs liquid culture were added to the medium for inoculation. The flask was then placed on an incubated shaker set at 240 RPM and 30 degrees Celsius. The crops were grown for six days and then stored at 4 degrees Celsius for future use.
Example 2. Test of cultivation and maturity of pepper
PPFM water resuspension
The PPFM strains to be tested were cultured as described in Example 1 in a liquid medium supplemented with a solid substance. In the biological hood, the desired amount of PPFM solution in conical tubes was pipetted (making sure to rotate / shake the bottle vigorously before pipetting to suspend in the form of particles). A centrifuge was used to rotate at 3,500 RPM for 15 minutes at 23qC. While the tubes were centrifuged, a volume of warm tap water was measured to bring the volume of each sample to the total volume.
15 Liquid was carefully poured from each tube, and the granule was carefully kept intact. The appropriate volume of tap water was added to each tube to match its initial volume of PPFM solution. Water resuspended PPFMs were used as quickly as possible.
Seed Preparation
20 100 seeds of Johnny’s (’Bangkok’s Selected Seed, Waterville, ME, USA) pepper seed were counted per group. The seeds were soaked in 30 ml of warm tap water for 30 minutes for the control group. The seeds were soaked in 30 ml of water-resuspended PPFMs, strain NLS0038, for the treated seed groups.
Plantation
25 A tray with caps of 2.54 cm (1 inch) of 96 cells per group was used. Two trays were filled with Farfard Super Fine Germination soilless media mix (from Sun Gro Horticulture, Agawam, MA, USA), leveled with the top of the cell, pressed with the thumbs in each cell to compact, then filled with new level with the top of the cell. They were irrigated, with well-tempered water, using the shower apparatus. Using pincitas, the seeds were planted at a depth of approximately 0.63 cm (% ”). The control group was planted first to avoid cross contamination. 1 seed was placed in each cell for a total of 96 seeds per group. The seeds were covered
and water was added slightly. The groups were marked with labels and humidity dips were placed above the flat boxes.
Cultivation and Development
Moisture dips were removed after germination occurred at 7 days. At 5 30 days of maturity, 8 plants in each group were transplanted from their 96 trays
11.43 cm (4.5 ”) green potted cells filled with Fafard 3B soilless media mix (from Sun Gro Horticulture, Agawam, MA, USA). At 81 days, the plants were large enough to fall. All plants were of a similar size, and several nodes were trimmed to lighten the plants. The control group was cut first, then the clippers were sterilized and the treated group was cut.
Results
At 92 days, more than half of the plants in the treated group showed ripe red fruit, while the plants in the control group did not exhibit ripe fruit. Both ripe and immature fruit were counted for all plants. Total fruit counts 15 for the group treated with NLS0038 were 15.8% higher. See Tables 2-4 below.
Table 2. Plants with observed ripe red fruit, 92 days of growth
 + NLS0038 Control
 Total  5/8 0/8
Table 3. Total fruit counts observed at 92 days of growth
 + NLS0038 Control
 29 25
 15 29
 30 31
 22 25
 35 21
 + NLS0038 Control
 44 29
 46 32
 31 23
 Total  252 215
Table 4. Total ripe fruit counts observed at 105 days of growth
 + NLS0038 Control
 15 20
 12 12
 5 20
 17 14
 14 9
 23 14
 23 14
 24 18
 Total  133 121
Conclusion
5 Pepper plants grown to maturity from seeds treated with PPFM, strain NLS0038, produced more ripe fruit and an increased total fruit count since initial observations of ripening fruit at 92 days. Thirteen days after the initial observations, the treated group continued to show a slight increase in ripe fruit counts.
io Example 3. Inoculation of ‘Rex’ lettuce seeds to identify PPFMs that increase
growth of rates and outbreaks.
Sowing
A 104-cell HorticubeXL ™ Oasis (grooved bottom, single planter; Srnithers-Oasis North America, Kent, OH, USA) was placed in a 1040 flat box without holes. Four cubes were removed in the center of the grid to allow watering the bottom. The Oasis HorticubeXL ™ spread so that it was fully saturated, the shower was used with warm water. One seed was placed in each cell for a total of 100 seeds per group.
Inoculation of lettuce seeds
The PPFM strains to be assayed as described in Example 1 were cultured in a liquid medium supplemented with a solid substance. In the biological hood, the desired amount of PPFM solution was pipetted into conical tubes (making sure to rotate / shake the bottle vigorously before pipetting to suspend in the form of particles). One centhfuga was used to rotate at 3,500 RPM for 15 minutes at 23qC. While the tubes were centrifuged, a volume of warm tap water was measured to bring the volume of each sample to the total volume.
Liquid was carefully poured from each tube, carefully to keep the granule intact. The appropriate volume of tap water was added to each tube to match its initial volume of PPFM solution. Water resuspended PPFMS were used as quickly as possible.
100 microliters of solution (PPFM solution for the treated groups and tap water for the control groups) were pipetted over the top of each seed. After every 3 rows, the tube was plugged and stirred to resuspend any PPFMs that might have settled in the bottom. Pipette tips were changed between each group to avoid cross contamination. Labels were marked and dated for each flat box and transparent moisture dips were placed above the flat box. The flat boxes were placed in a growth chamber with temperature settings at 20 ° C and 12-hour days with illumination of 200 micromoles.
Increase
30 After five to six days, the dome was removed after the seeds germinated. Flat boxes were irrigated at the bottom only, and fertilized with Jack’s ™ 16-16-17 (JR PETERS, Inc. Allentown, PA, USA) at each irrigation (approximately every two
days).
A daily relocation of the flat boxes was carried out to prevent potential effects on growth due to variations in the light conditions in the growth chamber.
5 Processing
Flat boxes were harvested between two and three weeks. Transparent moisture cups were placed on each flat box to prevent evapotranspiration during transport. The tickets were left in place until the box was processed. Each plant is cut directly below the cotyledons and is immediately weighed on an analog scale.
Observations
It was observed that some strains repeatedly showed an increase in biomass of shoots of lettuce shoots when a seed was treated at the time of planting. Visual observations of the mass and root development were also made, the treated groups showed more growth at the time of harvest. Due to the natural variance of the biological systems, all the sample sizes were 98 - 100 plants as a minimum, and nothing below a 12% difference was considered significant.
Conclusion
It was evident that strains NLS0017, NLS0020, NLS0066, NLS0065, and NLS0089 show an increase in the wet weight of lettuce stems after seed treatment. Strains NLS0069, NLS0037, NLS0038, and NLS0062 exhibited negligible increases in wet weight compared to controls. Also, along with an increase in the biomass of the shoots, there was a corresponding increase in root development.
25
Table 5. Effects of seed treatments on lettuce growth
 wet weight of the stem (mg)
 strain  Title experimental control difference (%) confidence interval
 NLS0017  2.7E + 08 226.18 306.36 35.45% 0.000
 NLS0017  1.4E + 08 298.27 353.34 18.46% 0.012
 NLS0017  2.7E + 08 169.56 176.68 4.20% 0.567
 NLS0017  1.1E + 09 98.92 167.51 69.34% 0.000
 NLS0020  7.2E + 08 226.18 274.46 21.35% 0.027
 NLS0020  1.2E + 09 98.92 157.11 58.83% 0.000
 NLS0020  1.2E + 09 462.20 614.72 33.00% 0.000
 NLS0021    462.20 539.39 16.70% 0.008
 NLS0037  3.0E + 08 226.18 258.68 14.37% 0.085
 NLS0038  5.2E + 07 462.20 514.99 11.42% 0.070
 NLS0042  2.1E + 08 226.18 310.85 37.44% 0.000
 NLS0042  1.1E + 08 169.56 189.46 11.73% 0.105
 NLS0046  1.8E + 09 462.20 511.78 10.73% 0.084
 NLS0062  1.8E + 08 169.56 187.62 10.65% 0.121
 NLS0064    169.56 157.67 -7.01% 0.275
 NLS0065  1.2E + 08 169.56 211.92 24.98% 0.001
 NLS0065  9.1E + 07 98.92 132.35 33.80% 0.000
 NLS0066  5.9E + 08 56.15 69.57 23.91% 0.000
 NLS0066  4.2E + 08 546.61 665.46 21.74% 0.000
 NLS0066  1.2E + 08 98.92 129.81 31.23% 0.000
 wet weight of the stem (mg)
 strain  Title experimental control difference (%) confidence interval
 NLS0068  3.1E + 08 213.52 234.95 10.04% 0.029
 NLS0069  5.6E + 07 226.18 244.25 7.99% 0.307
 NLS0069  5.6E + 07 298.27 332.53 11.49% 0.144
 NLS0089  1.5E + 08 98.92 146.99 48.60% 0.000
 NLS0089    462.20 600.82 29.99% 0.000
1 Each line represents data obtained from a flat box of plants obtained from treated seed versus control seed.
Example 4. Foliar application of lettuce ‘Rex’ to identify PPFMs that increase root and shoot growth.
5 Sowing
A 104-cell HorticubeXL Oasis (grooved bottom, single planter) was placed in a 1020 flat box without holes. Four cubes in the center of the rack were removed to allow the bottom to water. The Oasis HorticubeXL ™ spread so that it was fully saturated, the shower was used with warm water. One seed was placed in each cell for a total of 100 seeds per group. Labels were marked and dated for each flat box and
transparent humidity dome was placed above the flat box. The flat boxes were placed in a growth chamber with temperature settings at 20 ° C and 12-hour days with illumination of 200 micromoles.
Inoculation of lettuce stems
15 After five to six days, the dome was removed after the seeds had germinated. The plants were inoculated at this time, when only the cotyledons had emerged. The PPFM strains to be tested were grown as described in Example 1 in a liquid medium supplemented with a solid substance. The PPFM strains to be tested were cultured as described in Example 1 in a liquid medium supplemented with a solid substance. In the biological campaign, the quantity was pipetted
Desired solution of PPFM in conical tubes (after rotating / stirring the solution
vigorously before pipetting to suspend in the form of particles). A centrifuge was used to rotate at 3,500 RPM for 15 minutes at 23qC. While the tubes were centrifuged, a volume of warm tap water was measured to bring the volume of each sample to the total volume.
5 Liquid was carefully poured from each tube, carefully to keep the granule intact. The appropriate volume of tap water was added to each tube to match its initial volume of PPFM solution. Water resuspended PPFMS were used as quickly as possible.
100 mL of PPFM solution (tap water for control) was poured into a 1 L SoIo ™ 10 Handheld Sprayer (SoIo ™, Newport News, VA, USA). The flat box was removed from the group to avoid cross contamination. The finest ratio adjustment was used and a uniform layer of solution was sprayed on top of the stems, ensuring uniform coverage across the entire flat box. For each group this was repeated, using an appropriate treatment.
15 Growth
Flat boxes were irrigated at the bottom only and fertilized with Jack’s ™ 15-16-17 (JR PETERS, Inc. Allentown, PA, USA) at each irrigation (approximately every two days). A daily relocation of the flat boxes was carried out to prevent potential effects on growth due to variations in the light conditions in the growth chamber.
Processing
Flat boxes were harvested between two and weeks. Transparent moisture cups were placed on each flat box to prevent evapotranspiration during transport. The tickets were left in place until the box was processed. Each plant 25 is cut directly below the cotyledons and is immediately weighed on an analog scale.
Observations
It was observed that some strains repeatedly show an increase in biomass of shoots of lettuce shoots when the stem was treated in the cotyledon phase. There were also visual observations of the mass and development of the root, the treated groups showed more growth at the time of harvest. Due to the natural variance of the biological systems, all sample sizes were 98 - 100 plants as
minimal, and nothing below a 12% difference was considered significant.
Conclusion
It is evident that strains NLS0042, NLS0017, NLS0020, and NLS0068 show an increase in the wet weight of lettuce stems after foliar application. Strains 5 NLS0069, NLS0037, NLS0038, and NLS0062 exhibited negligible increases in weight
wet compared to controls. It was also noted that along with an increase in the biomass of the shoots there is a corresponding increase in root development.
Table 6. Effects of foliar treatments on lettuce growth
 wet weight of the stem (mg)
 strain  Titles experimental control difference (%) confidence interval
 NLS0017  1.4E + 08 197.04 213.76 8.49% 0.075
 NLS0017  1.1E + 09 157.72 211.03 33.81% 0.000
 NLS0020  2.2E + 08 104.41 145.95 39.79% 0.000
 NLS0020  7.2E + 08 205.34 247.12 20.34% 0.030
 NLS0020  1.2E + 09 280.84 260.95 -7.08% 0.224
 NLS0021  1.6E + 07 157.72 178.46 13.15% 0.021
 NLS0037    197.04 198.93 0.96% 0.846
 NLS0038  7.4E + 07 197.04 186.04 -5.58% 0.250
 NLS0042  9.3E + 07 103.36 127.05 22.92% 0.000
 NLS0042  2.1E + 08 205.34 235.92 14.89% 0.095
 NLS0042  6.4E + 07 298.27 331.62 11.18% 0.138
 NLS0042  1.1E + 08 157.72 196.12 24.35% 0.000
 NLS0046  1.8E + 09 157.72 195.03 23.66% 0.000
 NLS0062    280.84 243.09 -13.44% 0.018
5
 wet weight of the stem (mg)
 strain  Titles experimental control difference (%) confidence interval
 NLS0064    205.34 240.47 17.10% 0.042
 NLS0064    298.27 306.88 2.89% 0.691
 NLS0065  4.2E + 08 197.04 214.59 8.91% 0.077
 NLS0066  5.9E + 08 205.34 241.92 17.81% 0.035
 NLS0066  1.2E + 08 280.84 166.98 -40.54% 0.000
 NLS0068  1.7E + 08 104.41 204.26 95.65% 0.000
 NLS0068  1.6E + 08 205.34 288.46 40.47% 0.000
 NLS0068  3.1E + 08 298.27 296.68 -0.53% 0.944
 NLS0068  3.1E + 08 280.84 264.65 -5.76% 0.337
 NLS0068  3.1E + 08 157.72 183.84 16.56% 0.010
 NLS0069  4.5E + 07 99.85 103.54 3.70% 0.711
 NLS0089  1.3E + 09 280.84 282.94 0.75% 0.896
1 Each Imea represents data obtained from independent flat boxes of treated versus control plants.
Example 5. Additional pepper growth and maturity test
Pepper plants were subjected to additional tests essentially as described in Example 2, except that the strains of Methylobacterium NLS0037, NLS0042, and NLS0062 were tested. The results are presented in Table 7.
Table 7: Effects of foliar treatment on flower and pepper count
 Treatment  Flower count (average per plant) 46 days after planting Pepper count (average per plant) 64 days after planting
 Untreated  18.3 25.4
 NLS0037  24.8 (p = 0.08) 40.8 (p <0.05)
 NLS0042  25.1 (p = 0.09) 38.9 (p <0.05)
 NLS0062  24.1 (p = 0.06) 38.8 (p = 0.06)
It was clear from these experiments that the indicated Methylobacterium treatments had a positive effect on both flower and pepper counts.
5 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: 40254032.
io 2. Broekaert WF, Terras FR, Cammue BP, Vanderleyden J (1990) An automated quantitative assay for fungal growth inhibition. FEMS Microbiology Letters 69: 55-60.
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: 629-634.
15 4. Corpe, W.A., and D.V. Basile 1982. Methanol-utilizing bacteria associated with green plants.
Devel Industr. Microbiol 23: 483-493.
5. Corpe, W.A., and S. Rheem. 1989. Ecology of the methylotrophic bacteria on living leaf surfaces. FEMS Microbiol. EcoI. 62: 243-250.
6. Green, P.N. 2005. Methylobacterium. In Brenner, D.J., N.R. Krieg, and J.T. Staley (eds.). 20 "Bergey’s Manual of Systematic Bacteriology. Volume two, The Proteobacteria. Part C, The
alpha-, beta-, delta-, and epsilonproteobacteria. "Second edition. Springer, New York. Pages
567-571.
7. Green, P.N. 2006. Methylobacterium. In Dworkin, M., S. Falkow, E. Rosenberg, K.-H. Schleifer, and E. Stackebrandt (eds.). "The Prokaryotes. A Handbook on the Biology of Bacteria. Volume 5. Proteobacteria: Alpha and Beta Subclasses. "Third edition. Springer,
5 New York. Pages 257-265.
8. Holland, M.A. 1997. Methylobacterium and plants. Recent Res. Devel. in Plant Physiol. 1: 207-213.
9. Holland, M.A., and J.C. Polacco 1994. PPFMs and other covert contaminants: Is there more to plant physiology than just plant Annu Rev. Plant Physiol. Plant MoI. Biol. 45: 197-209.
io 10. Kutschera, U. 2007. Plant-associated methylobacteria as co-evolved phytosymbionts. To hypothesis. Plant Signal Behav. 2: 74-78.
11. Lidstrom, M.E. 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. Ecophysiology and biochemistry. "Third edition. Springer,
15 New York. Pages 618-634.
12. Madhaiyan, M., S. Poonguzhali, H.S. Lee, K. Hari, S.P. Sundaram, and T.M. Sa. 2005. Pink-pigmented facultative methylotrophic bacteria accelerate germination, growth and yield of sugarcane clone Co86032 (Saccharum officinarum L.) Biol. Fertil. Soils 41: 350-358.
13. Madhaiyan, M., S. Poonguzhali, M. Senthilkumar, S. Seshadri, H. Chung, J. Yang, S. 20 Sundaram, and T. Sa. 2004. Growth promotion and induction of systemic resistance in rice
cultivate CO-47 (Oryza sativa L.) by Methylobacterium spp. Bot. Bull. Acad. Without. 45: 315-324.
14. Madhaiyan, M., S. Poonguzhali, and T. Sa. 2OO7. Influence of plant species and environmental conditions on epiphytic and endophytic pink-pigmented facultative methylotrophic bacterial populations associated with field-grown rice cultivars. J Microbiol
25 Biotechnol. 2OO7 Oct; 17 (1O): 1645-54.
15. Stanier, R.Y., N.J. Palleroni, and M. Doudoroff. 1966. The aerobic pseudomonads: A taxonomic study. J. Gen. Microbiol. 43: 159-271.
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. 2OO1. Methylotrophic Methylobacterium
3O Bacteria Nodulate and Fix Nitrogen in Symbiosis with Legumes. Jour. Bacteriol 183 (1): 214-22O,
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. Appl. Environ. Microbiol 71: 7245-7252.
18. Vogel, H.J., and D.M. Bonner 1956. Acetylornithinase of Escherichia coli: Partial 5 purification and some properties. J. Biol. Chem. 218: 97-106.
19. Vogel, H. j. 1956. A convenient growth medium for Neurospora (Medium N). Microbial Genet Bull 13: 42-43
20. Whittenbury, R., S.L. Davies, and J.F. Wilkinson 1970. Enrichment, isolation and some properties of methane-utilizing bacteria. J. Gen. Microbiol. 61: 205-218.
io The inclusion of various references in this report is not to be interpreted as any admission by the applicants that the references constitute prior art. Applicants expressly reserve their right to rebut any allegations of non-patentability of the inventions described herein on the references included herein.
15 Having illustrated and described the principles of the present invention, it should be apparent to those skilled in the art that the invention can be modified in disposition and detail without departing from such principles.
Although the materials and methods of this invention have been described in terms of various embodiments and illustrative examples, it will be apparent to those skilled in the art that variations may be applied to the materials and methods described herein without departing from the concept, espmtu and scope of the invention. All such similar substitutes and obvious modifications to those skilled in the art are considered to be within the scope, scope and concept of the invention, defined by the appended claims.
25

权利要求:
Claims (43)
[1]
one.
5
10
[2]
2.
fifteen
[3]
3.
twenty
[4]
Four.
25 5.
[6]
6.
claims
A method for improving fruit production, said method comprising:
(a) applying a composition comprising Methylobacterium to a fruit producing plant or seed, wherein said composition comprises a solid substance with adherent Methylobacterium grown thereon or an emulsion having Methylobacterium grown therein; Y,
(b) harvest fruit from said plant or a plant grown from said seed, wherein said plant or plant grown from said seed exhibits a faster fruit set, fruit set increased, earlier maturation, and / or more uniform fruit ripening compared to an untreated control plant, thus obtaining an improved fruit production.
The method of claim 1, wherein said composition comprises Methylobacterium in a titer of about 1x106 colony forming units per gram (CFU / g) of solid at about 1x1014 CFU / g of solid for the composition containing solid or in a title from about 1x106 CFU / mL to about 1X1011 CFU / mL for the composition containing emulsion.
The method of claim 1, wherein said fruit producing plant is an apple, pear, grape, doric, melon, pepper, tomato, berry, kiwi, mango or banana plant.
The method of claim 1, wherein said composition is free of substances that promote the growth of resident microorganisms on said plant or seed.
The method of claim 1, wherein the Methylobacterium is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL B-50941), NLS0038 (NRRL B-50942),
NLS0042 (NRRL B-50932), NLS0046 (NRRL B-50929), NLS0062 (NRRL B-50937),
NLS0064 (NRRL B-50938), NLS0065 (NRRL B-50935), NLS0066 (NRRL B-50940),
NLS0068 (NRRL B-50934), NLS0069 (NRRL B-50936), NLS0089 (NRRL B-50933), and
derivatives thereof.
The method of claim 5, wherein the Methylobacterium is selected from the
5
10
fifteen
twenty
25
group consisting of NLS0037 (NRRL B-50941), NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932), NLS0062 (NRRL B-50937), and derivatives thereof.
[7]
7. The method of claim 1, wherein the solid substance with the adherent Methylobacterium cultured thereon is provided in a liquid or emulsion.
[8]
8. The method of any one of claims 1 to 7, wherein said composition partially covers or covers said plant or a part thereof, or said seed.
[9]
9. A method for improving fruit production, said method comprising:
(a) apply a composition comprising Methylobacterium to a plant or seed
fruit producer, wherein said composition comprises a Methylobacterium sp. selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL b-50941), NLS0038
(NRRL b-50942), NLS0042 (NRRL b-50932), NLS0046 (NRRL B-50929), NLS0062
(NRRL b-50937), NLS0064 (NRRL B-50938), NLS0065 (NRRL B-50935), NLS0066
(NRRL B-50940), NLS0068 (NRRL B-50934), NLS0069 (NRRL B-50936), NLS0089
(NRRL B-50933), and derivatives thereof, and an adjuvant, excipient, or combination thereof agriculturally acceptable; Y,
(b) harvest fruit from said plant or a plant grown from said seed, wherein said plant or plant grown from said seed exhibits a faster fruit set, fruit set increased, earlier maturation, and / or more uniform fruit ripening compared to an untreated control plant, thus obtaining an improved fruit production.
[10]
10. The method of claim 9, wherein said composition comprises the Methylobacterium in a titer of about 1x106 colony forming units per gram (CFU / g) of solid at about 1x1014 CFU / g of solid for a composition comprising a substance solid with adherent Methylobacterium grown thereon or in a titer of about 1x106 CFU / mL to about 1X1011 CFU / mL for a composition comprising an emulsion having the Methylobacterium grown therein.
[11]
11. The method of claim 9, wherein said fruit producing plant is an apple, pear, grape, citrus, melon, pepper, tomato, berry, kiwi, mango or banana plant.
[13]
13.
5
[14]
14.
10 15.
[16]
16.
fifteen
twenty
[17]
17.
25
[18]
18.
[19]
19.
30
[20]
twenty.
The method of claim 9, wherein said composition is free of substances that promote the growth of resident microorganisms on said plant or seed.
The method of claim 9, wherein the Methylobacterium is selected from the group consisting of NLS0037 (NRRL B-50941), NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932), NLS0062 (NRRL B-50937), and derivatives thereof.
The method of claim 13, wherein the Methylobacterium is selected from the group consisting of NLS0038 (NRRL b-50942), NLS0042 (NRRL b-50932), NLS0062 (NRRL b-50937), and derivatives thereof.
The method of any one of claims 9 to 14, wherein said composition partially covers or covers said plant or a part thereof, or said seed.
A method for improving fruit production, said method comprising:
(a) applying a composition comprising Methylobacterium to a fruit producing plant or seed, wherein said composition is free of substances that promote the growth of resident microorganisms on said plant or seed; Y,
(b) harvest fruit from said plant or a plant grown from said seed, wherein said plant or plant grown from said seed exhibits a faster fruit set, fruit set increased, earlier maturation, and / or more uniform fruit ripening compared to an untreated control plant, thus obtaining an improved fruit production.
The method of claim 16, wherein said fruit producing plant is an apple, pear, grape, titrico, melon, pepper, tomato, berry, kiwi, mango or banana plant.
The method of claim 16, wherein said composition comprises a solid substance with adherent Methylobacterium grown thereon.
The method of claim 18, wherein said composition comprises Methylobacterium in a titer of about 1x106 CFU / g to about 1x1014 CFU / g.
The method of claim 16, wherein said composition comprises a
5
10
fifteen
twenty
25
Emulsion that has Methylobacterium grown in it.
[21]
21. The method of claim 20, wherein said composition comprises Methylobacterium in a titer of about 1x106 CFU / mL to about 1X1011 CFU / mL.
[22]
22. The method of any one of claims 16 to 21, wherein said composition partially covers or covers said plant or a part thereof, or said seed.
[23]
23. A method for preparing a treatment composition of a plant or plant seed comprising Methylobacterium that is free of substances that promote the growth of resident microorganisms on a plant or seed, said method comprising:
(a) cultivating a mono-culture or co-culture of Methylobacterium in a medium comprising an aqueous phase, a liquid phase and a solid substance, or an emulsion, thereby obtaining a medium containing Methylobacterium;
(b) separating the Methylobacterium from at least one other portion of the medium containing Methylobacterium; Y
(c) reconstitute Methylobacterium into a matrix that lacks substances that promote the growth of resident bacteria on a plant or seed; producing in this way a treatment composition of a plant or seed comprising Methylobacterium that is free of substances that promote the growth of resident microorganisms on a plant or seed.
[24]
24. The method of claim 23, wherein said separation is effected by centrifugation, filtration, or sedimentation of the medium containing Methylobacterium and removal of excess liquid or emulsion thereof.
[25]
25. The method of claim 23, wherein said substance that promotes the growth of resident microorganisms on a plant or seed is selected from the group consisting of a carbon source, a nitrogen source, a phosphorus source, and combinations of the same.
[26]
26. The method of claim 23, wherein said matrix is a liquid, an emulsion, or one or more solids.
[27]
27. The method of claim 23, wherein the matrix comprises an adjuvant and / or
5
10
fifteen
twenty
25
agriculturally acceptable excipient.
[28]
28. The method of claim 23, wherein Methylobacterium is grown in a medium comprising a liquid phase and a solid substance, the solid substance with adherent Methylobacterium grown thereon is separated from the liquid phase of the medium containing Methylobacterium, and the solid substance with adherent Methylobacterium grown thereon is reconstituted in said matrix.
[29]
29. The method of claim 23, wherein said substance bound with adherent Methylobacterium is not a substance that promotes the growth of resident microorganisms on a plant or seed.
[30]
30. A method for treating a plant or seed with a composition comprising Methylobacterium, said method comprising:
(a) preparing said composition according to the method of any one of claims 23 to 29; Y
(b) apply said composition to a plant, a part thereof, or a seed, thus treating the plant or seed with a composition comprising Methylobacterium that is free of substances that promote the growth of resident microorganisms on a plant or seed
[31]
31. The method of claim 30, wherein said composition is applied to said plant, part thereof, or seed as a spray, or wherein said composition is applied to said plant, part thereof or seed by immersion.
[32]
32. The method of claim 30, wherein the applied composition partially covers or covers the plant, the part thereof, or the seed.
[33]
33. A method for treating a plant or seed with Methylobacterium, which comprises applying to a plant or seed a composition comprising a solid substance with adherent Methylobacterium grown thereon and free of substances that promote the growth of resident microorganisms on a plant or seed, thus treating the plant or seed with Methylobacterium.
[34]
34. The method of claim 33, wherein said composition is applied to said plant or a part thereof as a spray, or said composition is applied to said plant, part thereof or seed by immersion.
[35]
35. The method of claim 33, wherein the applied composition is coated or coated.
5
10
fifteen
twenty
25
partially the plant, the part of it, or the seed.
[36]
36. The method of claim 33, wherein the solid substance with adherent Methylobacterium is not a substance that promotes the growth of resident microorganisms on a plant or seed.
[37]
37. The method of any one of claims 33 to 36, wherein the composition comprises an adjuvant and / or excipient acceptable from the agricultural point of view.
[38]
38. A composition comprising a solid substance with adherent Methylobacterium grown thereon, wherein said composition is free of substances that promote the growth of resident microorganisms on a plant or seed.
[39]
39. The composition of claim 38, wherein said substance that promotes the growth of resident microorganisms on a plant or seed is selected from the group consisting of a carbon source, a nitrogen source, a phosphorus source, and combinations of the same.
[40]
40. The composition of claim 38, further comprising an adjuvant and / or excipient acceptable from the agricultural point of view.
[41]
41. The composition of claim 38, wherein the composition is a solid composition and the Methylobacterium are in a range of about 1x106 CFU / g to about 1x1014 CFU / g.
[42]
42. The composition of claim 41, wherein said adherent Methylobacterium are in a range of at least about 5 x 108 CFU / g to at least about 5 x 1013 CFU / g.
[43]
43. The composition of claim 38, wherein the composition is a liquid or an emulsion containing the solid substance and the Methylobacterium are in a range of about 1x106 CFU / mL to about 1X1011 CFU / mL.
[44]
44. The composition of claim 43, wherein the Methylobacteriums are in a range of about 5 x 108 CFU / mL to about 1X1011 CFU / mL.
[45]
45. The composition of claim 38, wherein the solid substance with adherent Methylobacterium grown thereon is not a substance that promotes the growth of resident microorganisms on a plant or seed.
[46]
46. The composition of any one of claims 38-45, wherein the Methylobacterium is selected from the group consisting of NLS0017 (NRRL B- 50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL B-
50941), NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932), NLS0046 (NRRL B-
5 50929), NLS0062 (NRRL B-50937), NLS0064 (NRRL B-50938), NLS0065 (NRRL B-
50935), NLS0066 (NRRL B-50940), NLS0068 (NRRL B-50934), NLS0069 (NRRL B-
50936), NLS0089 (NRRL B-50933), and derivatives thereof.

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