US 2016.007 3641A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0073641 A1 Allen et al. (43) Pub. Date: Mar. 17, 2016

(54) MICROBAL INOCULANT FORMULATIONS Publication Classification (71) Applicant: Newleaf Symbiotics, Inc., St. Louis, (51) Int. Cl. MO (US) AOIN 63/02 (2006.01) C05F II/08 (2006.01) (72) Inventors: Kimberly Allen, Ballwin, MO (US); CI2N I/04 (2006.01) Gregg Bogosian, Clarkson Valley, MO (52) U.S. Cl. (US) CPC A0IN 63/02 (2013.01); C12N I/04 (2013.01); C05F II/08 (2013.01) (21) Appl. No.: 14/856,020 (57) ABSTRACT Compositions comprising dried formulations of viable (22) Filed: Sep. 16, 2015 Methylobacterium as well as methods for making and using the formulations are provided. In particular the compositions Related U.S. Application Data provide for Methylobacterium formulations with improved (60) Provisional application No. 62/051,028, filed on Sep. viability, shelf-life, and plant or seed treatment characteris 16, 2014. tics. Patent Application Publication Mar. 17, 2016 Sheet 1 of 5 US 2016/0073641 A1

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US 2016/007 3641 A1 Mar. 17, 2016

MICROBAL INOCULANT FORMULATIONS growth, plant yield, seed germination, male fertility, and plant nutritional qualities has been disclosed in U.S. Pat. No. 5,512. CROSS-REFERENCE TO RELATED 069, U.S. Pat. No. 5,961,687, U.S. Pat. No. 6,174,837, U.S. APPLICATIONS Pat. No. 6,329,320, U.S. Pat. No. 7,435,878, and US Patent Application Pub. No. 2006/0228797. In addition, PPFM bac 0001. This U.S. Non-provisional patent application teria have been found to increase the yield of cultivated algae, claims the benefit of the U.S. Provisional Patent Application Suggesting their application to the production of algae-de No. 62/051,028, filed on Sep. 16, 2014 and incorporated rived biofuels (US Patent Application Pub. No. 2011/ herein by reference in its entirety. 0269219). BACKGROUND SUMMARY 0002 One-carbon organic compounds such as methane 0005 Provided herein are methods for efficient produc and methanol are found extensively in nature, and are utilized tion of large quantities of Methylobacterium. These methods as carbon Sources by bacteria classified as methanotrophs and can result in high titer Methylobacterium cultures where pro methylotrophs. Methanotrophic bacteria include species in duction time per batch is significantly reduced. The methods the genera Methylobacter, Methylomonas, Methylomicro of Methylobacterium production provided herein can also use bium, Methylococcus, Methylosinus, Methylocystis, Methy culture medium comprised of inexpensive and readily avail losphaera, Methylocaldum, and Methylocella (Lidstrom, able components. Also provide herein are useful fermentation 2006). Methanotrophs possess the enzyme methane broths, fermentation broth products, fermentation products, monooxygenase, that incorporates an atom of oxygen from and compositions comprising Methylobacterium. Methods of O into methane, forming methanol. All methanotrophs are using the fermentation broths, fermentation broth products, obligate one-carbon utilizers, unable to use compounds con fermentation products, and compositions comprising Methy taining carbon-carbon bonds. Methylotrophs, on the other lobacterium to treat plants or plant parts are also provided hand, can also utilize more complex organic compounds, herein. The methods and compositions provided herein can Such as organic acids, higher alcohols, Sugars, and the like. be used to produce large quantities of Methylobacterium for Thus, methylotrophic bacteria are facultative methylotrophs. application to plants or plant parts, for use as an inoculum in Methylotrophic bacteria include species in the genera Methy bioremediation, for production of useful products, and for lobacterium, Hyphomicrobium, Methylophilus, Methyloba production of recombinant proteins. Useful products obtain cillus, Methylophaga, Aminobacter, Methylorhabdus, Methy able by the methods and compositions provided herein lopilla, Methylosulfonomonas, Marinosulfonomonas, include, but are not limited to, poly-3-hydroxybutyric acid, Paracoccus, Xanthobacter, Ancylobacter (also known as 1,3-propanediol, and oxazopyrroloquinolines. Microcyclus). Thiobacillus, Rhodopseudomonas, Rhodo 0006 Provided herein are fermentation broths comprising bacter, Acetobacter, Bacillus, Mycobacterium, Arthobacter, a liquid phase and a solid phase that can be suspended therein, and Nocardia (Lidstrom, 2006). wherein the Solid phase comprises a Solid Substance wherein 0003 Most methylotrophic bacteria of the genus Methy a mono-culture or co-culture of Methylobacterium is adhered lobacterium are pink-pigmented. They are conventionally thereto and wherein the fermentation broth is essentially free referred to as PPFM bacteria, being pink-pigmented faculta of contaminating microorganisms. In certain embodiments, tive methylotrophs. Green (2005, 2006) identified twelve the broth can further comprise one or more microorganisms validated species in the genus Methylobacterium, specifically of pre-determined identity other than Methylobacterium. In M. aminovorans, M. chloromethanicum, M. dichlo certain embodiments, the Solid phase comprises at least about romethanicum, M. extorquens, M. filjisawaense, M. mesophi 0.02% to about 0.5% of the broth by mass. In certain embodi licum, M. Organophilum, M. radiotolerans, M. rhodesianum, ments, the Solid Substance is of animal, plant, microbial, M. rhodinum, M. thiocyanatum, and M. zatmanii. However, fungal, or mineral origin. In certain embodiments, the Solid M. nidulans is a nitrogen-fixing Methylobacterium that is not Substance is an agriculturally acceptable adjuvant or agricul a PPFM (Sy et al., 2001). Methylobacterium are ubiquitous in turally acceptable excipient. In certain embodiments, the nature, being found in soil, dust, fresh water, sediments, and Solid Substance is a polymer. In certain embodiments, the leaf Surfaces, as well as in industrial and clinical environ Solid Substance comprises a polysaccharide, diatomaceous ments (Green, 2006). earth, or a salt crystal. In certain embodiments, the polysac 0004. The existence of PPFM bacteria as colonizers of the charide is selected from the group consisting of a cellulosic leaf Surfaces of most (if not all) species of plants (ranging polysaccharide, a chitinous polysaccharide, and a galactan from algae, mosses and liverworts, and angiosperms and polysaccharide. In certain embodiments, the Methylobacte gymnosperms) suggests that PPFM bacteria may play an rium are at atiter of at least about 5x10 colony-forming units important role in plant physiology (Corpe and Rheem, 1989; per milliliter, at least about 1x10 colony-forming units per Holland and Polacco, 1994; Holland, 1997; Kutschera, milliliter, at least about 1x10" colony-forming units per mil 2007). The fact that plants produce and excrete methanol, liliter, or at least about 3x10" colony-forming units per mil probably as a waste product of pectin metabolism in growing liliter. In certain embodiments, the Methylobacterium are at a plant cell walls, Suggested to these researchers that a symbi titerofat least about 5x10 colony-forming units permilliliter otic relationship exists, with the PPFM bacteria feeding on to at least about 6x10' colony-forming units per milliliter. In the plant-produced methanol and in turn providing positive certain embodiments, at least one of the Methylobacterium is benefits to the plants. The suggested benefits of PPFM bac a Pink Pigmented Facultative Methylotroph (PPFM). In cer teria on plant physiology include positive effects on nitrogen tain embodiments, the Pink Pigmented Facultative Methy metabolism, seed germination, and stimulation of plant lotroph (PPFM) is selected from the group consisting of M. growth through the provision of PPFM-generated cytokinin aminovorans, M. chloromethanicum, M. dichloromethani plant hormones. The use of PPFM bacteria to improve plant cum, M. extorquens, M. fijisawaense, M. mesophilicum, M. US 2016/007 3641 A1 Mar. 17, 2016

Organophilum, M. radiotolerans, M. rhodesianum, M. rhodi growth of the Methylobacterium and wherein the media is num, M. thiocyancilium, M. cerastii, M. gossipicola, Methy essentially free of contaminating microorganisms. In certain lobacterium sp. strain LMG6378, M. phyllosphaerae, M. embodiments, the media further comprises one or more Olyzae, M. platani, M. populi, and M. zatmanii. In certain microorganisms of pre-determined identity other than Methy embodiments, at least one of the Methylobacterium is M. lobacterium. In certain embodiments, the Solid phase com nodulans. In certain embodiments of any of the aforemen prises at least about 0.02% to about 0.5% of the media by tioned broths, at least 10% of the Methylobacterium in the mass. In certain embodiments, the Solid Substance is an agri fermentation broth are Methylobacterium that are adhered to culturally acceptable adjuvant or agriculturally acceptable the solid substance. In certain embodiments of any of the aforementioned broths, the solid is not a photosynthetic excipient. In certain embodiments, the solid Substance pro microorganism. vides for adherent growth of the Methylobacterium. In certain embodiments, the Solid Substance is a polymer or is of animal, 0007 Also provided are fermentation broth products or plant, microbial, fungal, or mineral origin. In certain embodi fermentation products comprising a solid phase that can be ments, the Solid Substance comprises a polysaccharide, diato Suspended in liquid, wherein the Solid phase comprises a solid maceous earth, or a salt crystal. In certain embodiments, the Substance wherein a mono-culture or co-culture of Methyllo polysaccharide is selected from the group of a cellulosic bacterium is adhered thereto, and wherein the fermentation polysaccharide, a chitinous polysaccharide, and a galactan broth product or fermentation product is essentially free of polysaccharide. In certain embodiments, the growing com contaminating microorganisms. In certain embodiments, the prises the steps of inoculating the media with the Methylo fermentation broth product or fermentation product further bacterium and incubating the inoculated media under condi comprises one or more microorganisms of pre-determined tions sufficient to provide for growth of the identity other than Methylobacterium. In certain embodi Methylobacterium. In certain embodiments, the Methylobac ments, the Solid Substance comprises a plurality of Suspen terium are inoculated into the media at a titer of at least about sible particles with adherent Methylobacterium. In certain 5x10" colony-forming units per milliliter or at least about embodiments, the Solid Substance is an agriculturally accept 1x10 colony-forming units per milliliter. In certain embodi able adjuvant or agriculturally acceptable excipient. In cer ments, the Methylobacterium is selected from the group con tain embodiments, the Methylobacterium titer of the solid sisting of M. aminovorans, M. chloromethanicum, M. dichlo phase is at least about 5x10 colony-forming units per gram to romethanicum, M. extorquens, M. filjisawaense, M. at least about 6x10" colony-forming units of Methylobacte mesophilicum, M. Organophilum, M. radiotolerans, M. rho rium per gram of solid. In certain embodiments of any of the desianum, M. rhodinum, M. thiocyanatum, M. nodulans, M. aforementioned fermentation broth products or fermentation cerastii, M. gossipicola, Methylobacterium sp. Strain products, the Solid Substance is not a photosynthetic micro LMG6378. M. phyllosphaerae, M. oryzae, M. platani, M. organism. populi, and M. zatmanii. In certain embodiments, at least 10% 0008 Also provided are compositions comprising a plu of the viable Methylobacterium in the fermentation broth are rality of particles that can be suspended in a liquid, wherein adherent Methylobacterium. In certain embodiments, a titer each of the particles comprise a solid Substance wherein a of at least about 5x10 colony-forming units per milliliter to mono-culture or co-culture of Methylobacterium is adhered about 6x10" colony-forming units per milliliter is attained. thereto and wherein the solid substance is essentially free of In certain embodiments, the titer of at least about 5x10 contaminating microorganisms. In certain embodiments, the colony-forming units per milliliter to about 6x10" colony composition further comprises one or more microorganisms forming units per milliliter is attained within about 48 hours, of pre-determined identity other than Methylobacterium. In about 72 hours, or about 96 hours of inoculating the media certain embodiments, the Solid Substance comprises an agri with Methylobacterium. In certain embodiments of any of the culturally acceptable adjuvant oran agriculturally acceptable aforementioned methods, the method further comprises the excipient. In certain embodiments, the composition further step of harvesting the Methylobacterium. In certain embodi comprises at least one of an agriculturally acceptable adju ments of any of the aforementioned methods, the solid sub vant, an agriculturally acceptable excipient, and/or a pesti stance is not a photosynthetic microorganism. Also provided cide. In certain embodiments, the composition is an essen are Methylobacterium mono-cultures or co-cultures obtained tially dry product, an emulsion, or a suspension. In certain by any of the aforementioned methods. In certain embodi embodiments, each of the particles is a particle of about 2 ments, the mono-culture or co-culture of Methylobacterium microns to about 1000 microns in average length or average is essentially free of contaminating microorganisms. Also diameter. In certain embodiments, the Methylobacterium titer provided are Methylobacterium culture products obtained of the particles is at least about 5x10 colony-forming units from the Methylobacterium monoculture or co-culture per gram to at least about 6x10" colony-forming units of obtained by the methods, wherein the Methylobacterium cul Methylobacterium per gram of particles. In certain embodi ture product comprises a plurality of particles that can be ments of any of the aforementioned compositions, the density Suspended in a liquid and the particles comprise a solid Sub of adherent Methylobacterium on the particles is at least stance wherein a mono-culture or co-culture of Methylobac about 1 Methylobacterium/20 square micrometers of particle terium is adhered thereto. Also provided are compositions Surface area. In certain embodiments of any of the aforemen comprising the Methylobacterium culture product, fermenta tioned compositions, the solid Substance is not a photosyn tion broth product, or fermentation product. In certain thetic microorganism. embodiments, the culture product, fermentation broth prod 0009. Also provided are methods for culturing Methylo uct, or fermentation product comprises a mono-culture or bacterium comprising growing a mono-culture or co-culture co-culture of Methylobacterium that is essentially free of of Methylobacterium in media that comprises a liquid phase contaminating microorganisms. In certain embodiments, the and a solid phase that can be suspended therein, wherein the composition further comprises an agriculturally acceptable Solid phase comprises a solid Substance that provides for adjuvant, an agriculturally acceptable excipient, and/or a pes US 2016/007 3641 A1 Mar. 17, 2016

ticide. Also provided hereinare methods for treating a plant or tain embodiments of any of the aforementioned methods, the a plant part with Methylobacterium comprising the step of Solid Substance is not a photosynthetic microorganism. Also applying to the plant or plant parta Methylobacterium mono provided are plants or plant parts obtained by any of the cultures or co-culture, fermentation broth product, fermenta aforementioned methods, wherein the plant or plant part is at tion product, or composition obtained by any of the afore least partially coated with an exogenously applied solid Sub mentioned methods. In certain embodiments of any of the stance wherein a mono-culture or co-culture of Methylobac aforementioned Methylobacterium mono-cultures or co-cul terium is adhered thereto. In certain embodiments, the plant tures, fermentation broth products, fermentation products, or part is a seed, stem, root, flower, cotyledon, a coleoptile, or a compositions, the Solid Substance is not a photosynthetic leaf. Also provided are processed plant products obtained microorganism. from any of the aforementioned plants or plant parts, wherein the processed product contains an exogenous solid Substance 0010 Also provided are methods for treating a plant or a wherein a mono-culture or co-culture of Methylobacterium is plant part with Methylobacterium comprising the step of adhered thereto. In certain embodiments, the plant product is applying to the plant or plant part a composition comprising a meal, paste, flour, flake, or feed. In certain embodiments, a solid Substance wherein a mono-culture or co-culture of any of the aforementioned processed products is non-regen Methylobacterium is adhered thereto. In certain embodi erable. In certain embodiments of any of the aforementioned ments, the mono-culture or co-culture of Methylobacterium plants, plant parts, or processed products, the Solid Substance is essentially free of contaminating microorganisms. In cer is not a photosynthetic microorganism. tain embodiments, the Solid Substance is an agriculturally acceptable adjuvant oran agriculturally acceptable excipient. 0011. Also provided are methods for producing an indus In certain embodiments, the composition is an essentially dry trial product comprising growing a mono-culture or co-cul product, an emulsion, or a suspension. In certain embodi ture of Methylobacterium in media that comprises a liquid ments, the Solid Substance comprises a plurality of Suspen phase and a solid phase that can be suspended therein, sible particles. In certain embodiments, each of the Suspen wherein the Solid phase comprises a Solid Substance that sible particles is a particle of about 2 microns to about 1000 provides for growth of the Methylobacterium and wherein the microns in length or diameter. In certain embodiments, the media is essentially free of contaminating microorganisms, plant part is a seed and the composition has a Methylobacte and harvesting the industrial product from the Solid phase, the rium titer of at least about 5x10 colony-forming units per liquid phase, or the combination thereof after growing the gram to about 6x10" colony-forming units per gram of the Methylobacterium. In certain embodiments, the solid sub composition. In certain embodiments, the Methylobacterium stance provides for adherent growth of the Methylobacte titer of the composition is at least about 5x10 colony-form rium. In certain embodiments, the industrial product is a ing units per gram to at least about 6x10" colony-forming polymeric precursor, a biopolymer, a precursor of a medicinal units of Methylobacterium per gram of the composition. In compound, a medicinal compound, or a recombinant protein. certain embodiments, the plant part is a seed, stem, root, In certain embodiments of any of the aforementioned meth flower, cotyledon, a coleoptile, or a leaf. In certain embodi ods, the industrial product is poly-3-hydroxybutyric acid, ments, the plant is a corn, Brassica sp., alfalfa, rice, rye, 1,3-propanediol, a pyrroloquinolinequinone, or an oxazopy Sorghum, pearl millet, proso millet, foxtail millet, finger mil rroloquinoline. In certain embodiments of any of the afore let, Sunflower, safflower, soybean, tobacco, potato, peanuts, mentioned methods, the Solid Substance is not a photosyn cotton, Sweet potato, cassava, coffee, coconut, pineapple, thetic microorganism. citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, 0012. Also provided herein are methods for obtaining a olive, papaya, cashew, macadamia, almond, Sugar beet, Sug Methylobacterium preparation comprising growing a mono arcane, oat, barley, tomato, lettuce, green bean, lima bean, culture or co-culture of Methylobacterium in media that com pea, cucurbit, ornamental, or conifer plant. In certain embodi prises a liquid phase and a solid phase, wherein the Solid ments, the plant is a cereal plant and the part is a seed, a phase provides for increased yield of the Methylobacterium coleoptile, and/or a leaf. In certain embodiments, the plant is relative to yield obtained by growing the Methylobacterium a cereal plant, the part is a seed, and the composition is in liquid media alone. In certain embodiments, the methods applied in an amount Sufficient to provide for an increase in further comprise harvesting Methylobacterium grown in the nodal root growth in a cereal plant grown from the treated media. In certain embodiments, at least substantially all of the seed. In certain embodiments, the plant is a cereal plant, the Solid phase is suspended in the liquid phase, or at least Sub part is a coleoptile, and/or a leaf, and the composition is stantially all of the Solid phase is not suspended in the liquid applied in an amount Sufficient to provide for an increase in phase, or portions of the solid phase are Suspended in the nodal root growth in a cereal plant comprising the treated liquid phase and portions of the Solid phase are not suspended coleoptile and/or a leaf. In certain embodiments of any of the in the liquid phase. In certain embodiments, the media further aforementioned methods, the cereal plant is selected from the comprises one or more non-photosynthetic microorganisms group consisting of corn, barley, millet, oat, rice, rye, Sor of pre-determined identity other than Methylobacterium. In ghum, Triticale, and wheat. In certain embodiments, the plant certain embodiments, the Solid phase comprises at least about is a corn plant and the partis a seed, a coleoptile, and/or a leaf. 0.02% to about 20% of the media by mass. In certain embodi In certain embodiments, the plant is a corn plant, the part is a ments, the Solid phase is an agriculturally acceptable adjuvant seed, and the composition is applied in an amount Sufficient to or agriculturally acceptable excipient. In certain embodi provide for an increase in corn nodal root growth in a corn ments, the solid phase provides for adherent growth of the plant grown from the treated seed. In certain embodiments, Methylobacterium and/or the solid phase does not serve as a the plant is a corn plant, the part is a coleoptile, and/or a leaf. carbon source for the Methylobacterium. In certain embodi and the composition is applied in an amount Sufficient to ments, the Solid phase comprises a solid Substance selected provide for an increase in corn nodal root growth in a corn from the group consisting of a man-made material, a material plant comprising the treated coleoptile and/or a leaf. In cer of animal origin, a material of plant origin, a material of US 2016/007 3641 A1 Mar. 17, 2016 microbial origin, a material of fungal origin, a material of the composition further comprises an agriculturally accept mineral origin, and combinations thereof. In certain embodi able adjuvant or an agriculturally acceptable excipient. In ments, the Solid Substance is inanimate. In certain embodi certain embodiments of the methods, the composition is an ments, the Solid phase comprises a solid Substance selected essentially dry product, an emulsion, or a Suspension. In from the group consisting of a polysaccharide, a diatoma certain embodiments of the methods, the plant part is a seed ceous earth, a salt crystal, and combinations thereof. In cer and the composition has a Methylobacterium titer of at least tain embodiments, the Solid phase comprises a polysaccha about 5x10 colony-forming units per gram of the composi ride is selected from the group of a cellulosic polysaccharide, tion to about 6x10', 3x10', 5x10", 1x10', or 5x10' a chitinous polysaccharide, and a galactan polysaccharide. In colony-forming units per gram of the composition. In certain certain embodiments, growing the mono-culture or co-cul embodiments of the methods, the plant part is a seed, stem, ture of Methylobacterium comprises the steps of inoculating root, flower, cotyledon, a coleoptile, a fruit, or a leaf. In the media with the Methylobacterium and incubating the certain embodiments of the methods, the plant or plant part is inoculated media under conditions sufficient to provide for a corn, Brassica sp., alfalfa, rice, rye, Sorghum, pearl millet, growth of the Methylobacterium. In certain embodiments, proso millet, foxtail millet, finger millet, sunflower, safflower, either: (i) the solid phase comprises at least about 0.02% to Soybean, tobacco, potato, peanuts, cotton, Sweet potato, cas about 0.5% of the media and substantially all of the solid sava, coffee, coconut, pineapple, citrus trees, cocoa, tea, phase is suspended in the liquid phase; or (ii) the Solid phase banana, avocado, fig, guava, mango, olive, papaya, cashew, comprises at least about 0.02% to about 20% of the media macadamia, almond, Sugar beet, Sugarcane, oat, barley, and: (a) Substantially all of the solid phase is not Suspended in tomato, lettuce, green bean, lima bean, pea, cucurbit, orna the liquid phase; or (b) portions of the Solid phase are sus mental, or conifer plant part. pended in the liquid phase and portions of the Solid phase are 0015. Also provided are plants or plant parts obtained by not Suspended in the liquid phase. In certain embodiments, any of the aforementioned methods, wherein the plant or the Methylobacterium is selected from the group consisting plant part is at least partially coated with an exogenous solid of M. aminovorans, M. chloromethanicum, M. dichlo Substance wherein a mono-culture or co-culture of Methyllo romethanicum, M. extorquens, M. filjisawaense, M. mesophi bacterium is adhered thereto. Processed plant products licum, M. Organophilum, M. radiotolerans, M. rhodesianum, obtained from the plants or plant parts obtained by any of the M. rhodinum, M. thiocyanatum, M. nodulans, M. cerastii, M. aforementioned methods, wherein the processed product gossipicola. Methylobacterium sp. strain LMG6378. M. contains an exogenous solid Substance wherein a mono-cul phyllosphaerae, M. oryzae, M. platani, M. populi, and M. ture or co-culture of Methylobacterium is adhered thereto are Zatmanii. In certain embodiments, at least 10% of the viable also provided. In certain embodiments, the processed plant Methylobacterium in the fermentation broth are Methylobac product is a meal, paste, flour, flake, or feed. In certain terium that are adhered to the solid phase. In certain embodi embodiments, the processed plant product is non-regener ments, the Solid Substance is not a photosynthetic microor able. ganism, and/or the media is essentially free of contaminating 0016. Also provided are methods for obtaining a Methy microorganisms. In certain embodiments, the harvesting lobacterium preparation comprising: (i) growing a mono comprises recovering all or a portion of the Solid phase with culture or co-culture of Methylobacterium in either: (a) a Methylobacterium adhered thereto and/or recovering all or a culture vessel that comprises or contains one or more solid portion of non-adherent Methylobacterium from the liquid surfaces that provide for adherent growth of the Methylobac phase. In certain embodiments, the methods further comprise terium; or, (b) media that comprises a liquid phase and a solid disassociating some or all of the Solid phase with Methyllo phase, wherein the solid phase provides for increased yield of bacterium adhered thereto. In certain embodiments, the the Methylobacterium relative to yield obtained by growing methods further comprise drying the disassociated or par the Methylobacterium in liquid media alone; and, (ii) harvest tially disassociated material. In certain embodiments, the ing Methylobacterium adhered to the solid surface or the solid methods further comprise: i) drying the solid phase with phase. In certain embodiments of the methods, harvesting Methylobacterium adhered thereto that had been separated comprises removal of the Methylobacterium from the solid from the liquid phase; or, ii) drying the Solid phase with surface or the solid phase by of exposing the Methylobacte Methylobacterium adhered thereto and non-adherent Methy lobacterium that were recovered from the liquid phase. In rium to one or more of a physical and/or a chemical treatment. certain embodiments, the methods further comprise disasso 0017. In certain embodiments of the methods, the chemi ciating some or all of either: i) the dried solid phase with cal treatment comprises one or more of anionic strength shift, Methylobacterium adhered thereto; or, ii) the dried solid a pH shift, a detergent treatment, a solvent treatment, and/or phase with Methylobacterium adhered thereto and non-ad an enzymatic treatment. In certain embodiments of the meth herent Methylobacterium. ods, the enzymatic treatment comprises exposing Methyllo bacterium adhered to the solid surface or to the solid phase to 0013 Also provided are Methylobacterium preparations a protease, a lipase, a glucanase, or any combination thereof. obtained by any of the aforementioned methods, wherein the In certain embodiments of the methods, the detergent treat Methylobacterium preparation comprises the Solid Substance ment comprises exposing Methylobacterium adhered to the wherein a mono-culture or co-culture of Methylobacterium is Solid Surface or the solid phase to an ionic detergent, a non adhered thereto. In certain embodiments, the solid substance ionic detergent, or any combination thereof. In certain in the preparation is not a photosynthetic microorganism. embodiments of the methods, the physical treatment com 0014. Also provided are methods for treating a plant or a prises exposing Methylobacterium adhered to the Solid Sur plant part with Methylobacterium that comprise the step of face or to the Solid phase to Sonication, Scraping, a pressurized applying to the plant or plant part a composition comprising liquid, a pressurized slurry, heat, or any combination thereof. the Methylobacterium preparation made by any of the afore In certain embodiments of the methods: (i) at least substan mentioned methods. In certain embodiments of the methods, tially all of the Solid phase is suspended in the liquid phase; or US 2016/007 3641 A1 Mar. 17, 2016

(ii) at least substantially all of the solid phase is not suspended vant and/or an agriculturally acceptable excipient. In certain in the liquid phase; or (iii) portions of the Solid phase are embodiments, the solid Substance comprises a plurality of Suspended in the liquid phase and portions of the Solid phase particles with Methylobacterium adhered thereto. In certain are not suspended in the liquid phase. In certain embodiments embodiments, the particles comprise particles of about 2 of the methods, the solid phase provides for adherent growth microns to about 1000 microns in average length or average of the Methylobacterium and/or the solid phase does not serve diameter. In certain embodiments, the Methylobacterium titer as a carbon source for the Methylobacterium. In certain of the particles is at least about 5x10 colony-forming units embodiments of the methods, the solid phase comprises a per gram of particles to at least about 6x10', 3x10', 5x10', Solid Substance selected from the group consisting of a man 1x10", or 5x10" colony-forming units of Methylobacterium made material, a material of animal origin, a material of plant per gram of particles. In certain embodiments, the Solid Sub origin, a material of microbial origin, a material of fungal stance is inanimate. In certain embodiments, the Solid Sub origin, a material of mineral origin, and combinations thereof. stance is selected from the group consisting of a man-made In certain embodiments of the methods, the solid substance is material, a material of animal origin, a material of plant inanimate. In certain embodiments of the methods, the Solid origin, a material of microbial origin, a material of fungal phase comprises a solid Substance selected from the group origin, a material of mineral origin, and combinations thereof. consisting of a polysaccharide, a diatomaceous earth, a salt In certain embodiments, the solid substance is selected from crystal, and combinations thereof. In certain embodiments of the group consisting of a polysaccharide, a diatomaceous the methods, the polysaccharide is selected from the group of earth, a salt crystal, and combinations thereof. In certain a cellulosic polysaccharide, a chitinous polysaccharide, and a embodiments, the polysaccharide is selected from the group galactan polysaccharide. In certain embodiments of any of of a cellulosic polysaccharide, a chitinous polysaccharide, the aforementioned method, the methods can further com and a galactan polysaccharide. In certain embodiments, the prise the step of drying the harvested Methylobacterium. Solid Substance is essentially free of contaminating microor 0018. Also provided herein are fermentation products ganisms. In certain embodiments, the composition is essen comprising a solid Substance wherein a mono-culture or co tially free of contaminating microorganisms. In certain culture of Methylobacterium is adhered thereto, wherein the embodiments, the composition and/or the Solid Substance can Solid Substance is not a photosynthetic microorganism, and further comprise one or more microorganisms of pre-deter wherein the fermentation product is essentially free of con mined identity other than Methylobacterium. In certain taminating microorganisms. In certain embodiments, the fer embodiments, the Solid Substance comprises an agriculturally mentation product further comprises one or more microor acceptable adjuvant oran agriculturally acceptable excipient. ganisms of pre-determined identity other than In certain embodiments, the Solid Substance is not a photo Methylobacterium. In certain embodiments, the solid sub synthetic microorganism. In certain embodiments, the com stance comprises one or more of: (i) a plurality of suspensible position further comprises at least one pesticide and/or at particles with adherent Methylobacterium; (ii) a solid sub least one bacteriostatic agent. In certain embodiments, the stance that cannot be suspended infermentation broth; or (iii) pesticide is selected from the group consisting of an insecti a solid Substance wherein a portion of the Substance can be cide, a fungicide, a nematocide, and a bacteriocide, wherein suspended in fermentation broth and a portion of the sub the pesticide does not substantially inhibit growth of the stance cannot be suspended in fermentation broth. In certain Methylobacterium. In certain embodiments, the composition embodiments, the Solid Substance is inanimate. In certain is an essentially dry product, an emulsion, or a suspension. In embodiments, the Solid Substance is selected from the group certain embodiments, the density of adherent Methylobacte consisting of a man-made material, a material of animal ori rium on the solid substance is at least about 1 Methylobacte gin, a material of plant origin, a material of microbial origin, rium/20 square micrometers of particle Surface area. a material of fungal origin, a material of mineral origin, and 0020. Also provided herein are methods for treating a combinations thereof. In certain embodiments, the solid sub plant or a plant part with Methylobacterium comprising the stance is selected from the group consisting of a polysaccha step of applying to the plant or plant part any of the afore ride, a diatomaceous earth, a salt crystal, and combinations mentioned compositions. In certain embodiments, the plant thereof. In certain embodiments, the polysaccharide is part is a seed, stem, root, flower, cotyledon, a coleoptile, fruit, selected from the group of a cellulosic polysaccharide, a or a leaf. In certain embodiments, the plant or plant part is a chitinous polysaccharide, and a galactan polysaccharide. In corn, Brassica sp., alfalfa, rice, rye, Sorghum, pearl millet, certain embodiments, the solid Substance is an agriculturally proso millet, foxtail millet, finger millet, sunflower, safflower, acceptable adjuvant or agriculturally acceptable excipient. In Soybean, tobacco, potato, peanuts, cotton, Sweet potato, cas certain embodiments of any of the aforementioned fermenta sava, coffee, coconut, pineapple, citrus trees, cocoa, tea, tion products, the Methylobacterium titer of the solid phase is banana, avocado, fig, guava, mango, olive, papaya, cashew, at least about 5x10 colony-forming units per gram of solid to macadamia, almond, Sugar beet, Sugarcane, oat, barley, at least about 6x10', 3x10', 5x10', 1x10'', or 5x10' tomato, lettuce, green bean, lima bean, pea, cucurbit, orna colony-forming units of Methylobacterium per gram of solid. mental, or conifer plant or plant part. Also provided are plants In certain embodiments of any of the aforementioned fermen obtained by any of the aforementioned methods, wherein the tation products, the density of adherent Methylobacterium on plant is at least partially coated with the fermentation product the solid substance is at least about 1 Methylobacterium/20 of the composition. In certain embodiments, the plant is a square micrometers of particle Surface area. corn plant, the part is a seed, and the composition is applied in 0019. Also provided are compositions that comprise a fer an amount Sufficient to provide for an increase in corn nodal mentation product comprising a solid Substance wherein a root growth in a corn plant grown from the treated seed. In mono-culture or co-culture of Methylobacterium is adhered certain embodiments, the plant is a corn plant, the part is a thereto. In certain embodiments, the composition further coleoptile, and/or a leaf, and said composition is applied in an comprises at least one of an agriculturally acceptable adju amount Sufficient to provide for an increase in corn nodal root US 2016/007 3641 A1 Mar. 17, 2016 growth in a corn plant comprising the treated coleoptile and/ about 1000 microns in average length or average diameter. In or leaf. Also provided are plant parts obtained by any of the certain embodiments, the Methylobacterium titer of the par aforementioned methods, wherein the plant part is at least ticles is at least about 5x10 colony-forming units per gram of partially coated with the fermentation product of the compo particles to at least about 6x10', 3x10', 5x10'', 1x10', or sition. In certain embodiments, the plant part is a seed, stem, 5x10" colony-forming units of Methylobacterium per gram root, flower, cotyledon, a coleoptile, fruit, or a leaf. of particles. In certain embodiments, the density of adherent 0021. Also provided are plants that are at least partially Methylobacterium on the solid substance is at least about 1 coated with a fermentation product comprising a solid Sub Methylobacterium/20 square micrometers of particle surface stance wherein a mono-culture or co-culture of Methylobac area. In certain embodiments, the Solid Substance is inani terium is adhered thereto. In certain embodiments, the plant is mate. In certain embodiments, the solid Substance is selected selected from the group consisting of a corn, Brassica sp., from the group consisting of a man-made material, a material alfalfa, rice, rye, Sorghum, pearl millet, proso millet, foxtail of animal origin, a material of plant part origin, a material of millet, finger millet, Sunflower, safflower, soybean, tobacco, microbial origin, a material of fungal origin, a material of potato, peanuts, cotton, Sweet potato, cassava, coffee, coco mineral origin, and combinations thereof. In certain embodi nut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, ments, the Solid Substance is selected from the group consist guava, mango, olive, papaya, cashew, macadamia, almond, ing of a polysaccharide, a diatomaceous earth, a salt crystal, Sugar beet, Sugarcane, oat, barley, tomato, lettuce, greenbean, and combinations thereof. In certain embodiments, the lima bean, pea, cucurbit, ornamental, and conifer plant. In polysaccharide is selected from the group of a cellulosic certain embodiments, the Solid Substance comprises a plural polysaccharide, a chitinous polysaccharide, and a galactan ity of particles with adherent Methylobacterium. In certain polysaccharide. In certain embodiments, the Solid Substance embodiments, the particles are about 2 microns to about 1000 is essentially free of contaminating microorganisms. In cer microns in average length or average diameter. In certain tain embodiments, the Solid Substance further comprises one embodiments, the Methylobacterium titer of the particles is at or more microorganisms of pre-determined identity other least about 5x10 colony-forming units per gram of particles than Methylobacterium. In certain embodiments, the solid to at least about 6x10', 3x10', 5x10", 1x10', or 5x10' Substance comprises an agriculturally acceptable adjuvant or colony-forming units of Methylobacterium per gram of par an agriculturally acceptable excipient. In certain embodi ticles. In certain embodiments, the density of adherent Methy ments, the Solid Substance is not a photosynthetic microor lobacterium on the solid substance is at least about 1 Methy ganism. In certain embodiments, the composition further lobacterium/20 square micrometers of particle surface area. comprises at least one pesticide. In certain embodiments, the In certain embodiments, the Solid Substance is inanimate. In pesticide is selected from the group consisting of an insecti certain embodiments, the solid substance is selected from the cide, a fungicide, a nematocide, and a bacteriocide, wherein group consisting of a man-made material, a material of ani the pesticide does not inhibit the Methylobacterium. In cer mal origin, a material of plant origin, a material of microbial tain embodiments of any of the aforementioned embodi origin, a material of fungal origin, a material of mineral ments, the plant part is a seed. origin, and combinations thereof. In certain embodiments, 0023. Also provided are processed plant products the solid Substance is selected from the group consisting of a obtained from any of the aforementioned plants or plant parts, polysaccharide, a diatomaceous earth, a salt crystal, and com wherein the processed product contains a detectable amount binations thereof. In certain embodiments, the polysaccha of an exogenous solid Substance wherein a mono-culture or ride is selected from the group of a cellulosic polysaccharide, co-culture of Methylobacterium is adhered thereto. In certain a chitinous polysaccharide, and a galactan polysaccharide. In embodiments, the processed product is a meal, paste, flour, certain embodiments, the solid substance is essentially free of flake, or feed. In certain embodiments, the processed product contaminating microorganisms. In certain embodiments, the is non-regenerable. Solid Substance further comprises one or more microorgan 0024 Methods for obtaining a dried preparation of viable isms of pre-determined identity other than Methylobacte Methylobacterium comprising: (a) combining a fermentation rium. In certain embodiments, the Solid Substance comprises product comprising a solid Substance having a mono-culture an agriculturally acceptable adjuvant or an agriculturally or co-culture of Methylobacterium adhered thereto with at acceptable excipient. In certain embodiments, the Solid Sub least one cryoprotection agent to obtain a biphasic mixture stance is not a photosynthetic microorganism. that comprises the cryoprotection agent and a Solid phase 0022. Also provided are plant parts that are at least par comprising the fermentation product; and, (b) lyophilizing tially coated with a composition that comprises a fermenta the biphasic mixture, thereby obtaining the dried preparation tion product comprising a solid Substance wherein a mono of viable Methylobacterium are provided. In certain embodi culture or co-culture of Methylobacterium is adhered thereto. ments, the cryoprotection agent is selected from the group In certain embodiments, the plant part is selected from the consisting of disaccharide, poly Sucrose, hydroxyethylcellu group consisting of a corn, Brassica sp., alfalfa, rice, rye, lose, and combinations thereof. In certain embodiments the Sorghum, pearl millet, proso millet, foxtail millet, finger mil fermentation product has a Methylobacterium titer of 5x10, let, Sunflower, safflower, soybean, tobacco, potato, peanuts, 1x10, or 1x10" colony-forming units per gram of solid to cotton, Sweet potato, cassava, coffee, coconut, pineapple, about 5x10" colony-forming units of Methylobacterium per citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, gram of the solid Substance. In certain embodiments, the olive, papaya, cashew, macadamia, almond, Sugar beet, Sug disaccharide is selected from the group consisting of Sucrose, arcane, oat, barley, tomato, lettuce, green bean, lima bean, lactose, trehalose, and combinations thereof. In certain pea, cucurbit, ornamental, and conifer plant part. In certain embodiments, the poly Sucrose has a molecular weight of embodiments, the solid Substance comprises a plurality of about 60 to about 80 kilodaltons (kD) or about 300 to about particles with adherent Methylobacterium. In certain embodi 500 kilodaltons (kD). In certain embodiments, the cryopro ments, the particles comprise particles of about 2 microns to tection agent is Sucrose. In certain embodiments, the US 2016/007 3641 A1 Mar. 17, 2016 hydroxyethylcellulose has a molecular weight of about 600 product comprising a solid Substance having a mono-culture kilodaltons (kD) to about 700 kilodaltons (kD). In certain or co-culture of Methylobacterium adhered thereto with at embodiments, the methods can further comprise the addition least one cryoprotection agent to obtain a biphasic mixture ofaprotein of plant, animal or microbial originto the biphasic that comprises the cryoprotection agent and a Solid phase mixture. In certain embodiments, the Solid Substance is comprising the fermentation product; and, (b) lyophilizing selected from the group consisting of a man-made material, a the biphasic mixture, thereby obtaining the dried preparation material of animal origin, a material of plant origin, a material of viable Methylobacterium are provided. In certain embodi of microbial origin, a material of fungal origin, a material of ments, the cryoprotection agent is selected from the group mineral origin, and combinations thereof. In certain embodi consisting of disaccharide, poly Sucrose, hydroxyethylcellu ments, the Solid Substance is an agriculturally acceptable lose, and combinations thereof. In certain embodiments the adjuvant or agriculturally acceptable excipient. In certain fermentation product has a Methylobacterium titer of 5x10, embodiments, the Solid Substance is inanimate. In certain embodiments, the Solid Substance is selected from the group 1x10, or 1x10" colony-forming units per gram of solid to consisting of a polysaccharide, a diatomaceous earth, a salt about 5x10" colony-forming units of Methylobacterium per crystal, and combinations thereof. In certain embodiments, gram of the solid Substance. In certain embodiments, the the polysaccharide is selected from the group of a cellulosic disaccharide is selected from the group consisting of Sucrose, polysaccharide, a chitinous polysaccharide, a galactan lactose, trehalose, and combinations thereof. In certain polysaccharide, and combinations thereof. In certain embodi embodiments, the poly Sucrose has a molecular weight of ments of any of the aforementioned methods, the methods can about 60 to about 80 kilodaltons (kD) or about 300 to about further comprise converting the dried preparation to a pow 500 kilodaltons (kD). In certain embodiments, the cryopro der. In certain embodiments of any of the aforementioned tection agent is Sucrose. In certain embodiments, the methods, the methods can further comprise the addition of an hydroxyethylcellulose has a molecular weight of about 600 agriculturally acceptable adjuvant or agriculturally accept kilodaltons (kD) to about 700 kilodaltons (kD). In certain embodiments, the methods can further comprise the addition able excipient. In certain embodiments of any of the afore ofaprotein of plant, animal or microbial originto the biphasic mentioned methods, the methods can further comprise the mixture. In certain embodiments, the Solid Substance is step of applying the dried preparation of viable Methylobac selected from the group consisting of a man-made material, a terium to a plant or plant part. In certain embodiments of any material of animal origin, a material of plant origin, a material of the aforementioned methods, at least about 50%, 60%, or of microbial origin, a material of fungal origin, a material of 70% of Methylobacterium cells in the dried preparation are mineral origin, and combinations thereof. In certain embodi viable. In certain embodiments of any of the aforementioned ments, the Solid Substance is an agriculturally acceptable methods, the dried preparation is substantially free of water, adjuvant or agriculturally acceptable excipient. In certain essentially free of water, or essentially dry. Also provided are embodiments, the Solid Substance is inanimate. In certain compositions comprising the dried preparation of viable embodiments, the Solid Substance is selected from the group Methylobacterium made by any of the aforementioned meth consisting of a polysaccharide, a diatomaceous earth, a salt ods. Also provided are plants or plant parts that are at least crystal, and combinations thereof. In certain embodiments, partially coated with the compositions. In certain embodi the polysaccharide is selected from the group of a cellulosic ments, the plant part is selected from the group consisting of polysaccharide, a chitinous polysaccharide, a galactan a seed, stem, root, flower, cotyledon, a coleoptile, fruit, and a polysaccharide, and combinations thereof. In certain embodi leaf. In certain embodiments, the plant part is a seed. In ments of any of the aforementioned methods, the methods can certain embodiments, the seed is a lettuce seed and is coated further comprise converting the dried preparation to a pow with about 1x10 CFU of Methylobacterium per seed to about der. In certain embodiments of any of the aforementioned 1x107 CFU of the Methylobacterium per seed. In certain methods, the methods can further comprise the addition of an embodiments, the seed is a lettuce seed and is coated with agriculturally acceptable adjuvant or agriculturally accept about 1x10 CFU of Methylobacterium per seed to about able excipient. In certain embodiments of any of the afore 5x10 CFU of the Methylobacterium per seed. In certain mentioned methods, the methods can further comprise the embodiments, the Methylobacterium is heterologous to the step of applying the dried preparation of viable Methylobac plant or plant part that is at least partially coated with the terium to a plant or plant part. In certain embodiments of any composition. In certain embodiments, the Methylobacterium of the aforementioned methods, at least about 50%, 60%, or is heterologous to the seed, stem, root, flower, cotyledon, a 70% of Methylobacterium cells in the dried preparation are coleoptile, fruit, or leaf that is at least partially coated with the viable. In certain embodiments of any of the aforementioned composition. In certain embodiments, any of the aforemen methods, the dried preparation is substantially free of water, tioned compositions, plants or plant parts can comprise or be essentially free of water, or essentially dry. Also provided are at least partially coated with an isolated Methylobacterium compositions comprising the dried preparation of viable selected from the group consisting of NLS0017 (NRRL Methylobacterium made by any of the aforementioned meth B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL ods. Also provided are plants or plant parts that are at least B-50939), NLS0037 (NRRL B-50941), NLS0038 (NRRL partially coated with the compositions. In certain embodi B-50942), NLS0042 (NRRL B-50932), NLS0046 (NRRL ments, the plant part is selected from the group consisting of B-50929), NLS0062 (NRRL B-50937), NLS0064 (NRRL a seed, stem, root, flower, cotyledon, a coleoptile, fruit, and a B-50938), NLS0065 (NRRL B-50935), NLS0066 (NRRL leaf. In certain embodiments, the plant part is a seed. In B-50940), NLS0068 (NRRL B-50934), NLS0069 (NRRL certain embodiments, the seed is a lettuce seed and is coated B-50936), NLS0089 (NRRL B-50933), and derivatives with about 1x10 CFU of Methylobacterium per seed to about thereof. 1x107 CFU of the Methylobacterium per seed. In certain 0025 Methods for obtaining a dried preparation of viable embodiments, the seed is a lettuce seed and is coated with Methylobacterium comprising: (a) combining a fermentation about 1x10 CFU of Methylobacterium per seed to about US 2016/007 3641 A1 Mar. 17, 2016

5x10 CFU of the Methylobacterium per seed. In certain 0033. As used herein, the phrase "agriculturally accept embodiments, the Methylobacterium is heterologous to the able adjuvant” refers to a substance that enhances the perfor plant or plant part that is at least partially coated with the mance of an active agent in a composition for treatment of composition. In certain embodiments, the Methylobacterium plants and/or plant parts. In certain compositions, an active is heterologous to the seed, stem, root, flower, cotyledon, a agent can comprise a mono-culture or co-culture of Methy coleoptile, fruit, or leaf that is at least partially coated with the lobacterium. composition. In certain embodiments, any of the aforemen 0034. As used herein, the phrase "agriculturally accept tioned compositions, plants or plant parts can comprise or be able excipient” refers to an essentially inert substance that can at least partially coated with an isolated Methylobacterium be used as a diluent and/or carrier for an active agent in a selected from the group consisting of NLS0017 (NRRL composition for treatment of plants. In certain compositions, B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL an active agent can comprise a mono-culture or co-culture of B-50939), NLS0037 (NRRL B-50941), NLS0038 (NRRL Methylobacterium. B-50942), NLS0042 (NRRL B-50932), NLS0046 (NRRL 0035. As used herein, the term “algae' refers to any type of B-50929), NLS0062 (NRRL B-50937), NLS0064 (NRRL micro- or macroalgae. B-50938), NLS0065 (NRRL B-50935), NLS0066 (NRRL 0036. As used herein, the term “Methylobacterium” refers B-50940), NLS0068 (NRRL B-50934), NLS0069 (NRRL to bacteria that are facultative methylotrophs of the genus B-50936), NLS0089 (NRRL B-50933), and derivatives Methylobacterium. The term Methylobacterium, as used thereof. herein, thus does not encompass include species in the genera Methylobacter, Methylomonas, Methylomicrobium, Methylo BRIEF DESCRIPTION OF THE DRAWINGS coccus, Methylosinus, Methylocystis, Methylosphaera, 0026. The accompanying drawings, which are incorpo Methylocaldum, and Methylocella, which are obligate metha rated in and form a part of the specification, illustrate certain notrophs. embodiments of the present invention. In the drawings: 0037. As used herein, the phrase “co-culture of Methylo 0027 FIG. 1 is a photomicrograph of an aliquot of a fer bacterium” refers to a Methylobacterium culture comprising mentation product comprising liquid media, a Solid (diatom at least two strains of Methylobacterium or at least two spe shells), and Methylobacterium. The solid diatom shell and cies of Methylobacterium. adherent Methylobacterium are indicated by the labels in the 0038. As used herein, the phrase “contaminating microor photomicrograph. The Methylobacterium strain is ganism” refers to microorganisms in a culture, fermentation DSM-6343 Methylobacterium extorquens. broth, fermentation broth product, or composition that were 0028 FIG. 2 is a photomicrograph of an aliquot of a fer not identified prior to introduction into the culture, fermenta mentation product comprising liquid media, a Solid (diatom tion broth, fermentation broth product, or composition. shells), and Methylobacterium. The solid diatom shell, adher 0039. As used herein, the phrase “derivatives thereof, ent Methylobacterium, and non-adherent Methylobacterium when used in the context of a Methylobacterium isolate, are indicated by the labels in the photomicrograph. The refers to any strain that is obtained from the Methylobacte Methylobacterium strain is DSM-6343 Methylobacterium rium isolate. Derivatives of a Methylobacterium isolate extorquens. include, but are not limited to, variants of the strain obtained 0029 FIG. 3 A, B, C are photographs of test tubes con by selection, variants of the strain selected by mutagenesis taining liquid media with non-particulate Solid Substances and selection, and a genetically transformed strain obtained with adherent Methylobacterium. In 3A, liquid media con from the Methylobacterium isolate. taining cotton tufts with adherent Methylobacterium that 0040. As used herein, the phrase “essentially free of con impart a dark pink color to the cotton are shown. In 3B, liquid taminating microorganisms' refers to a culture, fermentation media containing glass wool with adherent Methylobacte broth, fermentation product, or composition where at least rium that impart a pink color to the glass wool are shown. In about 95% of the microorganisms present by amount or type 3C, liquid media containing body scrub material with adher in the culture, fermentation broth, fermentation product, or ent Methylobacterium that impart a pink color to the body composition are the desired Methylobacterium or other scrub material are shown. The Methylobacterium strain is desired microorganisms of pre-determined identity. DSM-6343 Methylobacterium extorquens. 0041 As used herein, the term "heterologous', when used 0030 FIG. 4 is a photomicrograph of a showing PPFM in the context of Methylobacterium that at least partially coats strain ATCC-35065 M.fijisawaense adhered to cotton fibers. a plant or plant part, refers to a Methylobacterium that is not The cotton fibers, adherent Methylobacterium, and non-ad naturally associated with a plant or plant part of the same herent Methylobacterium are indicated by the labels in the species as the plant or plant part that is at least partially coated photomicrograph. with the Methylobacterium. In certain embodiments, the het 0031 FIG. 5 is a high-magnification photograph of three erologous Methylobacterium that is used to at least partially lettuce seeds that are coated with a dried preparation of coat a plant or plant part of a first plant species is a Methyllo Methylobacterium that were adhered to diatomaceous earth bacterium that was isolated, or can be isolated, from a second (bottom 3 seeds) and an uncoated control seed (labelled “Non-powdered seed' at top of photo). and distinct plant species. 0042. As used herein, the phrase “inanimate solid sub DESCRIPTION stance” refers to a substance which is insoluble or partially soluble in water or aqueous Solutions and which is either Definitions non-living or which is not a part of a still-living organism 0032. As used herein, the phrases “adhered thereto' and from which it was derived. “adherent” refer to Methylobacterium that are associated with 0043. As used herein, the phrase “mono-culture of Methy a Solid Substance by growing, or having been grown, on a lobacterium” refers to a Methylobacterium culture consisting Solid Substance. of a single strain of Methylobacterium. US 2016/007 3641 A1 Mar. 17, 2016

0044 As used herein, a "pesticide” refers to an agent that Methylobacterium in liquid media with a particulate solid is insecticidal, fungicidal, nematocidal, bacteriocidal, or any Substance that can be suspended in the liquid by agitation combination thereof. under conditions that provide for Methylobacterium growth. 0045. As used herein, the phrase “bacteriostatic agent 99 In certain embodiments where particulate Solid Substances refers to agents that inhibit growth of bacteria but do not kill are used, at least substantially all of the solid phase canthus be the bacteria. Suspended in the liquid phase upon agitation. Such particulate 0046. As used herein, the phrase “pesticide does not sub Solid Substances can comprise materials that are about 1 mil stantially inhibit growth of said Methylobacterium” refers to limeter or less in length or diameter. In certain embodiments, any pesticide that when provided in a composition compris the degree of agitation is sufficient to provide for uniform ing a fermentation product comprising a solid Substance distribution of the particulate solid substance in the liquid wherein a mono-culture or co-culture of Methylobacterium is phase and/or optimal levels of culture aeration. However, in adhered thereto, results in no more than a 50% inhibition of other embodiments provided herein, at least substantially all Methylobacterium growth when the composition is applied to of the Solid phase is not suspended in the liquid phase, or a plant or plant part in comparison to a composition lacking portions of the solid phase are suspended in the liquid phase the pesticide. In certain embodiments, the pesticide results in and portions of the Solid phase are not Suspended in the liquid no more than a 40%, 20%, 10%, 5%, or 1% inhibition of phase. Non-particulate Solid Substances can be used in certain Methylobacterium growth when the composition is applied to biphasic media where the Solid phase is not Suspended in the a plant or plant part in comparison to a composition lacking liquid phase. Such non-particulate solid Substances include, the pesticide. but are not limited to, materials that are greater than about 1 0047. As used herein, the term “PPFM bacteria' refers millimeter in length or diameter. Such particulate and non without limitation to bacterial species in the genus Methyllo particulate Solid Substances also include, but are not limited bacterium other than M. nodulans. to, materials that are porous, fibrous, or otherwise configured 0048. As used herein, the phrase “solid substance' refers to provide for increased surface areas for adherent growth of to a substance which is insoluble or partially soluble in water the Methylobacterium. Biphasic media where portions of the or aqueous Solutions. Solid phase are Suspended in the liquid phase and portions of 0049. As used herein, the phrase “solid phase that can be the Solid phase are not suspended in the liquid phase can suspended therein” refers to a solid substance that can be comprise a mixture of particulate and non-particulate solid distributed throughout a liquid by agitation. Substances. Such particulate and non-particulate Solid Sub 0050. As used herein, the term “non-regenerable' refers to stances used in any of the aforementioned biphasic media also either a plant part or processed plant product that cannot be include, but are not limited to, materials that are porous, regenerated into a whole plant. fibrous, or otherwise configured to provide for increased sur 0051. As used herein, the phrase “substantially all of the face areas for adherent growth of the Methylobacterium. In Solid phase is Suspended in the liquid phase' refers to media certain embodiments, the methods can comprise obtaining a wherein at least 95%, 98%, or 99% of solid substance(s) biphasic culture media comprising the liquid, the Solid, and comprising the Solid phase are distributed throughout the Methylobacterium and incubating the culture under condi liquid by agitation. tions that provide for growth of the Methylobacterium. 0052. As used herein, the phrase “substantially all of the Biphasic culture medias comprising the liquid, the Solid, and Solid phase is not suspended in the liquid phase' refers to Methylobacterium can be obtained by a variety of methods media where less than 5%, 2%, or 1% of the solid is in a that include, but are not limited to, any of: (a) inoculating a particulate form that is distributed throughout the media by biphasic media comprising the liquid and the Solid Substance agitation. with Methylobacterium: (b) inoculating the solid substance 0053 As used herein, the term "yield', when used in ref with Methylobacterium and then introducing the solid sub erence to Methylobacterium obtained in a fermentation, stance comprising the Methylobacterium into the liquid refers to the numbers of Methylobacterium obtained. Meth media; (c) inoculating the Solid Substance with Methylobac ods for determining Such yield include, but are not limited to, terium, incubating the Methylobacterium on the solid sub determining the numbers of colony forming units (CFU) per stance, and then introducing the solid Substance comprising unit volume or unit mass of material obtained, determining a the Methylobacterium into the liquid media; or (d) any com wet weight of the Methylobacterium obtained, and/or deter bination of (a), (b), or (c). The methods can also further mining a dry weight of the Methylobacterium obtained. comprise the steps of harvesting the mono- or co-culture of 0054) To the extent to which any of the preceding defini Methylobacterium. Methods for harvesting the Methylobac tions is inconsistent with definitions provided in any patent or terium can include, but are not limited to, separating the non-patent reference incorporated herein by reference, any Methylobacterium from the liquid phase by filtration, cen patent or non-patent reference cited herein, or in any patent or trifugation, decanting, and the like. Harvested Methylobacte non-patent reference found elsewhere, it is understood that rium obtained by these methods can be Methylobacterium the preceding definition will be used herein. that are adhered to the solid substance, Methylobacterium that are not adhered to the Solid Substance, and combinations Methods for Culturing Methylobacterium, Compositions, thereof. and Uses Thereof 0056. Agitation methods that can be used include, but are 0055 Methods where Methylobacterium are cultured in not limited to, stirring, reciprocal shaking, rotary shaking, biphasic media comprising a liquid phase and a solid Sub and combinations thereof. In certain embodiments, agitation stance have been found to significantly increase the resultant can comprise placing liquid media containing the Solid Sub yield of Methylobacterium relative to methods where the stances on a rotary shaker that provides at least 25, 50, 100, Methylobacterium are cultured in liquid media alone. In cer 200, 250, 500, or 1000 revolutions per minute (RPM). Agi tain embodiments, the methods can comprise growing the tation equivalent to that provided by a rotary shaker set at least US 2016/007 3641 A1 Mar. 17, 2016

at 25, 50, 100, 200, 250, 500, or 1000 revolutions per minute solid substance with Methylobacterium adhered thereto. In (RPM) can also be obtained by stirring, reciprocal shaking, other embodiments, a solid substance with Methylobacterium and other methods. In certain embodiments, at least Substan adhered thereto, or a solid substance further comprising both tially all of the Solid phase, or a portion of the Solid phase, can adherent Methylobacterium and non-adherent Methylobacte be suspended in the liquid phase upon agitation equivalent to rium, can be dried and then dissociated. In certain embodi that provided by a rotary shaker set at least at 25, 50, 100, 200, ments where the solid substance with Methylobacterium 250, 500, or 1000 revolutions per minute (RPM). adhered thereto, or a solid substance further comprising both 0057. In certain embodiments, harvested material com adherent Methylobacterium and non-adherent Methylobacte prising a solid substance with Methylobacterium adhered rium, is dried and then disassociated, Solid Substances that thereto can be disassociated. Dissociation can be effected by become friable upon drying can be used as the Solid Substance any techniques that permit the Solid Substance with Methyllo in the Methylobacterium fermentation process. Examples of bacterium adhered thereto to be broken into smaller elements. Such solid Substances that become friable upon drying and Disassociation techniques including, but are not limited to, that can be used in methods provided herein include, but are macerating, grinding, crushing, Sonicating, and/or partially not limited to, certain materials of plant origin (e.g. certain dissolving the solid substance with Methylobacterium materials comprising cellulose, hemi-cellulose, and/or lig adhered thereto can be used to break the solid substance with nin), and the like. Methylobacterium adhered thereto into smaller elements. 0059 Biphasic fermentation broths used in the methods Such smaller elements include, but are not limited to, non provided hereincan be axenic cultures that are essentially free particulate solid substances with Methylobacterium adhered of contaminating microorganisms. In certain embodiments, thereto and particles of the solid substance with Methylobac at least about 95%, 98%, 99%, 99.5%, 99.8%, 99.9%, or terium adhered thereto. Such non-particulate and/or particu 100% of the microorganisms present by amount or type in the late solid substances with Methylobacterium adhered thereto culture, fermentation broth, fermentation product, or compo can either be directly applied to plants or plant parts or incor sitions provided herein are the desired Methylobacterium or porated into compositions that can be applied to plants or other desired microorganisms of pre-determined identity. plant parts. In certain embodiments, the Solid Substances with Desired Methylobacterium or other desired microorganisms Methylobacterium adhered thereto are broken into particles of pre-determined identity are microorganisms obtained from of about 1 millimeter in diameter or less. In certain embodi a pure culture. To provide for Such axenic cultures, the liquid ments, a harvested solid substance with Methylobacterium and solid components used in the biphasic culture media are adhered thereto is disassociated into particles of about 2 sterilized or obtained in an essentially sterile form prior to microns to about 1000 microns in average length or average inoculation of Methylobacterium and/or any additional diameter. In certain embodiments, a harvested Solid Sub desired microorganisms in the mono- or co-culture. Steriliza stance with Methylobacterium adhered thereto is disassoci tion of various solid and liquid components can be achieved ated into particles of about 1 microns to about 1000 microns by methods including, but not limited to, autoclaving, irra in average length or average diameter. In certain embodi diation, filter sterilization (for liquids), and the like. A culture, ments, a harvested solid substance with Methylobacterium fermentation broth, fermentation product, or composition adhered thereto is disassociated into particles of about 1, 2, 4, that is essentially free of contaminating microorganisms can 10, 20, or 40 microns to any of about 100, 200, 500, 750, or be obtained where the liquid and/or solid components of that 1000 microns in average length or average diameter. In cer culture, fermentation broth, fermentation product, or compo tain embodiments, the Methylobacterium titer of the particles sition were sterile prior to the inoculation or provision of the obtained by disassociation is at least about 5x10 colony desired microorganisms of pre-determined identity and Suit forming units per gram of particles to at least about 6x10", able steps are taken to avoid contamination of the culture 3x10', 5x10'', 1x10, or 5x10" colony-forming units of during growth of the desired microorganisms or contamina Methylobacterium per gram of particles. In certain embodi tion of the composition. ments, a solid substance with Methylobacterium adhered 0060 Methods provided herein where Methylobacterium thereto will also comprise non-adherent Methylobacterium. are cultured in biphasic media comprising a liquid phase and In certain embodiments, Solid Substances that further com a Solid Substance can be practiced in any of a batch-mode prising both adherent Methylobacterium and non-adherent fermentation, a fed-batch mode fermentation, or a continuous Methylobacterium can also be disassociated to obtain any of fermentation. Fermentation broths, fermentation broth prod the aforementioned fragments or particles. In still other ucts, and compositions provided herein can also be obtained embodiments, solid substances with Methylobacterium from any of a batch-mode fermentation, a fed-batch mode adhered thereto can be disassociated and non-adherent fermentation, or a continuous fermentation. In certain Methylobacterium can then be added to the disassociated embodiments, factors such as the pH and oxygen concentra Solid Substances comprising adherent Methylobacterium. tion can be controlled in any of the batch-mode fermentation, 0058 Solid substances with Methylobacterium adhered fed-batch mode fermentation, or continuous fermentation thereto can be disassociated when they are in either a wet or processes used in the methods provided herein. moist form or a dry form. Drying of the solid substance with 0061 Monocultures or co-cultures of Methylobacterium Methylobacterium can be effected by any technique that and resultant fermentation broths and fermentation broth maintains viability of the majority of the adherent Methylo products provided herein can comprise one or more Methy bacterium, and, when present, non-adherent Methylobacte lobacterium that include, but are not limited to, M. amino rium. Such drying techniques include, but are not limited to, vorans, M. chloromethanicum, M. dichloromethanicum, M. lyophilization, desiccation, heating, and combinations extorquens, M. fijisawaense, M. mesophilicum, M. Organo thereof. In certain embodiments, drying can be thus effected philum, M. radiotolerans, M. rhodesianum, M. rhodinum, M. after disassociation of a solid substance with Methylobacte thiocyanatum, M. nodulans, M. cerasti, M. gossiplicola, rium adhered thereto to obtain fragments or particles of the Methylobacterium sp. strain LMG6378 M. phyllosphaerae US 2016/007 3641 A1 Mar. 17, 2016

M. Oryzae, M. platani, M. populi, and M. zatmanii. In certain TABLE 1 embodiments, monocultures or co-cultures of Methylobacte rium and resultant fermentation broths and fermentation Representative Methylobacterium broth products provided herein can consist of one or more Methyliobacterium Depository Accession Numbers for Type Strain Methylobacterium. However, the methods provided herein Methyliobacterium AR27 - CCM 7305 - CECT 7069 = DSM can also be used on other Methylobacterium. Methylobacte adhaesivum 17169T = KCTC 22099T rium can also be obtained by various published methods Methyliobacterium DSM 19013 = JCM 164O6 =KACC 11766 (Madhaiyan et al., 2007). In certain embodiments, such other aerolatin Methylobacterium that can be used will be Methylobacterium Methyliobacterium ATCC 51358 = CIP 105328 = IFO (now aninovorans NBRC) 15686 = JCM8240 = VKM B-2145 having 16S RNA sequences of at least about 60%, 70%, 80%, Methyliobacterium CCM 7218 CECT 5998 = CIP 1083.33 = 90%, or 95% sequence identity to the 16S RNA sequences of aquatictim DSM 16371 other known Methylobacterium. Typing of Methylobacte Methyliobacterium DSM 19569 - NBRC 103629 - NCIMB 14379 brachiatum rium by use of 16S RNA sequence comparisons is at least Methyliobacterium DSM 21893 = LMG 24788 described by Cao et al., 2011. In certain embodiments, the builiatum Methyliobacterium CCM 7788 - CCUG 6004O = DSM 23679 mono-cultures or co-cultures and resultant products can com cerasii prise a Methylobacterium that can colonize plants and/or Methyliobacterium NCIMB 13688 - VKMB-2223 plant parts. Methylobacterium that can colonize plants and/or chioromethanicum plant parts include, but are not limited to, M. extorquens, M. Methyliobacterium CIP 106787 = DSM 6343 - VKMB-2191 dichloromethanicum nodulans, and M. mesophilicum. Methylobacterium that can Methyliobacterium ATCC 43645 = CCUG 2084 = DSM 1337 = IAM colonize plants and/or plant parts also include, but are not extorquiens 2631 = IFO (now NBRC) 15687 = JCM 2802 = limited to, Methylobacterium cerastii species (with a repre NCCB 78015 = NCIB (now sentative strain available as DSM 23679 from the Leibniz NCIMB) 9399 = VKM B-2064. Methyliobacterium ATCC 43884 = CIP 10377S = DSM5686 = Institute DSMZ-German Collection of Microorganisms and filisawaense FO (now NBRC) 15843 = JCM 10890 = Cell Cultures (“DSMZ), Braunschweig, Germany), Methy NCIB (now NCIMB) 12417 Methyliobacterium CCM7S72 = NRRLB-51692 lobacterium gossipicola species (with a representative strain gossipicola available as NRRL B-51692 from the USDA ARS, Peoria, Methyliobacterium DSM 19564 = NBRC 103626=NCIMB Ill., USA). Methylobacterium sp. strain LMG6378 (available gregains 4376 Methylobacterium GP34 = CCM 7219 = CECT 5997 = CIP from the Belgian Co-ordinated Collection of Micro-organ hispanictim O8332 = DSM 16372 isms/Laboratorium voor Microbiologie (“BCCLM) Ghent, Methylobacterium iners DSM 19015 = JCM 16407 = KACC 11765 Belgium), Methylobacterium phyllosphaerae species (with a Methyliobacterium CCM 73O4 = CECT 7068 isibiiense representative strain available as available as DSM 19779T Methyliobacterium KCTC 12671 = LMG 23639 from the DSMZ), Methylobacterium oryzae species (with a jeotgali representative strain available as DSM 18207T from the Methyliobacterium DSM 19563 = NBRC 103627 =NCIMB DSMZ), Methylobacterium nodulans species (with a repre komagatae 4377 Methyliobacterium CECT 78.06 = DSM 23933 sentative strain available as LMG21967 from the BCCLM), longtin Methylobacterium platani species (with a representative Methyliobacterium DSM 14457 =NCIMB 13779 - VKMB strain available as KCTC 12901 from the Korean Collection lusitantin 2239 for Type Cultures, Yusong-Ku, Taejon, K R (“KCTC), and Methyliobacterium CCUG 56108 = DSM 21328 Methylobacterium populi species (with a representative marchantiae Methyliobacterium ATCC 29983 = CCUG 16482 = CIP strainavailable as ATCC BAA-705 from the ATCC). Fermen mesophilicum O1129 = DSM 1708 - ICPB4095 IFO tation broths, fermentation broth products, compositions, (now NBRC) 15688 = JCM 2829 = LMG methods of making the same, and methods of using the same, 5275 = NCIB (now NCIMB) 11561 = including, but not limited to, methods of treating plants, NRRLB-14246 Methyliobacterium LMG 21967 = ORS 2060 where the Methylobacterium is a Methylobacterium that can nodulans colonize a plant and/or a plant part that is selected from the Methyliobacterium ATCC 27886 = CIP 101.049 = DSM76O = group consisting of M. extorquens. M. nodulans, M. mesophi organophilum HAMBI 2263 = IFO (now NBRC) 15689 = licum, M. cerastii, M. gossipicola, Methylobacterium sp. CM2833 = LMG 6083 = NCCB 78041 = strain LMG6378, M. phyllosphaerae, M. oryzae, M. platani, WKMB-2066 and M. populi are thus provided. Methods of isolating other Methyliobacterium DSM 182O7 = JCM 16405 = KACC 11585 = oryzae LMG 23582 Methylobacterium that can colonize plants and/or plant parts Methyliobacterium DSM 19562 = NBRC 103628 = NCIMB have been described in various publications and can also be persicinum 4378 used (see Madhaiyan et al., and references cited therein). Methyliobacterium DSM 19779 = JCM 16408 =KACC 11716 = Without seeking to be limited by theory, it is believed that the phyllosphaerae LMG 24361 methods of culturing Methylobacterium in a biphasic media Methyliobacterium CM 14648 = KCTC 12901 platani comprising a liquid and a Solid Substance provided hereincan Methyliobacterium ATCC BAA-547 = DSM 15083 be especially advantageous for growing Methylobacterium podarium that can colonize plants and/or plant parts or that were iso Methyliobacterium ATCC BAA-705 - NCIMB 13946 lated from the Surfaces of plants and/or plant parts. populi Methyliobacterium ATCC 27329 = CIP 101.128 = DSM 1819 = 0062 Representative Methylobacterium that can be used radiotoierans IFO (now NBRC) 15690 = JCM2831 = in the fermentation broths, fermentation broth products, com LMG 2269 = NCIB (now NCIMB) 10815 = positions and related methods provided herein include, but WKMB-2144 are not limited to, the Methylobacterium of Table 1. US 2016/007 3641 A1 Mar. 17, 2016

TABLE 1-continued TABLE 2-continued Representative Methyliobacterium Methylobacterium sp. isolates USDAARS Methyliobacterium Depository Accession Numbers for Type Strain NLS NRRL No.

Methyliobacterium ATCC 14821 = CIP 101127 = DSM 2163 = NLSOO69 NRRLB-SO936 rhodinum FO (now NBRC) 15691 = JCM 2811 = NLSOO89 NRRLB-SO933 LMG 2275 = NCIB (now NCIMB) 9421 = WKMB-206S Deposit number for strain deposited with the AGRICULTURAL RESEARCHSERVICE CULTURE COLLECTION (NRRL) of the National Center for Agricultural Utilization Methyliobacterium DSM 14458 = NCIMB 13778 = WKMB Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North Siioniense 2238 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 Methyliobacterium DSM 19566 - NBRC 103632 =NCIMB Procedure. Subject to 37 CFRS1.808(b), all restrictions imposed by the depositor on the tardum 438O availability to the public of the deposited material will be irrevocably removed upon the granting of any patent from this patent application, Methyliobacterium ATCC 7OO647 = DSM 11490 = JCM thiocyanati in O893 - VKMB-2197 Methyliobacterium CCM7281 = CECT 7045 = DSM 1696.1 0064. The use of certain Methylobacterium isolates pro variabile vided in Table 2 to provide useful traits in crop plants such as Methyliobacterium ATCC 43883 - CCUG 369.16 = CIP lettuce, corn, Soybean, fruit plants, and tomato is described in Zainani O3774 = DSM5688 = IFO (now NBRC) co-assigned International Patent Applications PCT/US14/ S845 = JCM 108.92=LMG 6087 - NCIB 68558, PCT/US14/68657, PCT/US14/68660, PCT/US14/ (now NCIMB) 12243 = VKM B-2161 68663, and PCT/US14/68611, respectively. Co-assigned International Patent Applications PCT/US14/68558, PCT/ Depository Key US14/68657, PCT/US14/68660, PCT/US14/68663, and ATCC: American Type Tissue Culture Collection, Manassas, VA, USA PCT/US14/68611, were filed on Dec. 4, 2014, designate the CCUG: Culture Collection, University of Göteborg, Sweden United States, and are all incorporated herein by reference in CDP: Collection de l’InstitutPasteur, Paris, FR their entireties. DSM: DSMZ-German Collection of Microorganisms and Cell Cultures (“DSMZ”), Braun schweig, Germany 0065. In certain embodiments, the mono-cultures or co JCM: Japan Collection of Microorganisms, Saitama, Japan cultures and resultant fermentation broths and fermentation LMG: Belgian Co-ordinated Collection of Micro-organisms Laboratorium voor Microbi ologie (BCCLM”) Ghent, Belgium broth products can comprise one or more Methylobacterium NBRC: Biological Resource Center (NBRC), Chiba, Japan isolates or mutants that produce increased levels of useful NCIMB: National Collections of Industrial, Food and Marine Bacteria, UK nutrients or plant growth regulators. U.S. Pat. No. 8,153,118 NRRL: USDA ARS, Peoria, IL., USA discloses various Methylobacterium isolates that produce increased levels of vitamin B-12 and amino acids that can be 0063 Also provided herein are compositions, methods of used in the methods and compositions provided herein. Fer making the compositions, methods of using the composi mentation broths, fermentation broth products, and compo tions, and plants or plant parts that are at least partially coated sitions that comprise one or more of the Methylobacterium Such as Methylobacterium mutant B12-11 having accession with the compositions, where the compositions or methods number ATCC PTA-1561 that overproduces vitamin B-12, comprise or use any of the following Methylobacterium sp. Methylobacterium rhodinum (ATCC #43282) that over-pro isolates provided in the following Table 1 or derivatives of the duces the amino acid threonine, Methylobacterium sp. isolates. In certain embodiments, such derivatives can include (ATCC #21371) that over-produces the amino acid variants. In certain embodiments, the Methylobacterium are L-glutamic acid, Methylobacterium sp. (ATCC #21372) that lyophilized with a with at least one cryoprotection agent. over-produces the amino acid L-glutamic acid, Methylobac terium sp. (ATCC #21926) over-produces the amino acid Such derivatives can include, but are not limited to, variants of L-lysine, Methylobacterium sp. (ATCC #21969) over-pro the isolates obtained by selection, variants of the isolates duces the amino acid L-glutamic acid, Methylobacterium sp. selected by mutagenesis and selection, and genetically trans (ATCC #21927) over-produces the amino acids L-lysine, formed isolates obtained from the isolates. L-aspartic acid, L-alanine, L-Valine, L-leucine, and L-argin ine, and/or Methylobacterium sp. (ATCC #21438) that pro TABLE 2 duces single-cell protein are also provided. Methylobacterium sp. isolates 0066. In certain embodiments, the fermentation broth, fer mentation broth product, or compositions provided herein USDA ARS can further comprise one or more introduced microorganisms NLS NRRL No. of pre-determined identity other than Methylobacterium. NLSOO17 NRRLB-SO931 Other microorganisms that can be added include, but are not NLSOO20 NRRLB-SO930 limited to, microorganisms that are biopesticidal or provide NLSOO21 NRRLB-SO939 Some other benefit when applied to a plant or plant part. NLSOO37 NRRLB-SO941 NLSOO38 NRRL B-50942 Biopesticidal or otherwise beneficial microorganisms thus NLSOO42 NRRLB-SO932 include, but are not limited to, various Bacillus sp., NLSOO46 NRRLB-SO929 Pseudomonas sp., Coniothyrium sp., Panioea sp., Streptomy NLSOO62 NRRLB-SO937 ces sp., and Trichoderma sp. Microbial biopesticides can be a NLSOO64 NRRLB-SO938 NLSOO6S NRRLB-SO935 bacterium, fungus, virus, or protozoan. Particularly useful NLSOO66 NRRLB-SO940 biopesticidal microorganisms include various Bacillus subli NLSOO68 NRRLB-SO934 lis, Bacillus thuringiensis, Bacillus pumilis, Pseudomonas syringae, Trichoderma harzianum, Trichoderma virens, and US 2016/007 3641 A1 Mar. 17, 2016

Streptomyces lydicus strains. Other microorganisms that are insoluble or only partially soluble carbonates, chromates, added can be genetically engineered or naturally occurring Sulfites, phosphates, hydroxides, oxides, and Sulfides. In cer isolates that are available as pure cultures. In certain embodi tain embodiments, the Solid Substance can be a microbial cell, ments, it is anticipated that the bacterial or fungal microor fungal cell, microbial spore, or fungal spore. In certain ganism can be provided in the fermentation broth, fermenta embodiments, the solid substance can be a microbial cell or tion broth product, or composition in the form of a spore. Still microbial spore wherein the microbial cell or microbial spore other microorganisms that can be added include, but are not is not a photosynthetic microorganism. In certain embodi limited to, microorganisms that are photosynthetic microor ments, the microbial cell or microbial spore is not a photo ganisms. Such photosynthetic organisms include, but are not synthetic microorganism, where the photosynthetic microor limited to, algae. Such algae can include, but are not limited ganism is selected from the group consisting of algae, to, algae of the genii of Prolococcus, Ulva, Codium, Enlero cyanobacteria, diatoms, Botryococcus braunii, Chlorella, morpha, Neochloris, and/or Chlamydomonas. Dunaliella tertiolecta, Gracilaria, Pleurochrysis carterae, 0067. In certain embodiments, the liquid culture medium Sargassum, and Ulva. In still other embodiments, the Solid is prepared from inexpensive and readily available compo Substance can be an inactivated (i.e. inviable) microbial cell, nents, including, but not limited to, inorganic salts such as fungal cell, microbial spore, or fungal spore. In still other potassium phosphate, magnesium Sulfate and the like, carbon embodiments, the Solid Substance can be a quiescent (i.e. Sources Such as glycerol, methanol, glutamic acid, aspartic viable but not actively dividing) microbial cell, fungal cell, acid, Succinic acid and the like, and amino acid blends such as microbial spore, or fungal spore. In still other embodiments, peptone, tryptone, and the like. Exemplary liquid media that the solid substance can be cellular debris of microbial origin. can be used include, but are not limited to, ammonium min In still other embodiments, the solid substance can be par eral salts (AMS) medium (Whittenbury et al., 1970), Vogel ticulate matter from any part of a plant. Plant parts that can be Bonner (VB) minimal culture medium (Vogel and Bonner, used to obtain the solid substance include, but are not limited 1956), and LB broth (“Luria-Bertani Broth'). to, cobs, husks, hulls, leaves, roots, flowers, stems, bark, seeds, and combinations thereof. Products obtained from pro 0068. In general, the solid substance used in the methods cessed plant parts including but not limited to, bagasse, wheat and compositions that provide for the efficient growth of bran, Soy grits, crushed seed cake, Stover, and the like can also Methylobacterium can be any suitable solid substance which be used. Such plant parts, processed plants, and/or processed is insoluble or only partially soluble in water or aqueous plant parts can be milled to obtain the solid material in a Solutions. Such Suitable solid Substances are also non-bacte particulate form that can be used. In certain embodiments, riocidal or non-bacteriostatic with respect to Methylobacte wood or a wood product including, but not limited to, wood rium when the solid substances are provided in the liquid pulp, sawdust, shavings, and the like can be used. In certain culture media. In certain embodiments, such suitable Solid embodiments, the Solid Substance can be a particulate matter Substances are also solid Substances that are readily obtained from an animal(s), including, but not limited to, bone meal, in sterile form or rendered sterile. Solid substances used gelatin, ground or powdered shells, hair, macerated hide, and herein can be sterilized by any method that provides for removal of contaminating microorganisms and thus include, the like. but are not limited to, methods such as autoclaving, irradia 0069. In certain embodiments, the solid substance is pro tion, chemical treatment, and any combination thereof. These vided in a particulate form that provides for distribution of the Solid Substances include natural Substances of animal, plant, Solid Substance in the culture media. In certain embodiments, microbial, fungal, or mineral origin, manmade Substances, or the Solid Substance is comprised of particle of about 2 microns combinations of natural and manmade Substances. In certain to about 1000 microns in average length or average diameter. embodiments, the Solid Substances are inanimate Solid Sub In certain embodiments, the Solid Substance is comprised of stances. Inanimate Solid Substances of animal, plant, micro particle of about 1 microns to about 1000 microns in average bial, or fungal origin can be obtained from animals, plants, length or average diameter. In certain embodiments, the Solid microbes, or fungi that are inviable (i.e. no longer living) or substance is a particle of about 1, 2, 4, 10, 20, or 40 microns that have been rendered inviable. Diatom shells are thus to any of about 100,200,500,750, or 1000 microns in average inanimate solid Substances when previously associated dia length or average diameter. Desirable characteristics of par tomalgae have been removed or otherwise rendered inviable. ticles used in the methods and compositions provided herein Since diatom shells are inanimate solid Substances, they are include suitable wettability such that the particles can be not considered to be photosynthetic organisms or photosyn Suspended throughout the media upon agitation. thetic microorganisms. In certain embodiments, solid Sub 0070. In certain embodiments, the solid substance can be a stances include, but are not limited to, sand, silt, Soil, clay, solid substance that provides for adherent growth of the ash, charcoal, diatomaceous earth and other similar minerals, Methylobacterium on the solid substance. Methylobacterium ground glass or glass beads, ground ceramic materials, that are adhered to a solid substance are Methylobacterium ceramic beads, bentonite, kaolin, talc, perlite, mica, Vermicu that cannot be substantially removed by simply washing the lite, silicas, quartz powder, montmorillonite, and combina solid substance with the adherent Methylobacterium with tions thereof. In certain embodiments, the Solid Substance can growth media whereas non-adherent Methylobacterium can be a polymer or polymeric beads. Polymers that can be used be substantially removed by washing the solid substance with as a Solid Substance include, but are not limited to, various liquid growth media. In this context, “substantially removed polysaccharides such as cellulosic polymers and chitinous means that at least about 30%, 40%, 50%, 60%, 70%, or 80% polymers which are insoluble or only partially soluble in the Methylobacterium present are removed when the solid water or aqueous solutions, agar (i.e. galactans), and combi substance is washed with three volumes of liquid growth nations thereof. In certain embodiments, the Solid Substance media. Such washing can be effected by a variety of methods can be an insoluble or only partially soluble salt crystal. Salt including, by not limited to, decanting liquid from a washed crystals that can be used include, but are not limited to, Solid phase or passing liquid through a solid phase on a filter US 2016/007 3641 A1 Mar. 17, 2016 that permits flow through of bacteria in the liquid. In certain milliliter, at least about 5x10 colony-forming units per mil embodiments, the adherent Methylobacterium that are asso liliter to at least about 4x10" colony-forming units per mil ciated with the solid can include both Methylobacterium that liliter, or at least about 5x10 colony-forming units per mil are directly attached to the solid and/or Methylobacterium liliter to at least about 6x10' colony-forming units per that are indirectly attached to the solid substance. Methylo milliliter. In certain embodiments, fermentation broths pro bacterium that are indirectly attached to the solid substance vided herein can comprise Methylobacterium at a titer of at include, but are not limited to, Methylobacterium that are least about 1x10 colony-forming units per milliliter to at attached to another Methylobacterium or to another microor least about 3x10" colony-forming units permilliliter, at least ganism that is attached to the solid Substance, Methylobacte about 1x10 colony-forming units per milliliter to at least rium that are attached to the solid substance by being attached about 4x10" colony-forming units per milliliter, or at least to another Substance that is attached to the solid Substance, about 1x10 colony-forming units per milliliter to at least and the like. In certain embodiments, at least 10%, 20%, 30%, about 6x10" colony-forming units per milliliter. In certain 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5% or embodiments, fermentation broths provided herein will com 99.9% of the Methylobacterium in the fermentation broth, prise Methylobacterium at a titer of at least about 1x10' fermentation broth product, or compositions are Methylobac colony-forming units per milliliter to at least about 3x10" terium that are adhered to the solid substance. In certain colony-forming units per milliliter, at least about 1x10' embodiments, adherent Methylobacterium can be present on colony-forming units per milliliter to at least about 4x10" the surface of the solid substance in the fermentation broth, colony-forming units per milliliter, or at least about 1x10" fermentation broth product, or composition at a density of at colony-forming units per milliliter to at least about 6x10' least about 1 Methylobacterium/20 square micrometers, of at colony-forming units per milliliter. In certain embodiments, least about 1 Methylobacterium/10 square micrometers, of at fermentation broths provided herein will comprise Methylo least about 1 Methylobacterium/10 square micrometers, of at bacterium at a titer of, at least about 3x10" colony-forming least about 1 Methylobacterium/5 square micrometers, of at units per milliliter to at least about 4x10' colony-forming least about 1 Methylobacterium/2 square micrometers, or of units per milliliter, or at least about 3x10" colony-forming at least about 1 Methylobacterium/square micrometer. In cer units per milliliter to at least about 6x10" colony-forming tain embodiments, adherent Methylobacterium can be units per milliliter. present on the surface of the solid substance in the fermenta tion broth, fermentation broth product, or composition at a 0072 Also provided herein are methods for obtaining a density of at least about 1 Methylobacterium/20 square Methylobacterium preparation where the Methylobacterium micrometers to about 1 Methylobacterium/square microme either are grown in a culture vessel that comprises or contains ter, of at least about 1 Methylobacterium/10 square microme one or more solid surfaces that provide for adherent growth of ters to about 1 Methylobacterium/square micrometer, of at the Methylobacterium or are grown in media that comprises a least about 1 Methylobacterium/10 square micrometers to liquid phase and a solid phase, and then harvested by removal about 1 Methylobacterium/square micrometer, of at least from the solid surface or the solid phase to which they have about 1 Methylobacterium/5 square micrometers to about 1 adhered. In certain embodiments where a solid surface that Methylobacterium/square micrometer, or of at least about 1 provides for adherent growth of the Methylobacterium is Methylobacterium/2 square micrometers to about 1 Methylo used, the solid surface can form part of the culture vessel bacterium/square micrometer. In certain embodiments, itself. In still other embodiments, a solid surface that is con adherent Methylobacterium can be present on the surface of tained in a culture vessel is a solid Surface that can be detached the solid substance in the fermentation broth, fermentation from the culture vessel, particularly after or during the course broth product, or composition at a density of at least about 1 of a fermentation run, to facilitate removal of the adherent Methylobacterium/20 square micrometers to about 1 Methy Methylobacterium. Exemplary and non-limiting Solid Sur lobacterium/2 square micrometers, of at least about 1 Methy faces include beads, rings, cylinder and other shapes that lobacterium/10 square micrometers to about 1 Methylobac provide for improved surface area to volume ratios. Solid terium 2 square micrometers, of at least about 1 Surfaces used in the culture vessel can be either porous or Methylobacterium/10 square micrometers to about 1 Methy Smooth. Exemplary Solid Surfaces used culture vessels can be lobacterium 2 square micrometers, or of at least about 1 made from materials that include, but are not limited to, Methylobacterium/5 square micrometers to about 1 Methylo coated or uncoated metals, glass, plastics, ceramics, or com bacterium/2 square micrometers. Biphasic fermentation binations thereofthat permit adherent growth of Methylobac broths provided herein can comprise a liquid phase that con terium. Following the culturing, Methylobacterium that have tains non-adherent Methylobacterium. In certain embodi adhered to the solid surface or the solid phase can be har ments, titers of non-adherent Methylobacterium in the liquid Vested by one or more of a physical and/or chemical treatment phase can be less than about 100,000, 10,000, or 1,000 CFU/ (s). In certain embodiments of these methods, non-adherent ml. Methylobacterium that have accumulated in the liquid phase can also be harvested. Chemical treatments used to harvest 0071 Biphasic culture methods provided can yield fer the Methylobacterium include, but are not limited to, expos mentation broths with Methylobacterium at a titer of greater ing the adherent Methylobacterium to a shift in ionic strength, than about 5x10 colony-forming units permilliliter, at atiter a shift in pH, a detergent treatment, a solvent treatment, an of greater than about 1x10 colony-forming units per millili enzymatic treatment, and combinations thereof. Enzymatic ter, at a titer of greater than about 1x10" colony-forming treatments used to harvest the Methylobacterium can include, units per milliliter, at a titer of at least about 3x10" colony but are not limited to, exposing Methylobacterium adhered to forming units per milliliter. In certain embodiments, fermen the solid Surface or to the Solid phase to a protease, a lipase, a tation broths provided herein can comprise Methylobacte glucanase, or any combination thereof. Detergent treatments rium at a titer of at least about 5x10 colony-forming units per used to harvest the Methylobacterium can include, but are not milliliter to at least about 3x10" colony-forming units per limited to, exposing Methylobacterium adhered to the solid US 2016/007 3641 A1 Mar. 17, 2016

Surface or the solid phase to an ionic detergent, a non-ionic culturally acceptable adjuvant or an agriculturally acceptable detergent, or any combination thereof. Physical treatments excipient is typically an ingredient that does not cause undue used to harvest the Methylobacterium can include, but are not phytotoxicity or other adverse effects when exposed to a plant limited to, exposing Methylobacterium adhered to the solid or plant part. In certain embodiments, the Solid Substance can Surface or to the solid phase to Sonication, scraping, a pres itself be an agriculturally acceptable adjuvant or an agricul Surized liquid, a pressurized slurry, heat, or any combination turally acceptable excipient so longas it is not bacteriocidal or thereof. In certain embodiments, non-adherent Methylobac bacteriostatic to the Methylobacterium. In other embodi terium can be harvested from the liquid in the culture vessel. ments, the composition further comprises at least one of an In still other embodiments, non-adherent Methylobacterium agriculturally acceptable adjuvant oran agriculturally accept can be harvested from the liquid in the culture vessel and able excipient. Any of the aforementioned compositions can adherent Methylobacterium harvested from the solid surface. also further comprise a pesticide. Pesticides used in the com 0073. Without seeking to be limited by theory, it is position include, but are not limited to, an insecticide, a fun believed that the solid in the biphasic culture media provides gicide, a nematocide, and a bacteriocide. In certain embodi a surface on which Methylobacterium can adhere to and grow ments, the pesticide used in the composition is a pesticide that upon. Such adherent growth on the solid in the biphasic does not substantially inhibit growth of the Methylobacte culture media is believed to be more rapid (i.e. provide for a rium. As Methylobacterium are gram negative bacteria, Suit decreased doubling time) than growth in the absence of the able bacteriocides used in the compositions can include, but solid. It is believed that both the number (i.e. colony forming are not limited to, bacteriocides that exhibit activity against units permilliliter) and density (Methylobacterium per square gram positive bacteria but not gram negative bacteria. Com micrometers) of Methylobacterium will increase during the positions provided herein can also comprise a bacteriostatic course of the fermentation until a maximum number and/or agent that does not substantially inhibit growth of the Methy density is reached. In certain embodiments, it is believed that lobacterium. Bacteriostatic agents suitable for use in compo daughter cells from an adherent mother cell can either grow sitions provided herein include, but are not limited to, those on the Solid Surface, grow on the adherent mother cell, and/or that exhibit activity against gram positive bacteria but not be shed into the liquid phase. It is also thus believed that the gram negative bacteria. Any of the aforementioned composi number (i.e. colony forming units per milliliter) of Methylo tions can also be an essentially dry product (i.e. having about bacterium in the liquid phase can increase until a maximum 5% or less water content), an emulsion, or a Suspension. number is reached. 0077 Agriculturally acceptable adjuvants used in the 0074 Solid substances with adherent Methylobacterium compositions include, but are not limited to, components that can be used to make various compositions useful for treating enhance product efficacy and/or products that enhance ease of plants or plant parts. Alternatively, fermentation broths or product application. Adjuvants that enhance product efficacy fermentation broth products comprising Solid Substances can include various wetters/spreaders that promote adhesion with adherent Methylobacterium can be used to treat plants or to and spreading of the composition on plant parts, Stickers plant parts. Plants, plant parts, and, in particular, plant seeds that promote adhesion to the plant part, penetrants that can that have been at least partially coated with the fermentation promote contact of the active agent with interior tissues, broth products or compositions are thus provided. Also pro extenders that increase the half-life of the active agent by vided are processed plant products that contain the fermen inhibiting environmental degradation, and humectants that tation broth products or compositions. Solid substances with increase the density or drying time of sprayed compositions. adherent Methylobacterium can be used to make various Wetters/spreaders used in the compositions can include, but compositions that are particularly useful for treating plant are not limited to, non-ionic Surfactants, anionic Surfactants, seeds. Seeds that have been at least partially coated with the cationic Surfactants, amphoteric Surfactants, organo-silicate fermentation broth products or compositions are thus pro Surfactants, and/or acidified Surfactants. Stickers used in the vided. Also provided are processed seed products, including, compositions can include, but are not limited to, latex-based but not limited to, meal, flour, feed, and flakes that contain the Substances, terpene/pinolene, and pyrrolidone-based Sub fermentation broth products or compositions provided stances. Penetrants can include mineral oil, vegetable oil, herein. In certain embodiments, the processed plant product esterified vegetable oil, organo-silicate surfactants, and acidi will be non-regenerable (i.e. will be incapable of developing fied Surfactants. Extenders used in the compositions can into a plant). In certain embodiments, the solid Substance include, but are not limited to, ammonium Sulphate, or men used in the fermentation product or composition that at least thene-based substances. Humectants used in the composi partially coats the plant, plant part, or plant seed or that is tions can include, but are not limited to, glycerol, propylene contained in the processed plant, plant part, or seed product glycol, and diethyl glycol. Adjuvants that improve ease of comprises a Solid Substance and associated or adherent product application include, but are not limited to, acidifying/ Methylobacterium that can be readily identified by compar buffering agents, anti-foaming/de-foaming agents, compat ing a treated and an untreated plant, plant part, plant seed, or ibility agents, drift-reducing agents, dyes, and water condi processed product thereof. tioners. Anti-foaming/de-foaming agents used in the 0075) Fermentation broths, fermentation broth products, compositions can include, but are not limited to, dimethopol fermentation products, or other compositions comprising ysiloxane. Compatibility agents used in the compositions can solid substances with adherent Methylobacterium can be used include, but are not limited to, ammonium sulphate. Drift to produce industrial products or recombinant proteins or in reducing agents used in the compositions can include, but are bioremediation. not limited to, polyacrylamides, and polysaccharides. Water 0076 Compositions useful for treating plants or plant conditioners used in the compositions can include, but are not parts that comprise the solid substance with adherent Methy limited to, ammonium Sulphate. lobacterium can also comprise an agriculturally acceptable 0078 Methods of treating plants and/or plant parts with adjuvant or an agriculturally acceptable excipient. An agri the fermentation broths, fermentation broth products, and US 2016/007 3641 A1 Mar. 17, 2016 16 compositions are also provided herein. Treated plants, and provided that comprise the solid substance with Methylobac treated plant parts obtained therefrom, include, but are not terium and air drying the resulting product. Air drying can be limited to, corn, Brassica sp. (e.g., B. napus, B. rapa, B. accomplished at any temperature that is not deleterious to the juncea), alfalfa, rice, rye, Sorghum, millet (e.g., pearl millet seed or the Methylobacterium, but will typically not be (Pennisetum glaucum), proso millet (Panicum miliaceum), greater than 30 degrees Centigrade. The proportion of coating foxtail millet (Setaria italica), finger millet (Eleusine cora that comprises a solid Substance and Methylobacterium cana)), Sunflower, safflower, soybean, tobacco, potato, pea includes, but is not limited to, a range of 0.1 to 25% by weight nuts, cotton, Sweet potato (Ipomoea batatus), cassava, coffee, of the seed, 0.5 to 5% by weight of the seed, and 0.5 to 2.5% coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, by weight of seed. In certain embodiments, a Solid Substance fig, guava, mango, olive, papaya, cashew, macadamia, used in the seed coating or treatment will have Methylobac almond, Sugar beets, Sugarcane, oats, barley, tomatoes let terium adhered thereon. In certain embodiments, a solid sub tuce, greenbeans, lima beans, peas, cucurbits such as cucum stance used in the seed coating or treatment will be associated ber, cantaloupe, and muskmelon, ornamentals, and conifers. with Methylobacterium and will be a fermentation broth, Plant parts that are treated include, but are not limited to, fermentation broth product, or composition obtained by the leaves, stems, flowers, roots, seeds, fruit, tubers, coleoptiles, methods provided herein. Various seed treatment composi and the like. Ornamental plants and plant parts that can be tions and methods for seed treatment disclosed in U.S. Pat. treated include, but are not limited to azalea, hydrangea, Nos. 5,106,648, 5.512,069, and 8,181,388 are incorporated hibiscus, roses, tulips, daffodils, petunias, carnation, poinset herein by reference in their entireties and can be adapted for tia, and chrysanthemum. Conifer plants and plant parts that use with an active agent comprising the fermentation broths, can be treated include, but are not limited to, pines such as fermentation broth products, or compositions provided loblolly pine, slash pine, ponderosa pine, lodgepole pine, and herein. In certain embodiments, the composition used to treat Monterey pine; Douglas-fir, Western hemlock; Sitka spruce: the seed can contain agriculturally acceptable excipients that redwood; true firs such as silver fir and balsam fir, and cedars include, but are not limited to, woodflours, clays, activated such as Western redcedar and Alaska yellow-cedar. Turfgrass carbon, diatomaceous earth, fine-grain inorganic Solids, cal plants and plant parts that can be treated include, but are not cium carbonate and the like. Clays and inorganic Solids that limited to, annual bluegrass, annual ryegrass, Canada blue can be used with the fermentation broths, fermentation broth grass, fescue, bentgrass, wheatgrass, Kentucky bluegrass, products, or compositions provided herein include, but are orchardgrass, ryegrass, redtop, Bermuda grass, St. Augustine not limited to, calcium bentonite, kaolin, china clay, talc, grass, and Zoysia grass. Seeds or other propagules of any of perlite, mica, Vermiculite, silicas, quartz powder, montmoril the aforementioned plants can be treated with the fermenta lonite and mixtures thereof. Agriculturally acceptable adju tion broths, fermentation broth products, fermentation prod vants that promote sticking to the seed that can be used ucts, and/or compositions provided herein. include, but are not limited to, polyvinyl acetates, polyvinyl 0079. In certain embodiments, plants and/or plant parts acetate copolymers, hydrolyzed polyvinyl acetates, polyvi are treated by applying the fermentation broths, fermentation nylpyrrolidone-vinyl acetate copolymer, polyvinyl alcohols, broth products, fermentation products, and compositions as a polyvinyl alcohol copolymers, polyvinyl methyl ether, poly spray. Such spray applications include, but are not limited to, vinyl methyl ether-maleic anhydride copolymer, waxes, latex treatments of a single plant part or any combination of plant polymers, celluloses including ethylcelluloses and methyl parts. Spraying can be achieved with any device that will celluloses, hydroxy methylcelluloses, hydroxypropylcellu distribute the fermentation broths, fermentation broth prod lose, hydroxymethylpropylcelluloses, polyvinyl pyrroli ucts, fermentation products, and compositions to the plant dones, alginates, dextrins, malto-dextrins, polysaccharides, and/or plant part(s). Useful spray devices include a boom fats, oils, proteins, karayagum, jaguar gum, tragacanth gum, sprayer, a hand or backpack sprayer, crop dusters (i.e. aerial polysaccharide gums, mucilage, gum arabics, shellacs, spraying), and the like. Spraying devices and or methods vinylidene chloride polymers and copolymers, soybean providing for application of the fermentation broths, fermen based protein polymers and copolymers, lignoSulfonates, tation broth products, fermentation products, and composi acrylic copolymers, starches, polyvinylacrylates, Zeins, gela tions to either one or both of the adaxial surface and/or abaxial tin, carboxymethylcellulose, chitosan, polyethylene oxide, Surface can also be used. Plants and/or plant parts that are at acrylimide polymers and copolymers, polyhydroxyethyl least partially coated with any of a biphasic fermentation acrylate, methylacrylimide monomers, alginate, ethylcellu broth, a fermentation broth product, fermentation product, or lose, polychloroprene and syrups or mixtures thereof. Other compositions that comprise a solid Substance with Methyllo useful agriculturally acceptable adjuvants that can promote bacterium adhered thereto are also provided herein. Also coating include, but are not limited to, polymers and copoly provided herein are processed plant products that comprise a mers of vinyl acetate, polyvinylpyrrolidone-vinyl acetate solid substance with Methylobacterium adhered thereto. copolymer and water-soluble waxes. Various Surfactants, dis 0080. In certain embodiments, seeds are treated by expos persants, anticaking-agents, foam-control agents, and dyes ing the seeds to the fermentation broths, fermentation broth disclosed herein and in U.S. Pat. No. 8,181,388 can be products, fermentation products, and compositions provided adapted for use with an active agent comprising the fermen herein. Seeds can be treated with the fermentation broths, tation broths, fermentation broth products, or compositions fermentation broth products, and compositions provided provided herein. herein by methods including, but not limited to, imbibition, 0081. Use of the fermentation broths, fermentation broth coating, spraying, and the like. Seed treatments can be products, and compositions provided hereinto promote nodal effected with both continuous and/or a batch seed treaters. In root formation in cereal plants is also provided herein. Early certain embodiments, the coated seeds may be prepared by development of a vigorous nodal root system is important in slurrying seeds with a coating composition containing a fer establishing stands of cereal plant crops that include, but are mentation broth, fermentation broth product, or compositions not limited to, corn, barley, millet, oat, rice, rye, Sorghum, US 2016/007 3641 A1 Mar. 17, 2016

triticale, and wheat. The first roots that emerge from a cereal milliliter to at least about 6x10" colony-forming units per plant seed (the radicle and seminal roots) function mainly in milliliter. In certain embodiments, an amount of a fermenta the uptake of water from the soil. The radicle seminal roots do tion broth product provided herein that is sufficient to provide not provide other nourishment, which early in the growth of for an increase in nodal root growth in a cereal plant can be a the seedling is provided by energy and nutrient reserves in the fermentation broth product with a Methylobacterium titer of kernel. When nodal roots emerge from the cereal plant stem, the solid phase of that product is at least about 5x10 colony growth of the seminal roots slows dramatically and they con forming units per gram to at least about 6x10" colony-form tribute little to the season-long maintenance of the cereal ing units of Methylobacterium per gram of the Solid phase. In plant. Instead, the nodal root system plays this role. Thus, the certain embodiments, an amount of a composition provided early and vigorous establishment of a nodal root system plays herein that is sufficient to provide for an increase in nodal root a key role in the development of a uniform stand of a cereal growth in a cereal plant can be a composition with a Methy plant crop. Failure to do so results in Stunted plants and other lobacterium titer of at least about 5x10 colony-forming units deficiencies that end in lower yields at harvest. per gram to at least about 6x10' colony-forming units of 0082 Provided herein are fermentation broths, fermenta Methylobacterium per gram of particles in the composition tion broth products, and compositions that increase nodal root containing the particles that comprise a solid Substance growth in cereal plants relative to untreated cereal plants that have not been exposed to the fermentation broths, fermenta wherein a mono-culture or co-culture of Methylobacterium is tion broth products, and compositions. In certain embodi adhered thereto. ments, cereal plant parts, including, but not limited to, a seed, I0084. Lyophilized and shelf stable compositions compris a leaf, or a coleoptile can be treated with the fermentation ing solid substances with viable Methylobacterium adhered broths, fermentation broth products, and compositions to thereto and methods of preparing Such compositions are also increase cereal plant nodal root growth. Treatments or appli provided herein. In general, the methods comprise combining cations can include, but are not limited to, spraying, coating, a fermentation product comprising a solid Substance having a partially coating, immersing, and/or imbibing the cereal plant mono-culture or co-culture of Methylobacterium adhered or cereal plant parts with the fermentation broths, fermenta thereto with at least one cryoprotection agent to obtain a tion broth products, and compositions provided herein. In biphasic mixture that comprises the cryoprotection agent and certain embodiments, seeds can be immersed and/or imbibed a Solid phase comprising the fermentation product; and (b) with a fermentation broth, with a fermentation broth product lyophilizing the biphasic mixture, thereby obtaining the dried that has been partially or completely resuspended in a liquid, preparation of viable Methylobacterium. In certain embodi or with a liquid, semi-liquid, or slurry of a composition pro ments the liquid phase of the biphasic mixture is an aqueous vided herein. Such seed immersion or imbibition can be suf liquid. In certain embodiments, the Solid Substance is selected ficient to provide for an increase in nodal root growth in a from the group consisting of a man-made material, a material cereal plant in comparison to nodal root growth in a mock or of animal origin, a material of plant origin, a material of untreated cereal plant. Such increases in nodal root growth microbial origin, a material of fungal origin, a material of include increases in the numbers, length, dry weight, and/or mineral origin, and combinations thereof. In certain embodi wet weight of the nodal roots in treated cereal plants relative ments, the Solid Substance can be any of: (a) an agriculturally to untreated cereal plants. In certain embodiments, cereal acceptable adjuvant or agriculturally acceptable excipient; plant seeds can be immersed and/or imbibed for at least 1, 2, (b) an inanimate Solid Substance; (c) solid Substance is 3, 4, 5, or 6 hours. Such immersion and/or imbibition can, in selected from the group consisting of a polysaccharide, a certain embodiments, be conducted at temperatures that are diatomaceous earth, a salt crystal, and combinations thereof. not deleterious to the cereal plant seed or the Methylobacte or (d) a polysaccharide is selected from the group of a cellu rium. In certain embodiments, the seeds can be treated at losic polysaccharide, a chitinous polysaccharide, a galactan about 15 to about 30 degrees Centigrade or at about 20 to polysaccharide, and combinations thereof. Lyophilization about 25 degrees Centigrade. In certain embodiments, seed can be carried out until the dried preparation is substantially imbibition and/or immersion can be performed with gentle free of water, essentially free of water, or essentially dry. agitation. Dried preparations that are substantially free of water have 0.083 Amounts of the fermentation broths, fermentation about 5%, 4%, 3%, 2%, or 1% to 0.2% residual moisture broth products, and compositions Sufficient to provide for an content. Dried preparations that are essentially free of water increase in nodal root growth in a cereal plant can thus be have less than 0.2% residual moisture content to about determined by measuring any or all of an increase in the 0.001% residual moisture content. Dried preparations that are number, length, dry weight, and/or wet weight of the nodal essentially dry have less than 0.001% residual moisture con roots in treated cereal plants relative to untreated cereal tent. Residual moisture content is the percent of the total plants. In certain embodiments, an amount of a fermentation weight of a given material that is water. Residual moisture broth provided herein that is sufficient to provide for an content can be calculated by a variety of methods. In certain increase in nodal root growth in a cereal plant can be a embodiments, residual moisture content is calculated by a fermentation broth with Methylobacterium at atiter of at least gravimetric method by determining the wet weight and the about 5x10 colony-forming units permilliliter, at least about dry weight at the end of the freeze-drying procedure. Gravi 1x10 colony-forming units per milliliter, at least about metric procedures for determining residual moisture contents 1x10" colony-forming units per milliliter, or at least about can in certain embodiments be performed as described by Nei 3x10' colony-forming units per milliliter. In certain embodi et al. (Cryobiology 5:276-279, 1966). In certain embodi ments, an amount of a fermentation broth provided herein that ments of Such methods, the residual moisture content (A- is Sufficient to provide for an increase in nodal root growth in B)/Bx100; where A is the weight of the sample immediately a cereal plant can be a fermentation broth with Methylobac after freeze drying and B is the weight of the dry matter, terium at a titer of about 5x10 colony-forming units per obtained by drying for 3 h at 60° C. in a vacuum of 10 US 2016/007 3641 A1 Mar. 17, 2016 mmHg (1 mm Hg =133.3 Pa) (de Valdez et al. Appl. Environ. 5x10'', 1x10", 5x10", 1x10', or 5x10" colony-forming Microb. 49(2):413-415, 1985). units of Methylobacterium per gram of the Solid Substance. 0085. In practicing methods for preparing lyophilized and I0087. A variety of other additives can be added to the biphasic mixture prior to lyophilization and/or to the resultant shelf stable compositions comprising Solid Substances with dried preparation of viable Methylobacterium. In certain viable Methylobacterium provided herein, the components of embodiments, the method can comprise the addition of a the biphasic mixture can be combined in any order and in any protein of plant, animal or microbial origin to the biphasic form. In certain embodiments, at least one fermentation prod mixture and/or resultant dried preparation. Useful proteins uct is provided as a biphasic mixture comprising the Solid that can be added to the biphasic mixture and/or resultant substance with Methylobacterium adhered thereto or as just dried preparation include, but are not limited to, casein, Soy the solid substance with Methylobacterium adhered thereto protein, whey protein, wheat protein, corn protein, plant glu and is combined with an aqueous solution containing the tens, yeast protein, and the like. In certain embodiments, the dissolved cryoprotection agent. In certain embodiments, at protein can function as an anti-caking agent. In certain least one fermentation product in a biphasic mixture compris embodiments, anti-caking agents including, but not limited ing the solid substance with Methylobacterium adhered to, various desiccants, can be added to the biphasic mixture thereto and a liquid is combined with at least one cryoprotec and/or resultant dried preparation. In certain embodiments, tion agent in Solid form, which is then dissolved in the liquid agriculturally acceptable excipients that include, but are not phase of the biphasic mixture. In certain embodiments, the limited to, Woodflours, clays, activated carbon, diatomaceous solid substance with Methylobacterium adhered thereto is earth, fine-grain inorganic Solids, calcium carbonate and the harvested from at least one fermentation broth and resus like can be added to the biphasic mixture and/or resultant pended in an aqueous solution or an aqueous solution con dried preparation. Clays and inorganic Solids that can be taining at least one dissolved cryoprotection agent, separated added to the biphasic mixture and/or resultant dried prepara from the liquid phase, and resuspended in an second aqueous tions include, but are not limited to, calcium bentonite, kaolin, Solution containing at least one dissolved cryo protection china clay, talc, perlite, mica, Vermiculite, silicas, quartz pow agent. In certain embodiments, the Solid Substance with der, montmorillonite and mixtures thereof. Agriculturally Methylobacterium adhered thereto in a fermentation broth is acceptable adjuvants that promote sticking to the seed that combined with at least one cryoprotection agent in an aque can be added to the biphasic mixture and/or resultant dried ous solution to provide a biphasic mixture for lyophilization preparations include, but are not limited to, polyvinyl or for subsequent resuspension of the solid substance with acetates, polyvinyl acetate copolymers, hydrolyzed polyvinyl Methylobacterium adhered thereto in another aqueous Solu acetates, polyvinylpyrrolidone-vinyl acetate copolymer, tion containing at least one cryoprotection agent. In certain polyvinyl alcohols, polyvinyl alcohol copolymers, polyvinyl embodiments, the solid substance with Methylobacterium methyl ether, polyvinyl methyl ether-maleic anhydride adhered thereto in a fermentation broth is combined with at copolymer, waxes, latex polymers, celluloses including eth least one cryoprotection agent in Solid form, which is then ylcelluloses and methylcelluloses, hydroxy methylcellulo dissolved in the liquid phase of the fermentation broth to ses, hydroxypropylcellulose, hydroxymethylpropylcellulo provide a biphasic mixture for lyophilization or for subse ses, polyvinyl pyrrolidones, alginates, dextrins, malto quent resuspension of the Solid Substance with Methylobac dextrins, polysaccharides, fats, oils, proteins, karaya gum, terium adhered thereto in another aqueous Solution contain jaguar gum, tragacanth gum, polysaccharide gums, mucilage, ing at least one cryoprotection agent. gum arabics, shellacs, vinylidene chloride polymers and I0086) Cryoprotection agents can comprise one or more copolymers, soybean-based protein polymers and copoly compounds such as a disaccharide, poly Sucrose, a polyol. mers, lignoSulfonates, acrylic copolymers, starches, polyvi dextran, or a Sucrose-like polymers. In certain embodiments, nylacrylates, Zeins, gelatin, carboxymethylcellulose, chito the polyol is polyethylene glycol (PEG) oradonitol. In certain san, polyethylene oxide, acrylimide polymers and embodiments, the cryoprotection agent is a disaccharide, copolymers, polyhydroxyethyl acrylate, methylacrylimide poly Sucrose, hydroxyethylcellulose, or any combination monomers, alginate, ethylcellulose, polychloroprene and Syr thereof. In certain embodiments, the poly Sucrose has a ups or mixtures thereof. Other useful agriculturally accept molecular weight of about 60 to about 80 kilodaltons (kD) or able adjuvants that can promote coating that can be added to about 300 to about 500 kilodaltons (kD). In certain embodi the biphasic mixture and/or resultant dried preparations ments, the poly Sucrose has an average molecular weight of include, but are not limited to, polymers and copolymers of about 70 kDoran average molecular weight of about 400 kD. vinyl acetate, polyvinylpyrrolidone-vinyl acetate copolymer In certain embodiments, the hydroxyethylcellulose has a and water-soluble waxes. molecular weight of about 600 kilodaltons (kD) to about 700 I0088. In any of the aforementioned methods for preparing kilodaltons (kD). In certain embodiments, the disaccharide is lyophilized and shelf stable compositions comprising Solid Sucrose, lactose, trehalose, or any combinations thereof. In substances with viable Methylobacterium, the methods can certain embodiments, the cryoprotection agent is Sucrose. further comprise converting the dried preparation to a pow Sucrose can be provided in the biphasic mixture at a concen der. Methods of converting a dried preparation to a powder tration of about 5%, 7%, 8%, or 9% (w/v) to any of about include, but are not limited to, grinding, shaking, sieving, and 22%, 24%, or 25% (w/v). In certain embodiments, sucrose any combination thereof. can also be provided in the biphasic mixture at a concentra I0089. In certain embodiments, the dried preparations pro tion of about 5%, 7%, 8%, or 9% (w/v) to any of about 12%, duced by any of the aforementioned methods can comprise 14%, or 20% (w/v). In certain embodiments, a fermentation preparations wherein at least about 50%, 60%, 70%, 80%, product used in the methods has a Methylobacterium titer of 90%. 95%, or 98% of Methylobacterium cells in the compo about 5x10, 1x10, 5x10 or 1x10' colony-forming units sition or that were originally present in the biphasic mixture per gram of the solid substance to any of about 1x10', are viable. The percentage of viable Methylobacterium cells US 2016/007 3641 A1 Mar. 17, 2016 can be determined by a variety of methods including, but not weight (w/w). In certain embodiments, the compositions pro limited to, reconstituting the preparation and staining the cells vided herein comprise sucrose at about 63% to about 67% by with one or more agents that identify viable and inviable cells. weight (w/w). In other embodiments, the cryoprotection Examples of Such staining techniques include, but are not agent is a Sucrose-like polymer. Useful Sucrose like polymers limited to, (i) use of a membrane-permeant SYTO 9TM dye include polysucrose, hydroxyethylcellulose, or any combina that labels live bacteria with green fluorescence and a mem tion thereof. In certain embodiments, the poly Sucrose has a brane-imperimeant propidium iodide that labels membrane molecular weight of about 60 to about 80 kilodaltons (kD) or compromised bacteria with red fluorescence; (ii) use of mem about 300 to about 500 kilodaltons (kD). In certain embodi brane-permeant DAPITM that labels live bacteria with blue ments, the poly Sucrose has an average molecular weight of fluorescence and a membrane-impermeant SYTOXTM Green about 70 kDoran average molecular weight of about 400 kD. nucleic acid stain that labels bacteria with compromised In certain embodiments, the hydroxyethylcellulose has a membranes with green fluorescence. Reagents and kits for molecular weight of about 600 kilodaltons (kD) to about 700 selective staining of viable and inviable bacterial cells that kilodaltons (kD). In certain embodiments, the cryopro can be used are commercially available from the Molecular tectants can include a polyol or dextran. In certain embodi ProbesTM division of Life Technologies, Inc. (Carlsbad, ments, the polyol is polyethylene glycol (PEG) or adonitol. Calif.). Alternatively, the total count of viable and inviable Combinations of the aforementioned cryoprotection agents cells can be obtained, for example, by counting all cells under can also be used in the compositions. Compositions provided a microscope, and then obtaining a viable cell count by plat herein can be substantially free of water, essentially free of ing the cells on media. water, or essentially dry. Compositions that are substantially 0090 Compositions comprising the dried preparation of free of water have about 5%, 4%, 3%, 2%, or 1% to 0.2% viable Methylobacterium made by any of the aforementioned residual moisture content. Compositions that are essentially methods are also provided herein. free of water have less than 0.2% to about 0.001% residual 0091 Dried preparations of viable Methylobacterium pro moisture content. Compositions that are essentially dry have vided herein can also be applied to a plant or plant part. The less than 0.001% residual moisture content. plant or plant part can be either partially or completely coated 0093. Also provided herein are plants and plant parts that with the composition. In certain embodiments, the coated or are at least partially coated with any of the aforementioned partially coated plant part is selected from the group consist compositions. In certain embodiments, isolated plant parts ing of a seed, stem, root, flower, cotyledon, a coleoptile, fruit, that are at least partially coated with any of the aforemen and a leaf. Coated and partially coated plant seeds are pro tioned compositions are provided. In certain embodiments, vided. In certain embodiments, the seed can be any of a corn, the plant part is selected from the group consisting of a seed, Soybean, rice, wheat, cotton, or vegetable crop seed. In certain stem, root, flower, cotyledon, a coleoptile, fruit, and a leaf. embodiments, the seed is a lettuce seed and is at least partially Treated plants and treated plant parts, include, but are not coated with about 7x10 CFU of Methylobacterium per seed limited to, corn, Brassica sp. (e.g., B. napus, B. rapa, B. to about 4x10 CFU of the Methylobacterium per seed. juncea), alfalfa, rice, rye, Sorghum, millet (e.g., pearl millet 0092 Compositions that comprise a solid substance hav (Pennisetum glaucum), proso millet (Panicum miliaceum), ing a mono-culture or co-culture of Methylobacterium foxtail millet (Setaria italica), finger millet (Eleusine cora adhered thereto, and a cryoprotection agent, wherein the cana)), Sunflower, safflower, soybean, tobacco, potato, pea composition is substantially free of water and wherein at least nuts, cotton, Sweet potato (Ipomoea batatus), cassava, coffee, about 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 98% of coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, Methylobacterium cells in the composition are viable are also fig, guava, mango, olive, papaya, cashew, macadamia, provided herein. In certain embodiments, the cryoprotection almond, Sugar beets, Sugarcane, oats, barley, tomatoes let agent is selected from the group consisting of a disaccharide, tuce, greenbeans, lima beans, peas, cucurbits such as cucum poly Sucrose, hydroxyethylcellulose, and combinations ber, cantaloupe, and muskmelon, ornamental, turfgrass, and thereof. In certain embodiments, the Solid Substance has a conifer plants and plant parts. In certain embodiments, the at Methylobacterium titer of 5x10, 1x10, or 1x10" colony least partially coated plant part is a seed. In certain embodi forming units per gram of the solid substance to about 1x10', ments, the seed is selected from the group consisting of a 5x10'', 1x10", 5x10", 1x10', or 5x10" colony-forming corn, soy, wheat, cotton, tomato, and lettuce seed. In certain units of Methylobacterium per gram of the solid substance. In embodiments, the seed is a lettuce seed and is coated with certain embodiments, the dried preparation has a Methyllo about 7x10 CFU of Methylobacterium per seed to about bacterium titer of 5x10, 1x10, or 1x10' colony-forming 4x10 CFU of the Methylobacterium per seed. units per gram of the dried preparation to about 5x10'. 1x10", or 5x10" colony-forming units of Methylobacterium Examples per gram of the dried preparation. In certain embodiments, the solid substance with Methylobacterium adhered thereto is 0094. The following examples are included to demon an agriculturally acceptable adjuvant or agriculturally accept strate preferred embodiments of the invention. It will be able excipient. In certain embodiments, the composition appreciated by those of skill in the art that the techniques comprising a solid Substance with Methylobacterium adhered disclosed in the following examples represent techniques dis thereto further comprises an agriculturally acceptable adju covered by the Applicants to function well in the practice of vant or agriculturally acceptable excipient. In certain embodi the invention, and thus can be considered to constitute pre ments, the cryoprotection agent is a disaccharide. Useful ferred modes for its practice. However, those of skill in the art disaccharide cryoprotection agents used in the compositions should, in light of the instant disclosure, appreciate that many include, but are not limited to. Sucrose and trehalose. In cer changes can be made in the specific embodiments that are tain embodiments, the compositions provided herein com disclosed, while still obtaining like or similar results, without prise sucrose at about 63% or 64% to about 66% or 67% by departing from the scope of the invention. US 2016/007 3641 A1 Mar. 17, 2016 20

Example 1 preparation, preventing the formation of insoluble magne sium phosphate and calcium phosphate crystals. Growth of PPFM Bacteria on Solid Agar Plate 0099] To prepare one liter of solidagar plate media with an Media AMS base, 15 grams of agar were added to 940 ml of distilled water, and this mixture was autoclaved. After autoclaving, 20 0095 For the growth of PPFM bacteria on solidagar plate ml each of stock solutions I, II, and III were added, along with media, a variety of Standard media were tested. one ml of the filter-sterilized trace metals stock solution. 0096. One medium used was ammonium mineral salts 0100 If other medium components, such as a carbon (AMS) medium (Whittenbury et al., 1970). AMS medium source, were to be incorporated, for the most part these were contains, per liter, 700 milligrams of dibasic potassium phos added to the water and agar mixture before autoclaving. The phate anhydrous, 540 milligrams of monobasic potassium one exception to this was methanol, which was filter-steril phosphate anhydrous, one gram of magnesium Sulfate hep ized through a 0.2 micrometer filter apparatus and added after tahydrate, 500 milligrams of ammonium chloride anhydrous, the base medium had been autoclaved. 200 milligrams of calcium chloride dehydrate, 4 milligrams 0101. A second medium used was Vogel-Bonner (VB) offerric sulfate heptahydrate, 100 micrograms of zinc sulfate minimal culture medium (Vogel and Bonner, 1956). VB heptahydrate, 30 micrograms of manganese chloride tetrahy medium contains, per liter, 298 milligrams of magnesium drate, 300 micrograms of boric acid anhydrous, 200 micro sulfate heptahydrate, 14.93 grams of anhydrous dibasic grams of cobalt chloride hexahydrate, 10 micrograms of cop potassium phosphate, 5.22 grams of sodium ammonium per chloride dehydrate, 20 micrograms of nickel chloride phosphate tetrahydrate, and 2.73 grams of anhydrous citric hexahydrate, and 60 micrograms of sodium molybdate dehy acid (the free acid form). drate. 0102) Vogel-Bonner minimal medium was prepared from a 25x stock solution of the salts and citric acid. This 25x stock 0097 AMS medium was prepared from four stock solu solution was prepared by dissolving in one liter of distilled tions, listed below water the following amounts of each ingredient, in the order listed, and making Sure each one was completely dissolved before adding the next one: 7.46 grams of magnesium Sulfate Stock solution I: for one liter at 50x concentration heptahydrate, 68.23 grams of anhydrous citric acid, 373.13 dibasic potassium phosphate, anhydrous 35 grams grams of anhydrous dibasic potassium phosphate, and 130.60 monobasic potassium phosphate, anhydrous 27 grams grams of sodium ammonium phosphate tetrahydrate. By first dissolving the magnesium sulfate and then adding the citric acid, the magnesium ions were chelated by the citrate ions, preventing the formation of insoluble magnesium phosphate Stock solution II: for one liter at 50x concentration crystals when the phosphate salts are added. This insured the magnesium sulfate heptahydrate 50 grams preparation of a water-clear culture medium with all ingredi 25 grams ents in solution. ammonium chloride, anhydrous 0103) To prepare one liter of solidagar plate media with a VB base, 15 grams of agar were added to 960 ml of distilled water and this mixture was autoclaved. After autoclaving, 40 ml of the 25x VB salts stock solution were added. Stock solution III: for one liter at 50x concentration 0104. If other medium components, such as a carbon calcium chloride dihydrate 10 grams source, were to be incorporated, for the most part these were added to the water and agar mixture before autoclaving. The one exception to this was methanol, which was filter-steril ized through a 0.2 micrometer filter apparatus and added after Trace metals stock solution: for one liter at 1000x concentration the base medium had been autoclaved. 0105. A third medium used was LB broth. LB broth con ferric sulfate heptahydrate 4 grams tains, per liter, 10 grams of tryptone, 5 grams of yeast extract, Zinc sulfate heptahydrate 100 milligrams manganese chloride tetrahydrate 30 milligrams and 10 grams of sodium chloride. All components were dis boric acid, anhydrous 300 milligrams solved in one liter of distilled water and autoclaved. This cobalt chloride hexahydrate 200 milligrams medium was water-clear, with all ingredients in solution. copper chloride dihydrate 10 milligrams 0106 To prepare one liter of solidagar plate media with an nickel chloride hexahydrate 20 milligrams sodium molybdate dihydrate 60 milligrams LB base, 15 grams of agar were added to one liter of LB broth, and this mixture was autoclaved. 0107 Corpe and Basile (1982) conducted a systematic 0098 Stock solutions I, II, and III were autoclaved sepa survey of the growth of various strains of PPFM bacteria in rately. The trace metals stock solution could not be auto AMS media containing various carbon sources. Many of the claved, as most of the salts precipitated out during the auto tested substances supported little to no growth of PPFM bac claving step, and so it was filter-sterilized through a 0.2 teria. Corpe and Basile reported that glycerol and glutamate micrometer filter apparatus. These steps were necessary to were relatively good carbon sources for PPFM bacteria, and insure the preparation of a water-clear AMS culture medium that methanol, glucose, aspartate. Succinate and malate were with all ingredients in solution. As originally described by intermediate as carbon sources for PPFM bacteria. Whittenbury et al. (1970), the phosphate-containing compo 0108. The Applicants measured the growth of Methylo nents of the AMS medium were segregated from the other bacterium extorquens on LB plates, as well as on AMS and components until the final finishing steps of the medium VB plates supplemented with various carbon sources. The US 2016/007 3641 A1 Mar. 17, 2016

carbon sources, listed below, were all added to the AMS or VB 0113. A striking aspect of these results is the very poor base salts media at 10 grams per liter. In addition, Some media growth of the PPFM bacteria in all of these water-clear liquid compositions were tested that included peptone at 10 grams media, in the presence of the exact same nutrients as were per liter. The Methylobacterium extorquens was streaked out present in the Solid agar plate forms of these media on which on the various agar plates, and they were incubated at 30 the PPFM bacteria grew rapidly and abundantly (as described degrees C. for up to two weeks. Growth was measured as the in Example 1). Indeed, in all of these flasks, there was little or number of days of incubation required for the colonies to no visible turbidity (the classical indication of microbial become full-sized (about 2 millimeters in diameter); for those growth) and no hint of a pink hue whatsoever. growth conditions where full-sized colonies did not form even after prolonged incubation, the colonies were scored as Example 3 medium-sized (about 1 millimeters in diameter) or small sized (about 0.5 millimeters in diameter or smaller). All of the Growth of PPFM Bacteria in a Biphasic Culture colonies observed were of a deep, saturated pink color, as is Media Containing Insoluble Salt Crystals characteristic of PPFM bacteria. 0114 For the preparation of the biphasic culture media, 0109. The results were as follows: liquid AMS plus glycerol and peptone medium was made turbid (i.e. provided with a solid substance) by deliberately forming insoluble crystals of magnesium phosphate and/or VB plus aspartate Small-sized in 9 days calcium phosphate. To deliberately form insoluble crystals in VB plus succinate Small-sized in 10 days VB plus malate Small-sized in 10 days the media, the preparation method described in Example 1 LB full-sized in 9 days was altered as follows. All components except the trace met AMS plus glucose full-sized in 9 days als stock solution were mixed together before autoclaving. VB plus glucose full-sized in 14 days AMS plus methanol full-sized in 6 days That is, to 940 ml of distilled water were added 20 ml each of VB plus methanol medium-sized in 10 days stock Solutions I, II, and III, along with 10 grams of glycerol AMS plus glutamate and peptOne full-sized in 5 days and 10 grams of peptone. After autoclaving, the medium was AMS plus glycerol and peptone full-sized in 5 days completed by the addition of one ml of filter-sterilized trace VB plus glycerol and peptOne full-sized in 6 days metals stock solution. The autoclaving of the components of stock solutions I, II, and III, mixed together before autoclav 0110. The fastest and most abundant growth of the PPFM ing, resulted in the formation of insoluble salt crystals, pre bacterium Methylobacterium extorquens on the tested solid Sumably primarily magnesium phosphate dibasic and/or cal agar plate media was on AMS plus glycerol and peptone or cium phosphate dibasic. After autoclaving, the AMS plus AMS plus glutamate and peptone, followed closely by AMS glycerol and peptone medium made by this preparation plus methanol or VB plus glycerol and peptone. Growth on method yielded a liquid medium that was very turbid with the other tested media was significantly slower. these salt crystals. This new liquid medium was designated “turbid AMS plus glycerol and peptone'. Example 2 0115 To a flask containing 100 milliliters of the turbid AMS plus glycerol and peptone, an inoculum of the PPFM Growth of PPFM Bacteria in Clear, Monophasic bacterium Methylobacterium extorquens was added to give Liquid Media an initialtiter of about 1x10 colony-forming units (CFU) per milliliter. The flask was placed on a rotary shaker incubator 0111 For those four solid agar plate media found in set and grown for 3 days at 30 degrees C., and 250 rpm. After Example 1 to have Supported the fastest and most abundant just two days, the flask had developed a deep, Saturated pink growth of the PPFM bacterium Methylobacterium turbidity, indicating fast and abundant growth of PPFM bac extorquens, the corresponding liquid versions (that is, no teria. At both 2 days and 3 days after inoculation, the titers of added agar) were prepared and tested. These four liquid PPFM bacteria in the flask were determined. The results media, prepared as described in Example 1 (with the sole Were exception being that they did not contain any agar) were all water-clear liquids, with all ingredients in solution. 0112 To flasks containing 100 milliliters of these four initial tilter titer of PPFM titer of PPFM liquid media, an inoculum of the PPFM bacterium Methylo liquid medium of PPFM after 2 days after 3 days bacterium extorquens was added to give an initial titer of turbid AMS plus 1.7 x 10 1.3 x 108 1.7 x 109 about 1x10 colony-forming units (CFU) per milliliter. The glycerol and peptone flasks were placed on a rotary shaker incubator set and grown for 5 days at 30 degrees C., and 250 rpm. At the end of the 5 0116. Two striking aspects of this result were the very fast days of incubation, the titers of PPFM bacteria in the flasks growth of the PPFM bacteria, and their growth to titers were determined. The results were: approaching 10,000-fold higher than achieved in clear AMS plus glycerol and peptone liquid medium (as shown in Example 2). initial tilter titer of PPFM liquid medium of PPFM after 5 days Example 4 AMS plus glycerol and peptone 1.4 x 10 4.5 x 10 AMS plus glutamate and peptOne 2.0 x 10 3.8 x 10 Growth of PPFM Bacteria in Liquid Media AMS plus methanol 1.1 x 10 2.1 x 10 Containing Agar VB plus glycerol and peptOne 1.7 x 10 1.3 x 10 0117 For the preparation of solidagar plate media, agar is typically added at about 15 grams per liter of medium. To test US 2016/007 3641 A1 Mar. 17, 2016 22 whether lower amounts of agar, at levels too low to gel or shaker incubator set and grown for 3 days at 30 degrees C., solidify the medium, would be effective at promoting the fast and 250 rpm. After just two days, the flasks had all developed and abundant growth of PPFM bacteria, small amounts of a deep, Saturated pink turbidity, indicating fast and abundant agar were added to AMS plus glycerol and peptone liquid growth of PPFM bacteria. At both 2 days and 3 days after medium. inoculation, the titers of PPFM bacteria in the flasks were 0118. This liquid medium was prepared as described in determined. The results were: Example 1, that is, by the preparation method designed to prevent the formation of insoluble salt crystals of magnesium phosphate and calcium phosphate. The agar, as described in initial tilter tilter of PPFM titer of PPFM Example 1, was added to the water before autoclaving. The liquid medium of PPFM after 2 days after 3 days amounts of agar tested were, per liter, 750 milligrams, 1.5 AMS plus glycerol and 1.0 x 10 1.8 x 10 1.1 x 10 grams, and 3 grams. These new liquid media were designated peptone and 500 mg diatomaceous earth AMS plus glycerol and peptone and agar. AMS plus glycerol and 1.8 x 10 3.0 x 108 8.4 x 108 0119) To flasks containing 100 milliliters of the AMS plus peptone and 1 gram glycerol and peptone and agar, an inoculum of the PPFM diatomaceous earth AMS plus glycerol and 1.4 x 10 4.0 x 108 1.7 x 109 bacterium Methylobacterium extorquens was added to give peptone and 1.5 grams an initialtiter of about 1x10 colony-forming units (CFU) per diatomaceous earth milliliter. The flasks were placed on a rotary shaker incubator AMS plus glycerol and 1.7 x 10 3.4 x 108 2.0 x 109 set and grown for 3 days at 30 degrees C., and 250 rpm. After peptone and 2 grams just two days, the flasks had all developed a deep, saturated diatomaceous earth pink turbidity, indicating fast and abundant growth of PPFM bacteria. At both 2 days and 3 days after inoculation, the titers I0123. Two striking aspects of this result are the very fast of PPFM bacteria in the flasks were determined. The results growth of the PPFM bacteria, and their growth to titers Were approaching 10,000-fold higher than achieved in clear AMS plus glycerol and peptone liquid medium (as shown in initial tilter tilter of PPFM tilter of PPFM Example 2). The data acquired after 2 days of growth also liquid medium of PPFM after 2 days after 3 days indicate that increased amounts of growth are correlated to increased amounts of agar within the range of 0.5 grams to 1.5 AMS plus glycerol and 1.3 x 10 6.4x 10' 8.0 x 107 peptone and 750 mg agar grams per 100 ml of culture. AMS plus glycerol and 1.3 x 10 3.1 x 10' 2.0 x 10 peptone and 1.5 grams agar Example 6 AMS plus glycerol and 1.2 x 10 1.8 x 108 5.1 x 108 peptone and 3 grams agar Growth of PPFM Bacteria in a Controlled Bioreactor 0120) Two striking aspects of this result are the very fast 0.124. The growth of bacteria in flasks, incubated on a growth of the PPFM bacteria, and their growth to titers rotary shaker, is limited by changes in pH brought about by approaching 1000-fold higher than achieved in clear AMS the metabolism of the carbon source(s) in the growth plus glycerol and peptone liquid medium (as shown in medium. Controlled bioreactors (also known as “fermenters' Example 2). The data acquired after 3 days of growth also or “fermentation vessels') avoid this limitation by maintain indicate that increased amounts of growth are correlated to ing the pH at the desired level through the controlled addition increased amounts of agar. of acids or bases, as appropriate. Another factor which can limit the growth of bacteria is the availability of dissolved Example 5 oxygen. Controlled bioreactors avoid this limitation by main taining adequate levels of dissolved oxygen through the con Growth of PPFM Bacteria in Liquid Media trolled adjustment of airflow into the reactor vessel, the agi Containing Diatomaceous Earth tation rate of the vessel, and the air pressure within the vessel. 0121 To test whether diatomaceous earth would be effec 0.125. When grown in a controlled bioreactor containing tive at promoting the fast and abundant growth of PPFM the AMS plus glycerol and peptone liquid medium described bacteria, Small amounts of diatomaceous earth were added to in Example 1, further supplemented with solid substances AMS plus glycerol and peptone liquid medium. This liquid Such as insoluble salts, agar, or diatomaceous earth as medium was prepared as described in Example 1, that is, by described in Examples 3, 4 and 5, the final titer of PPFM the preparation method designed to prevent the formation of bacteria achieved will be at least 30-fold higher than that insoluble salt crystals of magnesium phosphate and calcium achieved in flasks, specifically at least 3x10" colony-form phosphate. The diatomaceous earth was added to the water ing units per milliliter. before autoclaving. The amounts of diatomaceous tested were, per liter, 500 milligrams, 1 gram, 1.5 grams, and 2 Example 7 grams. These new liquid media were designated “AMS plus glycerol and peptone and diatomaceous earth'. Growth of Various Methylobacterium in the Presence 0122) To flasks containing 100 milliliters of the AMS plus and Absence of Various Solids in the Media glycerol and peptone and diatomaceous earth, an inoculum of the PPFM bacterium Methylobacterium extorquens was 0.126 Various Methylobacterium listed in the following added to give an initial titer of about 1x10 colony-forming Table were obtained from the indicated depository organiza units (CFU) per milliliter. The flasks were placed on a rotary tions, purified as single colony isolates, and cultured in the US 2016/007 3641 A1 Mar. 17, 2016 presence and absence of Solids in AMS plus glycerol plus preceding Examples 3-6. Typically, the Methylobacterium peptone media. The solids were sterilized by autoclaving in are cultured to a high titer (i.e. at least about 5x10 colony the media. forming units per gram of solid). Adherent Methylobacterium associated with the solid are then harvested either with or TABLE 3 without any non-adherent Methylobacterium present in the culture. Harvesting can be achieved by filtration, centrifuga Depository Accession No. Methyliobacterium species tion, decanting, and combinations thereof. Harvested mate DSM-6343 Methyliobacterium extorquens rial can be applied directly to seeds or plants in certain DSM-1819 Methyliobacterium radiotolerans instances. In other instances, the harvested material is dried DSM-13060 Methyliobacterium extorquens by lyophilization or spray drying and the like prior to appli DSM-18172 Methyliobacterium organophilum DSM-1708 Methyliobacterium mesophilicum cation. Dried material can also be reconstituted with liquids DSM-182O7 Methyliobacterium oryzae as necessary or desired prior to application to plants or seed. DSM-19779 Methyliobacterium phyllosphaerae In certain cases, the solid materials with the adherent Methy ATCC-14718 Methyliobacterium extorquens lobacterium can be disassociated as described herein and ATCC-14821 Methyliobacterium rhodinum ATCC-21611 Methyliobacterium rhodesianum either applied directly to the seeds or plants or first dried and ATCC-3S06S Methyliobacterium fujisawaense then applied to seeds or plants. Solid materials with the adher ATCC-43883 Methyliobacterium zatmani ent Methylobacterium can also be first dried, then disassoci ATCC-S1358 Methyliobacterium aminovorans ated as described herein and applied directly to the seeds of ATCC-7OO647 Methyliobacterium thiocyanatum plants or used as active ingredients in other compositions for ATCC: American Type Tissue Culture Collection, Manassas, VA, USA plant or seed treatment. It is also possible to add additional DSM: DSMZ-German Collection of Microorganisms and Cell Cultures (“DSMZ”), Braun agriculturally acceptable excipients and/or adjuvants to any schweig, Germany of the harvested and/or dissociated solid materials with adher 0127. All of the Methylobacterium in the preceding Table ent Methylobacterium. Added excipients can include woodlf 3 grew very poorly or not at all in water-clear AMS plus lours, clays, activated carbon, diatomaceous earth, fine-grain glycerol plus peptone medium, and all grew very well in the inorganic solids, calcium carbonate and the like. Clays and same medium amended (at 2 grams per liter) with one fol inorganic Solids that can be added as excipients in the com lowing Solids: diatomaceous earth, agar, turbid medium positions include calcium bentonite, kaolin, china clay, talc, (made so as to be cloudy with insoluble crystals of magne perlite, mica, Vermiculite, silicas, quartz powder, montmoril sium phosphate as described in Example 3), bone meal, flax lonite and mixtures thereof. Agriculturally acceptable adju seed meal, “Rare Earth' (a mixture of pyrophyllitic silicate vants that promotesticking to the seed or other plant parts that clay and leonardite from General Hydroponics of Sebastopol, can be added to the compositions include polyvinyl acetates, Calif., USA), “White Hermit Crab Sand” (a mixture of cal polyvinyl acetate copolymers, hydrolyzed polyvinyl acetates, cium carbonate and magnesium carbonate from Zoo-Med polyvinylpyrrolidone-vinyl acetate copolymer, polyvinyl Laboratories, Inc., of San Luis Obispo, Calif., USA), dried alcohols, polyvinyl alcohol copolymers, polyvinyl methyl and powdered coconut meat, and crushed egg shell. ether, polyvinyl methyl ether-maleic anhydride copolymer, waxes, latex polymers, celluloses including ethylcelluloses Example 8 and methylcelluloses, hydroxy methylcelluloses, hydrox ypropylcellulose, hydroxymethylpropylcelluloses, polyvinyl Photomicroscopy of Methylobacterium Grown in the pyrrolidones, alginates, dextrins, malto-dextrins, polysaccha Presence of a Solid in the Media rides, fats, oils, proteins, karayagum, jaguar gum, tragacanth 0128 Methylobacterium were purified as single colony gum, polysaccharide gums, mucilage, gum arabics, shellacs, isolates and cultured in the presence of diatom shells in liquid vinylidene chloride polymers and copolymers, soybean growth media. The diatom shells were sterilized by autoclav based protein polymers and copolymers, lignoSulfonates, ing in the liquid media prior to inoculation of the thus steril acrylic copolymers, starches, polyvinylacrylates, Zeins, gela ized media with Methylobacterium. tin, carboxymethylcellulose, chitosan, polyethylene oxide, 0129. The results of the photomicrographic analysis of the acrylimide polymers and copolymers, polyhydroxyethyl cultures are shown in FIGS. 1 and 2. In FIG. 1, some portions acrylate, methylacrylimide monomers, alginate, ethylcellu of the latticed diatom shell are exposed while other portions lose, polychloroprene and syrups or mixtures thereof. Other of the diatom shell are obscured by the adherent Methylobac useful agriculturally acceptable adjuvants that can promote terium cells. FIG. 1 also shows that there are very few Methy coating of seeds or other plant parts include polymers and lobacterium cells in this culture that are not adherent. FIG. 2 copolymers of vinyl acetate, polyvinylpyrrolidone-vinyl shows a diatom shell that is almost completely coated with acetate copolymer and water-soluble waxes. These composi adherent Methylobacterium cells and a few apparently non tions can be maintained in a dry or semi dry form or can be adherent Methylobacterium cells in the liquid media. formulated into slurries by addition of liquids as desired. The compositions can then be used to spray or coat the plants or Example 9 seeds to obtain beneficial effects associated with application of Methylobacterium to plants. Plant Seed or Foliar Treatment Compositions Comprising Solid Substances with Adherent Example 10 Methylobacterium 0130. To obtain compositions suitable for plant seed or Titers of Various Methylobacterium in the Presence foliar treatments, Methylobacterium are cultured in liquid and Absence of Various Solids in the Media media containing a solid Substance by any of the methods I0131 Fourteen strains in the genus Methylobacterium disclosed or claimed herein, or as described in any of the were purchased from the DSMZ (Braunschweig, Germany) US 2016/007 3641 A1 Mar. 17, 2016 24 and the ATCC (Manassas, Va., USA). These 14 strains consist -continued of 12 different species, as there were three M. extorquens in the set: Growth of PPFM strains in turbid AMS-GP liquid medium 0132 1. DSM-6343 Methylobacterium extorquens 0.133 22. DSM-1819 Methylobacterium radiololerans initial titer tilter after 3 days 0134 33. DSM-13060 Methylobacterium extorquens ATCC-35065 M. filisawaense 8.2 x 10 2.3 x 108 0135 44. DSM-18172 Methylobacterium organophilum ATCC-43883 M. zaimani 1.8 x 10 6.2 x 10 0.136 5. DSM-1708 Methylobacterium mesophilicum ATCC-51358 M. aminovorans 6.8 x 10 1.7 x 109 0137 6.6 DSM-18207 Methylobacterium oryzae ATCC-7OO647 M. thiocyanatum 4.3 x 10 6.5 x 10 0138 77. DSM-19779 Methylobacterium phyllosphaerae 0139 8.8 ATCC-14718 Methylobacterium extorquens 0140) 9. ATCC-14821 Methylobacterium rhodinum 0141 10. ATCC-21611 Methylobacterium rhodesianum Growth of PPFM strains in AMS-GP liquid medium plus 0142 11. ATCC-35065 Methylobacterium filjisawaense diatonaceous earth (at 2 grams per liter 0143) 12. ATCC-43883 Methylobacterium Zalmanii 0144 13. ATCC-51358 Methylobacterium aminovorans initial titer tilter after 3 days (0145 14. ATCC-700647 Methylobacterium thiocyanatum DSM-6343 M. extorquiens 2.1 x 10 8.7 x 108 DSM-1819 M. radiotoierans 1.1 x 10 3.3 x 108 0146 For the tests below, the inocula came from cultures DSM-13060 M. extorquiens 6.8 x 10 8.4 x 108 grown in water-clear AMS-GP medium. These cultures were DSM-18172 M. organophilum 3.1 x 10 8.1 x 108 grown in 200 ml of the water-clear AMS-GP medium, titered, DSM-1708 M. mesophilicum 1.4 x 10 2.4 x 10 and then concentrated ten-fold. These PPFM cultures, with DSM-182O7 M. oryzae 2.1 x 10 3.1 x 109 DSM-19779 M. phyllosphaerae 9.6 x 10 3.6 x 108 no solid Substrates present, were used to inoculate test tubes ATCC-14718 M. extorquiens 1.8 x 10 4.8 x 10 containing 10 ml of water-clear AMS-GP medium, or AMS ATCC-14821 M. rhodinum 8.0 x 10' 5.9 x 108 GP medium with various added solid substrates. 20 mg of the ATCC-21611 M. rhodesianum 3.5 x 10 7.7 x 108 various solid substrates were added to each 10 ml tube, yield ATCC-35065 M. filisawaense 1.5 x 10 9.2 x 108 ATCC-43883 M. zaimani 6.5 x 10' 9.6 x 10 ing a solid Substrate concentration equivalent to 2 grams per ATCC-51358 M. aminovorans 3.0 x 10 2.7 x 109 liter. Titers are expressed throughout as Colony Forming ATCC-7OO647 M. thiocyanatum 1.4 x 10 2.3 x 109 Units per milliliter (CFU/mL). The target initial titer in each tube was about 1x10 PPFM colony forming units per ml. The inoculated test tubes were placed on a rotary shaker set and grown for three days at 30 degrees C., and 250 rpm. After Growth of PPFM strains in AMS-GP liquid medium plus three days of growth, the PPFM cultures were titered. agar (at 2 grams per liter initial titer tilter after 3 days Growth of PPFM strains in water-clear AMS-GP liquid medium DSM-6343 M. extorquiens 1.5 x 10 1.6 x 108 initial titer titer after 3 days DSM-1819 M. radiotoierans 1.3 x 10 3.7 x 108 DSM-13060 M. extorquiens 9.2 x 10' 1.7 x 108 DSM-6343 M. extorquiens 3.5 x 10 1.2 x 10 DSM-18172 M. organophilum 8.1 x 10' 9.3 x 107 DSM-1819 M. radiotoierans 1.8 x 10 1.3 x 10 DSM-1708 M. mesophilicum 5.2 x 10 2.8 x 108 DSM-13060 M. extorquiens 3.1 x 10 3.2 x 10 DSM-182O7 M. oryzae 1.9 x 10 5.0 x 108 DSM-18172 M. organophilum 1.4 x 10 1.8 x 10 DSM-19779 M. phyllosphaerae 1.0 x 10 5.3 x 108 DSM-1708 M. mesophilicum 3.3 x 10 1.0 x 10 ATCC-14718 M. extorquiens 2.4 x 10 4.7 x 10 DSM-182O7 M. oryzae 1.1 x 10 2.2 x 10 ATCC-14821 M. rhodinum 1.5 x 10 5.1 x 108 DSM-19779 M. phyllosphaerae 2.1 x 10 1.1 x 10 ATCC-21611 M. rhodesianum 8.1 x 10 4.3 x 108 ATCC-14718 M. extorquiens 2.0 x 10 1.7 x 10 ATCC-35065 M. filisawaense 1.0 x 10 4.4 x 10 ATCC-14821 M. rhodinum 4.4 x 10 2.9 x 10 ATCC-43883 M. zaimani 1.3 x 10 5.6 x 10 ATCC-21611 M. rhodesianum 1.8 x 10 1.4 x 10 ATCC-51358 M. aminovorans 4.5 x 10 6.4x 108 ATCC-35065 M. filisawaense 1.3 x 10 1.9 x 10 ATCC-7OO647 M. thiocyanatum 3.6 x 10 1.7 x 108 ATCC-43883 M. zaimani 5.0 x 10 1.9 x 10 ATCC-51358 M. aminovorans 9.5 x 10' 1.7 x 10 ATCC-7OO647 M. thiocyanatum 1.8 x 10 7.6 x 10

Growth of PPFM strains in AMS-GP liquid medium plus owdered kelp (at 2 grams per liter Growth of PPFM strains in turbid AMS-GP liquid medium initial titer tilter after 3 days initial titer titer after 3 days DSM-6343 M. extorquiens 3.8 x 10 4.3 x 108 DSM-1819 M. radiotoierans 9.1 x 10' 4.1 x 10 DSM-6343 M. extorquiens 2.5 x 10 2.1 x 10 DSM-13060 M. extorquiens 4.0 x 10 9.7 x 107 DSM-1819 M. radiotoierans 7.6 x 10 6.8 x 108 DSM-18172 M. organophilum 2.2 x 10 2.8 x 108 DSM-13060 M. extorquiens 1.9 x 10 4.4 x 108 DSM-1708 M. mesophilicum 1.2 x 10 1.0 x 108 DSM-18172 M. organophilum 1.1 x 10 1.9 x 10 DSM-182O7 M. oryzae 9.4 x 10' 6.4 x 10 DSM-1708 M. mesophilicum 1.4 x 10 9.3 x 10 DSM-19779 M. phyllosphaerae 7.7 x 10 3.8 x 10 DSM-182O7 M. oryzae 7.3 x 10' 2.4 x 108 ATCC-14718 M. extorquiens 3.0 x 10 4.7 x 108 DSM-19779 M. phyllosphaerae 1.8 x 10 7.5 x 108 ATCC-14821 M. rhodinum 8.4 x 10 1.6 x 108 ATCC-14718 M. extorquiens 9.3 x 10' 2.0 x 10 ATCC-21611 M. rhodesianum 9.7 x 10 4.1 x 10 ATCC-14821 M. rhodinum 7.1 x 10' 7.2 x 108 ATCC-35065 M. filisawaense 8.2 x 10' 3.4 x 10 ATCC-21611 M. rhodesianum 3.9 x 10 6.8 x 108 ATCC-43883 M. zaimani 9.7 x 10 4.0 x 108 US 2016/007 3641 A1 Mar. 17, 2016 25

-continued -continued Growth of PPFM strains in AMS-GP liquid medium plus Growth of PPFM strains in AMS-GP liquid medium plus powdered kelp (at 2 grams per liter bone meal (at 2 grams per liter initial titer tilter after 3 days initial titer tilter after 3 days ATCC-51358 M. aminovorans 2.6 x 10 6.3 x 10 ATCC-21611 M. rhodesianum 1.6 x 10 8.1 x 10' ATCC-7OO647 M. thiocyanatum 3.5 x 10 5.2 x 108 ATCC-35065 M. fujisawaense 6.9 x 10 2.0 x 108 ATCC-43883 M. zatmanii 5.7 x 10' 6.9 x 107 ATCC-51358 M. aminovorans 9.9 x 10' 4.3 x 10 ATCC-7OO647 M. thiocyanatum 1.9 x 10 1.5 x 108

Growth of PPFM strains in AMS-GP liquid medium plus coconut husk fiber (at 2 grams per liter initial titer titer after 3 days Growth of PPFM strains in AMS-GP liquid medium plus blood neal (at 2 grams per liter DSM-6343 M. extorquiens 2.0 x 10 3.4 x 108 DSM-1819 M. radiotoierans 2.1 x 10 6.0 x 108 initial titer tilter after 3 days DSM-13060 M. extorquiens 8.9 x 10' 2.5 x 108 DSM-18172 M. organophilum 8.5 x 10' 7.5 x 107 DSM-6343 M. extorquiens 7.7 x 10 7.0 x 108 DSM-1708 M. mesophilicum 6.0 x 10 6.0 x 108 DSM-1819 M. radiotoierans 7.9 x 10' 8.8 x 107 DSM-182O7 M. oryzae 1.9 x 10 3.2 x 108 DSM-13060 M. extorquiens 4.4 x 10 6.7 x 107 DSM-19779 M. phyllosphaerae 9.6 x 10' 1.2 x 10 DSM-18172 M. organophilum 3.1 x 10 8.4 x 10' ATCC-14718 M. extorquiens 2.8 x 10 9.7 x 107 DSM-1708 M. mesophilicum 1.0 x 10 4.0 x 10 ATCC-14821 M. rhodinum 3.5 x 10 8.9 x 107 DSM-182O7 M. oryzae 3.9 x 10 5.0 x 107 ATCC-21611 M. rhodesianum 7.4 x 10 8.2 x 107 DSM-19779 M. phyllosphaerae 2.5 x 10 8.0 x 107 ATCC-35065 M. filisawaense 6.1 x 10' 7.0 x 107 ATCC-14718 M. extorquiens 9.5 x 10' 9.5 x 107 ATCC-43883 M. zaimani 8.0 x 10' 5.3 x 108 ATCC-14821 M. rhodinum 6.8 x 10' 8.6 x 10' ATCC-51358 M. aminovorans 3.3 x 10 2.5 x 108 ATCC-21611 M. rhodesianum 7.4x 10 4.9 x 107 ATCC-7OO647 M. thiocyanatum 3.3 x 10 7.3 x 107 ATCC-35065 M. filisawaense 2.6 x 10 2.0 x 108 ATCC-43883 M. zaimani 3.2 x 10 4.3 x 10 ATCC-51358 M. aminovorans 7.0 x 10' 1.8 x 10 0147 The coconut husk fiber was a product called “Her- ATCC-700647 M. thiocyanatum 3.2 x 10 5.5 x 107 mit Soil', with the sole ingredient listed as “coconut fiber substrate' and sold by Zoo-Med Laboratories, Inc., of San Luis Obispo, Calif. Growth of PPFM strains in AMS-GP liquid medium plus Sand (at 2 grams per liter Growth of PPFM strains in AMS-GP liquid medium plus cottonseed neal (at 2 grams per liter initial titer tilter after 3 days initial titer titer after 3 days DSM-6343 M. extorquiens 1.8 x 10 3.2 x 108 DSM-1819 M. radiotoierans 1.4 x 10 1.9 x 108 DSM-6343 M. extorquiens 1.0 x 10 4.0 x 10 DSM-13060 M. extorquiens 1.1 x 10 6.0 x 10 DSM-1819 M. radiotoierans 1.3 x 10 5.5 x 108 DSM-18172 M. organophilum 2.8 x 10 4.8 x 10 DSM-13060 M. extorquiens 8.9 x 10 4.7 x 108 DSM-1708 M. mesophilicum 7.8 x 10 1.6 x 108 DSM-18172 M. organophilum 2.0 x 10 8.8 x 107 DSM-182O7 M. oryzae 5.2 x 10 3.2 x 108 DSM-1708 M. mesophilicum 7.9 x 10 5.8 x 108 DSM-19779 M. phyllosphaerae 2.5 x 10 9.7 x 107 DSM-182O7 M. oryzae 1.0 x 10 2.8 x 108 ATCC-14718 M. extorquiens 9.7 x 10 4.4 x 10 DSM-19779 M. phyllosphaerae 4.1 x 10 9.0 x 107 ATCC-14821 M. rhodinum 2.7 x 10 9.2 x 107 ATCC-14718 M. extorquiens 9.5 x 10' 7.9 x 107 ATCC-21611 M. rhodesianum 9.7 x 10' 6.4x 108 ATCC-14821 M. rhodinum 5.4 x 10 5.7 x 108 ATCC-35065 M. filisawaense 5.7 x 10 9.1 x 10' ATCC-21611 M. rhodesianum 5.1 x 10 2.7 x 108 ATCC-43883 M. zaimani 8.2 x 10 3.9 x 108 ATCC-35065 M. filisawaense 8.1 x 10 5.9 x 108 ATCC-51358 M. aminovorans 8.8 x 10 1.8 x 108 ATCC-43883 M. zaimani 7.6 x 10' 3.9 x 10 ATCC-7OO647 M. thiocyanatum 9.8 x 10 6.7 x 108 ATCC-51358 M. aminovorans 2.7 x 10 8.3 x 107 ATCC-7OO647 M. thiocyanatum 4.6 x 10 4.4 x 108 (0.148. The sand was a product called “White Hermit Crab Sand, with the ingredients listed as calcium carbonate and magnesium carbonate, and sold by Zoo-Med Laboratories, Growth of PPFM strains in AMS-GP liquid medium plus Inc., of San Luis Obispo, Calif. bone meal (at 2 grams per liter initial titer titer after 3 days Growth of PPFM strains in AMS-GP liquid medium plus a silica-micaceous clay (at 2grams per liter DSM-6343 M. extorquens 9.6 x 10 1.4 x 108 DSM-1819 M. radiotolerans 4.7 x 10 5.0 x 108 initial titer tilter after 3 days DSM-13060 M. extorquens 8.5 x 10' 4.7 x 10 DSM-18172 M. organophilum 5.9 x 10' 3.9 x 10 DSM-6343 M. extorquiens 9.8 x 10' 9.4 x 10 DSM-1708 M. mesophilicum 8.2 x 10 2.6 x 108 DSM-1819 M. radiotoierans 2.4 x 10 1.2 x 108 DSM-182O7 M. oryzae 3.6 x 10 4.5 x 108 DSM-13060 M. extorquiens 5.4 x 10 2.8 x 108 DSM-19779 M. phyllosphaerae 5.4 x 10' 8.0 x 107 DSM-18172 M. organophilum 2.3 x 10 6.0 x 10 ATCC-14718 M. extorquens 1.5 x 10 9.1 x 107 DSM-1708 M. mesophilicum 8.3 x 10' 5.5 x 108 ATCC-14821 M. rhodinum 9.9 x 10 7.0 x 107 DSM-182O7 M. oryzae 9.7 x 10' 3.2 x 108 US 2016/007 3641 A1 Mar. 17, 2016 26

-continued Growth of PPFM strains in AMS-GP liquid medium Growth of PPFM strains in AMS-GP liquid medium plus lus crushed egg shell (at 2 grams per liter a silica-micaceous clay (at 2grams per liter initial titer tilter after 3 days initial titer titer after 3 days DSM-6343 M. extorquiens 2.8 x 10 2.9 x 109 DSM-19779 M. phyllosphaerae 6.6 x 10' 9.8 x 10 DSM-1819 M. radiotoierans 2.1 x 10 7.4 x 10 ATCC-14718 M. extorquiens 1.2 x 10 5.5 x 108 DSM-13060 M. extorquens 3.3 x 10 6.4 x 10 ATCC-14821 M. rhodium 4.8 x 10 6.5 x 108 DSM-18172 M. organophilum 4.5 x 10 8.7 x 108 ATCC-21611 M. rhodesianun 2.0 x 10 5.2 x 108 DSM-1708 M. mesophilicum 4.3 x 10 2.5 x 109 ATCC-35065 M. filisawaense 6.2 x 10' 1.0 x 10 DSM-18207 M. oryzae 2.3 x 10 9.3 x 10 ATCC-43883 M. zaimani 3.1 x 10 9.0 x 107 DSM-19779 M. phyllosphaerae 3.1 x 10 1.7 x 10° ATCC-51358 M. aminovorans 4.3 x 10 3.2 x 108 ATCC-14718 M. extorquens 2.4 x 10 3.3 x 109 ATCC-7OO647 M. thiocyanatum 7.6 x 10' 8.0 x 10 ATCC-14821 M. rhodinum 4.2 x 10 7.8 x 108 ATCC-21611 M. rhodesianum 8.4 x 10' 5.7 x 108 ATCC-35065 M.fijisawaense 1.1 x 10 2.8 x 10 ATCC-43883 M. Zailinarii 3.2 x 10 6.9 x 108 014.9 The micaceous clay was a product called “Profile'. ATCC-51358 M. afirovojans 2.8 x 10 6.7 x 108 with the ingredients listed as a blend of silica and illite. Illite ATCC-700647 M. thiocyanatum 8.9 x 10' 9.2 x 10 is a micaceous clay. Profile is sold by Profile Products, LLC, of Buffalo Grove, Ill. 0152 The crushed shell was obtained from chicken egg shells. Growth of PPFM strains in AMS-GP liquid medium 0153. Four other solid substrates were tested with two lus a silicate-mineraloid clav (at 2 grams per liter PPFM strains. While all four of these solid Substrates enabled the two PPFM strains to grow to higher titers than in water initial titer titer after 3 days clear AMS-GP medium, the growth was relatively light com DSM-6343 M. extorquiens 3.5 x 10 4.0 x 108 pared to the other solid substrates tested above. The ground DSM-1819 M. radiotoierans 2.5 x 10 5.0 x 108 wheat and ground barley were very coarse, which could have DSM-13060 M. extorquens 5.2 x 10' 8.1 x 10 contributed to this relatively lightgrowth due to the relatively DSM-18172 M. organophilum 4.3 x 10 5.6 x 10 low surface area of a coarsely ground solid Substrate. DSM-1708 M. mesophilicum 1.0 x 10 2.0 x 109 0154 Because of this relatively light growth, these four DSM-18207 M. oryzae 2.2 x 10 8.2 x 108 solid substrates were not tested with the other PPFM strains. DSM-19779 M. phyllosphaerae 3.6 x 10 6.6 x 10 ATCC-14718 M. extorquens 2.4 x 10 4.1 x 10 ATCC-14821 M. rhodinum 3.4 x 10 1.3 x 109 ATCC-21611 M. rhodesianum 3.8 x 10 7.0 x 108 Growth of PPFM strains in AMS-GP liquid medium ATCC-35065 M.fijisawaense 5.5 x 10 5.4 x 10 plus flaxseed neal (at 2 grams per liter ATCC-43883 M. Zainarii 6.9 x 10 3.0 x 108 ATCC-51358 M. afirovoiars 7.8 x 10 1.9 x 109 initial titer tilter after 3 days ATCC-700647 M. thiocyanatum 4.2 x 10 4.8 x 108 DSM-6343 M. extorquens 3.4 x 10 6.4 x 10 DSM-1708 M. mesophilicum 9.9 x 10 1.2 x 107 0150. The mineraloid clay was a product called “Rare Earth', with the ingredients listed as a blend of a pyrophyllitic silicate clay and leonardite. Leonardite is a mineraloid com posed of oxidized lignite; it is high in humic acid. Rare Earth Growth of PPFM strains in AMS-GP liquid medium is sold by General Hydroponics of Sebastopol, Calif. plus ground wheat (at 2 grams per liter initial titer tilter after 3 days Growth of PPFM strains in AMS-GP liquid medium DSM-6343 M. exiorquens 2.1 x 10 9.7 x 106 plus an aluminum phyllosilicate clay (at 2 grams per liter DSM-1708 M. mesophilicum 4.3 x 10 3.3 x 107 initial titer titer after 3 days DSM-6343 M. extorquiens 3.5 x 10 8.0 x 10 DSM-1819 M. radiotoierans 2.5 x 10 6.1 x 10 Growth of PPFM strains in AMS-GP iquid medium DSM-13060 M. extorquens 5.2 x 10 5.5 x 108 lus ground barle at 2 grams per liter DSM-18172 M. organophilum 4.3 x 10 8.2 x 107 DSM-1708 M. mesophilicum 1.0 x 10 1.3 x 10 initial titer tilter after 3 days DSM-18207 M. oryzae 2.2 x 10 9.6 x 10 DSM-19779 M. phyllosphaerae 3.6 x 10 2.7 x 108 DSM-6343 M. extorquens 2.2 x 10 3.4 x 107 ATCC-14718 M. extorquens 3.0 x 10 7.8 x 108 DSM-1708 M. mesophilicum 9.2 x 10 1.4 x 107 ATCC-14821 M. rhodinum 3.4 x 10 6.5 x 10 ATCC-21611 M. rhodesianum 2.4 x 10 2.8 x 10 ATCC-35065 M.fijisawaense 9.9 x 10 2.2 x 109 ATCC-43883 M. Zainarii 2.3 x 10 7.2 x 108 ATCC-51358 M. afirovoiars 3.8 x 10 6.3 x 10 Growth of PPFM strains in AMS-GP liquid medium ATCC-700647 M. thiocyanatum 8.1 x 10' 8.8 x 10 plus dried shrimp (at 2 grams per liter initial titer tilter after 3 days 0151. The aluminum phyllosilicate clay was a product DSM-6343 M. extorquens 3.0 x 10 7.5 x 106 called “Bentonite', with the ingredient listed as an aluminum DSM-1708 M. mesophilicum 7.1 x 10 3.7 x 107 phyllosilicate clay. Bentonite is sold by L. D. Carlson Co. of Kent, Ohio. US 2016/007 3641 A1 Mar. 17, 2016 27

0155 The dried shrimp were powdered brine shrimp, sold particulate solid Substrate concentration equivalent to 2 by OmegaSea Ltd, of Sitka, Ak. grams per liter. The target initial titer in each tube was about 1x10 PPFM cells per ml. The inoculated test tubes were Example 11 placed on a rotary shaker set and grown for three days at 30 degrees C., and 250 rpm. Utilization of PPFM Bacteria to Promote Plant Growth and Early Development 0156 The establishment of a vigorous and uniform stand Growth of PPFM strains in AMS-GP liquid medium of corn plants early in the growing season is essential to a high plus tufts of cotton (at 2 grams per liter) yielding crop, and is mainly dependent on the development of titer after 3 a vigorous nodal root system. The first roots that emerge from initial titer days (CFU/mL) a corn seed (the radicle and seminal roots) function mainly in the uptake of water from the soil. The radicle seminal roots do DSM-6343 M. exiorgiiens 6.6 x 10 3.7 x 108 not provide other nourishment, which early in the growth of DSM-1819 M. radiotoierans 2.4 x 10 1.2 x 10 the seedling is provided by energy and nutrient reserves in the DSM-13060 M. extorquens 1.7 x 10 5.7 x 107 DSM-18207 M. oryzae 1.6 x 10 9.4 x 107 kernel. When nodal roots emerge from the corn stem, growth DSM-19779 M. phyllosphaerae 8.3 x 10' 4.7 x 10 of the seminal roots slows dramatically and they contribute ATCC-14718 M. extorquens 7.0x 10 8.1 x 107 little to the season-long maintenance of the corn plant. ATCC-21611 M. rhodesianum 3.5 x 10 6.5 x 107 Instead, the nodal root system plays this role. Thus, the early ATCC-35065 M.fijisawaense 3.3 x 10 2.5 x 108 and vigorous establishment of a nodal root system playSakey ATCC-51358 M. afirovojans 4.2 x 10 7.0 x 107 role in the development of a uniform stand of corn. Failure to ATCC-700647 M. thiocyanatum 2.1 x 10 7.4 x 107 do so results in stunted plants and other deficiencies that end in lower yields at harvest. (O157 Cultures of PPFM bacteria produced by the method 0160. After three days of growth, the cotton tufts were a of Example 3 (i.e. by growing the PPFM bacteria in liquid dark brilliant pink in color, being coated with adhered PPFM media containing insoluble salt crystals) were used to treat cells (FIG.3A). These adhered PPFM were removed by vig corn seeds. To 72 corn seeds, 20 milliliters of the PPFM orous vortexing, and the resulting suspensions of PPFM cells culture were added, in a container Such that the corn seeds were titered. The titers of PPFM cells attained were higher were completely immersed in the PPFM culture. As a control, than those attained by growing the PPFM in clear AMS-GP an equal number of corn seeds were immersed in the PPFM liquid medium (see representative results of Example 10 free culture medium of Example 3. The corn seeds were soaked in these solutions for 4 hours, at room temperature where PPFM grown for 3 days in water clear AMS-GP media (about 22 degrees C.), with gentle agitation. At the end of this did not exceed 10° colony forming units per ml). A photomi soaking period, the seeds were planted in potting soil and crograph showing PPFM strain ATCC-35065 M. allowed to germinate and grow for 8 days. At that time, the fiujisawaense adhered to cotton fibers is provided in FIG. 4. corn seedlings were dug up, rinsed to remove the soil, and the nodal roots counted and measured. The results are shown a. Growth of PPFM strains in AMS-GP liquid medium below. plus tufts of glass wool (at 2 grams per liter titer after 3 control PPFM treated initial titer days (CFU/mL) number of nodal roots 2.56 2.88. 13% more DSM-6343 M. extorquiens 3.4 x 10 2.0 x 10 per plant, in milligrams DSM-1819 M. radioioierans 1.9 x 10 2.3 x 10 average length of nodal roots 2.33 3.44 48% longer DSM-13060 M. extorquens 8.0 x 10 5.6 x 107 per plant in centimeters DSM-18207 M. oryzae 9.4 x 10 1.8 x 108 DSM-19779 M. phyllosphaerae 3.8 x 10 6.5 x 107 ATCC-14718 M. extorquens 7.4 x 10' 3.9 x 10 ATCC-21611 M. rhodesianum 3.2 x 10 8.0 x 107 0158. These results indicate that contacting corn seeds ATCC-35065 M.fijisawaense 6.8 x 10 1.0 x 108 with the high-titer PPFM culture provided by the instant ATCC-51358 M. afirovojans 1.1 x 10 6.5 x 107 invention results in the earlier emergence and more rapid ATCC-700647 M. thiocyanatum 1.7 x 10 3.0 x 10 growth of nodal roots. Example 12 0.161. After three days of growth, the glass wool tufts were brilliant pink in color, being coated with adhered PPFM cells Growth of Methylobacterium in Liquid Media with (FIG.3B). These adhered PPFM were removed by vigorous Non-Particulate Solid Substances vortexing, and the resulting suspensions of PPFM cells were 0159. Ten distinct Methylobacterium (PPFM) strains were titered. The titers of PPFM cells attained were higher than grown in 200 ml of the water-clear AMS-GP medium, titered, those attained by growing the PPFM in clear AMS-GP liquid and then concentrated ten-fold. These PPFM cultures, with medium (see representative results of Example 2). The titers no solid Substances present, were used to inoculate test tubes of PPFM cells attained were higher than those attained by containing 10 ml of water-clear AMS-GP medium with vari growing the PPFM in clear AMS-GP liquid medium (see ous added non-particulate Solid Substances. For the non-par representative results of Example 10 where PPFM grown for ticulate solids, 20 mg of the various non-particulate Solid 3 days in water clear AMS-GP media did not exceed 10° Substances were added to each 10 ml tube, yielding a non colony forming units per ml). US 2016/007 3641 A1 Mar. 17, 2016 28

(0171 Holland, M.A., and J. C. Polacco. 1994. PPFMs and b. Growth of PPFM strains in AMS-GP liquid medium other covert contaminants: Is there more to plant physiol plus tufts of synthetic Sponge (at 2 grams per liter ogy than just plant? Annu. Rev. Plant Physiol. Plant Mol. titer after 3 Biol. 45: 197-209. initial titer days (CFU/mL) 0172 Kutschera, U. 2007. Plant-associated methylobac teria as co-evolved phytosymbionts. A hypothesis. Plant DSM-6343 M. extorquiens 8.6 x 10' 8.4 x 107 DSM-1819 M. radiotoierans 4.0 x 10 4.4 x 107 Signal Behav. 2: 74-78. DSM-13060 M. extorquens 8.4 x 10 2.9 x 108 (0173 Lidstrom, M. E., 2006. Aerobic methylotrophic DSM-18207 M. oryzae 9.2 x 10 3.4 x 108 prokaryotes. In Dworkin, M., S. Falkow, E. Rosenberg, DSM-19779 M. phyllosphaerae 9.0 x 10' 5.7 x 107 ATCC-14718 M. extorquens 7.5 x 10 3.2 x 10 K.-H. Schleifer, and E. Stackebrandt (eds.). “The Prokary ATCC-21611 M. rhodesianum 8.5 x 10 1.6 x 108 otes. A Handbook on the Biology of Bacteria. Volume 2. ATCC-35065 M.fijisawaense 3.4 x 10 7.5 x 107 Ecophysiology and biochemistry.” Third edition. Springer, ATCC-51358 M. afirovoiars 7.8 x 10' 7.8 x 107 New York. Pages 618-634. ATCC-700647 M. thiocyanatum 2.4 x 10 9.5 x 107 0.174 Madhaiyan, M., S. Poonguzhali, H. S. Lee, K. Hari, S. P. Sundaram, and T. M. Sa. 2005. Pink-pigmented fac 0162 The synthetic sponge used was "Body Scrub”. ultative methylotrophic bacteria accelerate germination, manufactured by Compac Industries, Inc. of Decatur, Ga. The growth and yield of sugarcane clone Co86032 (Saccharum synthetic sponge is made of a polyester polymeric material. officinarum L.) Biol. Fertil. Soils 41: 350-358. 0.175. Madhaiyan, M., S. Poonguzhali, M. Senthilkumar, 0163. After three days of growth, the synthetic sponge S. Seshadri, H. Chung, J. Yang, S. Sundaram, and T. Sa. tufts were brilliant pink in color, being coated with adhered 2004. Growth promotion and induction of systemic resis PPFM cells (FIG. 3C). These adhered PPFM were removed tance in rice cultivar CO-47 (Oryza saliva L.) by Methylo by vigorous Vortexing, and the resulting Suspensions of bacterium spp. Bot. Bull. Acad. Sin. 45: 315-324. PPFM cells were titered. The titers of PPFM cells attained (0176) Madhaiyan, M., S. Poonguzhali, and T. Sa. 2007. were higher than those attained by growing the PPFM in clear Influence of plant species and environmental conditions on AMS-GP liquid medium (see representative results of epiphytic and endophytic pink-pigmented facultative Example 10 where PPFM grown for 3 days in water clear methylotrophic bacterial populations associated withfield AMS-GP media did not exceed 10° colony forming units per grown rice cultivars. J Microbiol Biotechnol. 2007 Octo ml). ber; 17(10): 1645-54. 0177 Stanier, R. Y., N. J. Palleroni, and M. Doudoroff. REFERENCES 1966. The aerobic pseudomonads: A taxonomic study. J. 0164. Abanda-Nkpwatt, D., M. Musch, J. Tschiersch, M. Gen. Microbiol. 43: 159-271. Boettner, and W. Schwab. 2006. Molecular interaction 0.178 Sy, A., Giraud, E., Jourand, P., Garcia, N., Willems, between Methylobacterium extorquens and seedlings: A., De Lajudie, P., Prin, Y., Neyra, M., Gillis, M., Boivin growth promotion, methanol consumption, and localiza Masson, C., and Dreyfus, B. 2001. Methylotrophic Methy tion of the methanol emission site. J. Exp. Bot. 57: 4025 lobacterium Bacteria Nodulate and Fix Nitrogen in Sym 4032. biosis with Legumes. Jour. Bacteriol. 183(1):214-220, 0.165 Cao, Y-R, Wang, Q., Jin, R-X. Tang, S-K. He, (0179 Sy, A., A. C.J. Timmers, C. Knief, and J. A. Vorholt. W-X., Lai, H-X, Xu, L-H., and C-L Jiang. 2011. Methylo 2005. Methylotrophic metabolism is advantageous for bacterium Solisp. nov. a methanol-utilizing bacterium iso Methylobacterium extorquens during colonization of lated from the forest soil. Antonie van Leeuwenhoek Medicago truncatula under competitive conditions. Appl. Environ. Microbiol. 71: 7245-7252. (2011) 99:629-634. 0180 Vogel, H. J., and D. M. Bonner. 1956. Acetylorni (0166 Corpe, W. A., and D. V. Basile. 1982. Methanol thinase of Escherichia coli: Partial purification and some utilizing bacteria associated with green plants. Devel. properties. J. Biol. Chem. 218: 97-106. Industr. Microbiol. 23: 483-493. 0181. Whittenbury, R., S. L. Davies, and J. F. Wilkinson. (0167 Corpe, W.A., and S. Rheem. 1989. Ecology of the 1970. Enrichment, isolation and some properties of meth methylotrophic bacteria on living leaf surfaces. FEMS ane-utilizing bacteria. J. Gen. Microbiol. 61: 205-218. Microbiol. Ecol. 62: 243-250. (0168 Green, P. N. 2005. Methylobacterium. In Brenner, Example 13 D.J., N. R. Krieg, and J.T. Staley (eds.). “Bergey’s Manual of Systematic Bacteriology. Volume two. The Proteobac Growth of Methylobacterium on a Solid and teria. Part C. The alpha-, beta-, delta-, and epsilonproteo Preparation of Dry Powder PPFM Formulation from bacteria.” Second edition. Springer, New York. Pages 567 the Same 571. 0182. The growth medium was a liquid base medium (0169 Green, P. N. 2006. Methylobacterium. In Dworkin, amended with a solid Substance. Thus, the medium was com M. S. Falkow, E. Rosenberg, K.-H. Schleifer, and E. prised of both a liquid phase and a solid phase. Stackebrandt (eds.). “The Prokaryotes. A Handbook on the 0183 The liquid base medium was Ammonium Mineral Biology of Bacteria. Volume 5. Proteobacteria: Alpha and Salts (AMS) containing peptone, glutamate as the carbon Beta Subclasses.” Third edition. Springer, New York. Source, and diatomaceous earth as the Solid Substance. Pages 257-265. 0184. To prepare this medium, three stock solutions were (0170 Holland, M.A. 1997. Methylobacterium and plants. made as follows. Stock solution I was made by dissolving 35 Recent. Res. Devel. in Plant Physiol. 1: 207-213. grams of anhydrous dibasic potassium phosphate and 27 US 2016/007 3641 A1 Mar. 17, 2016 29 grams of anhydrous monobasic potassium phosphate in a (0200 Dry Powder PPFM Formulation. A 2.2x10e&cfu per final volume of 1 liter of distilled water. Stock solution I was ml at a 50x concentration. 0185. Stock solution II was made by dissolving 50 grams 0201 Dry Powder PPFM Formulation B 1.9x10e&cfu per of magnesium Sulfate heptahydrate and 25 grams of anhy ml drous ammonium chloride in a final volume of 1 liter of (0202 Dry Powder PPFM Formulation C1.6x10e&cfu per distilled water. Stock Solution II was at a 50x concentration. ml 0186 Stock solution III was made by dissolving 10 grams calcium chloride dihydrate in one liter of distilled water. (0203 The post-lyophilization survival rates for the PPFM Stock Solution III was at 50x concentration. cells in the three culture suspensions were about 70% for Dry 0187 To prepare one liter of growth medium, 20 ml of Powder PPFM Formulation A, about 60% for Dry Powder stock solution I, 20 ml of stock solution II, 20 ml of stock PPFM Formulation B, and about 50% for Dry Powder PPFM Solution III, 10 grams of peptone, 15 grams of sodium Formulation C. glutamate, and 2 grams of diatomaceous earth were added to 940 ml of distilled water. The growth medium was autoclaved before use. Example 15 0188 A shake flask of this growth medium was inoculated with NewLeaf Symbiotics PPFM strain NLS0020 (NRRL Stimulation of Early Growth of Lettuce Seedlings by Deposit No. B-50930) and incubated on a rotary shaker at 250 the Dry Powder PPFM Formulation rpm and 30 degrees C. for 3 days. The final titer of this culture was 3.0x10e8 colony forming units (cfu) per ml. 0204 The lettuce seeds were planted in Oasis Hor ticubesTM. The control groups were not treated. There were Example 14 about 90-100 plants in each group, due to slight differences in germination rates. The lettuce seedlings were grown for 10 Preparation of Dry Powder PPFM Formulation days in agreenhouse, and the wet weights of the above ground (0189 The culture of PPFM strain NLS0020 was amended portions of the plants were measured. The test was run three with skim milk (at 0.9% V/v) and various concentration of times. Sucrose, before drying. (0190. Stock solutions of skim milk (10% w/v) and sucrose TABLE 4 (50% w/v) were prepared and autoclaved before use. (0191) To 8 ml of the NLS0020 culture of Example 1 were Test hi1 added 2 ml of the 50% sucrose stock solution and 1 ml of the average wet 10% skim milk stock solution, to yield a culture solution weight in mg per percent increase confidence containing 9.1% sucrose (w/v) and 0.9% skim milk (v/v). treatinent lettuce seedling over control interval (0192 To 7 ml of NLS0020 culture were added 3 ml of the control 78 50% sucrose stock solution and 1 ml of the 10% skim milk formulation A 104 33% >99% stock solution, to yield a culture solution containing 13.6% formulation B 105 35% >99% Viv sucrose and 0.9% V/v skim milk. formulation C 94 21% >99% 0193 To 6 ml of NLS0020 culture were added 4 ml of the 50% sucrose stock solution and 1 ml of the 10% skim milk stock Solution, to yield a culture Suspension containing 18.2% sucrose and 0.9% skim milk. TABLE 5 0194 The culture suspensions were frozen, and then Test i2 placed into a LabconcoTM 12 L console lyophilizer for four average wet days at -45° C., and <1 millibar psi. This yielded three dry weight in mg per percent increase confidence powder PPFM formulations, each containing different treatinent lettuce seedling over control interval amounts of Sucrose. control 88 0.195 The yield of powder for these various formulations formulation A 105 1996 >99% was about 160 mg per ml of the 9.1% w/v sucrose starting formulation B 99 13% >99% suspension, about 230 mg per ml of the 13.6%% w/v sucrose formulation C 96 9% >99% starting suspension, and about 300 mg per ml of the 18.2%% w/v Sucrose starting Suspension. (0196. The powder obtained from the 9.1% sucrose culture suspension was designated “Dry Powder PPFM Formulation TABLE 6 A. Test i3 (0197) The powder obtained from the 13.6% sucrose cul ture suspension was designated “Dry Powder PPFM Formu average wet lation B. weight in mg per percent increase confidence treatinent lettuce seedling over control interval (0198 The powder obtained from the 18.2% sucrose cul ture suspension was designated “Dry Powder PPFM Formu control 114 lation C. formulation A 154 35% >99% formulation B 135 18% >99% (0199 Samples of these three dry powder PPFM formula formulation C 130 14% >99% tions were resuspended in sterile 0.9% (w/v) saline to the original culture volume and titered. The results were: US 2016/007 3641 A1 Mar. 17, 2016 30

TABLE 7 0218. A sample of this dry powder PPFM formulation was resuspended insterile 0.9% (w/v) saline to the original culture Averages across all three tests Volume and titered. The results were: average wet 0219) Dry Powder PPFM Formulation D 5.4x10e9 cfu per weight in mg per percent increase confidence gram of powder. treatinent lettuce seedling over control interval 0220 100 mg samples of this powder were aliquoted into control 93 individual 1.7 ml conical microfuge tubes. These tubes were formulation A 121 30% >99% then placed into a 250 ml Corning disposable clear bottle and formulation B 113 22% >99% stored at room temperature. The tubes were not protected formulation C 107 15% >99% from the light. One tube was pulled from the bottle at 75 days and another pulled at 311 days and titered as described above. The results were: Example 16 0221) Dry Powder PPFM Formulation D4.4x10e9 cfu per gram of powder at 75 days. Shelf-Life Experiment 0222 Dry Powder PPFM Formulation D5.1x10e9 cfu per 0205 The growth medium was a liquid base medium gram of powder at 311 days. amended with a solid Substance. Thus, the medium was com 0223. These results indicate that this dry powder PPFM prised of both a liquid phase and a solid phase. formulation is very stable, with no detectable decline in the 0206. The liquid base medium was Ammonium Mineral titer of the powder over a period of 311 days of storage at Salts (AMS) containing peptone, glutamate as the carbon room temperature and in the light. Source, and diatomaceous earth as the Solid Substance. 0224 Having illustrated and described the principles of 0207 To prepare this medium, three stock solutions were the present invention, it should be apparent to persons skilled made as follows. in the art that the invention can be modified in arrangement 0208 Stock solution I was made by dissolving 35 grams of and detail without departing from Such principles. anhydrous dibasic potassium phosphate and 27 grams of 0225. Although the materials and methods of this inven anhydrous monobasic potassium phosphate in a final Volume tion have been described in terms of various embodiments of 1 liter of distilled water. Stock solution I was at a 50x and illustrative examples, it will be apparent to those of skill concentration. in the art that variations can be applied to the materials and 0209 Stock solution II was made by dissolving 50 grams methods described herein without departing from the con of magnesium Sulfate heptahydrate and 25 grams of anhy cept, spirit and scope of the invention. All Such similar Sub drous ammonium chloride in a final volume of 1 liter of stitutes and modifications apparent to those skilled in the art distilled water. Stock Solution II was at a 50x concentration. are deemed to be within the spirit, scope and concept of the 0210 Stock solution III was made by dissolving 10 grams invention as defined by the appended claims. calcium chloride dihydrate in one liter of distilled water. What is claimed is: Stock Solution III was at 50x concentration. 1. A method for obtaining a dried preparation of viable 0211 To prepare one liter of growth medium, 20 ml of Methylobacterium comprising: stock solution I, 20 ml of stock solution II, 20 ml of stock (a) combining a fermentation product comprising a solid Solution III, 10 grams of peptone, 15 grams of sodium Substance having a mono-culture or co-culture of glutamate, and 2 grams of diatomaceous earth were added to Methylobacterium adhered thereto with a cryoprotec 940 ml of distilled water. The growth medium was autoclaved tion agent selected from the group consisting of disac before use. charide, poly Sucrose, hydroxyethylcellulose, and com 0212. A shake flask of this growth medium was inoculated binations thereof to obtain a biphasic mixture that with NewLeaf Symbiotics PPFM strain NLS0020 (NRRL comprises the cryoprotection agent and a solid phase Deposit No. B-50930) and incubated on a rotary shaker at 250 comprising the fermentation product; and, rpm and 30 degrees C. for 3 days. The final titer of this culture (b) lyophilizing the biphasic mixture, thereby obtaining the was 7.2x10e8 colony forming units (cfu) per ml. dried preparation of viable Methylobacterium. 0213. The culture of PPFM strain NLS0020 was amended 2. The method of claim 1, wherein the fermentation prod with skim milk (at 0.9% V/v) and various concentration of uct has a Methylobacterium titer of 5x10, 1x10, or 1x10' Sucrose, before drying. colony-forming units per gram of solid to about 5x10' 0214 Stock solutions of skim milk (10% V/v) and sucrose colony-forming units of Methylobacterium per gram of the (50% w/v) were prepared and autoclaved before use. Solid Substance. 0215. To 9 ml of the NLS0020 culture of Example 1 were 3. The method of claim 1, wherein the disaccharide is added 1 ml of the 50% sucrose stock solution and 1 ml of the selected from the group consisting of Sucrose, lactose, treha 10% skim milk stock solution, to yield a culture solution lose, and combinations thereof. containing 4.6% sucrose (w/v) and 0.9% skim milk (v/v). 4. The method of claim 1, wherein the polysucrose has a 0216. The culture suspensions were frozen, and then molecular weight of about 60 to about 80 kilodaltons (kD) or placed into a LabconcoTM 12 L console lyophilizer for three about 300 to about 500 kilodaltons (kD). days at -45°C., and <1 millibar psi. This yielded dry powder 5. The method of claim 1, wherein the cryoprotection agent PPFM formulation D. is Sucrose. 0217. The yield of powder for these various formulations 6. The method of claim 5, wherein the hydroxyethylcellu was about 100 mg per ml of the 4.6% w/v sucrose starting lose has a molecular weight of about 600 kilodaltons (kD) to Suspension. about 700 kilodaltons (kD). US 2016/007 3641 A1 Mar. 17, 2016

7. The method of claim 6, further comprising the addition 22. The plant part of claim 21, wherein the seed is a lettuce ofaprotein of plant, animal or microbial originto the biphasic seed and is coated with about 1x10 CFU of Methylobacte mixture. rium per seed to about 1x10 CFU of the Methylobacterium 8. The method of claim 1, wherein the solid substance is per seed. 23. The plant part of claim 22, wherein the seed is a lettuce selected from the group consisting of a man-made material, a seed and is coated with about 1x10 CFU of Methylobacte material of animal origin, a material of plant origin, a material rium per seed to about 5x10 CFU of the Methylobacterium of microbial origin, a material of fungal origin, a material of per seed. mineral origin, and combinations thereof. 24. A composition that comprises a Solid Substance having 9. The method of claim 1, wherein the solid substance is an a mono-culture or co-culture of Methylobacterium adhered agriculturally acceptable adjuvant or agriculturally accept thereto, and a cryoprotection agent selected from the group able excipient. consisting of a disaccharide, poly Sucrose, hydroxyethylcel 10. The method of claim 1, wherein said solid substance is lulose, and combinations thereof, wherein the composition is inanimate. substantially free of water, essentially free of water, or essen 11. The method of claim 1, wherein the solid substance is tially dry, and wherein at least about 50%, 60%, or 70% of selected from the group consisting of a polysaccharide, a Methylobacterium cells in the composition are viable. 25. The composition of claim 24, wherein the solid sub diatomaceous earth, a salt crystal, and combinations thereof. stance has a Methylobacterium titer of 5x10, 1x10, or 12. The method of claim 1, wherein said polysaccharide is 1x10" colony-forming units per gram of the solid substance selected from the group of a cellulosic polysaccharide, a to about 5x10" colony-forming units of Methylobacterium chitinous polysaccharide, a galactan polysaccharide, and per gram of the Solid Substance. combinations thereof. 26. The composition of claim 24, wherein the solid sub 13. The method of claim 1, further comprising converting stance is an agriculturally acceptable adjuvant or agricultur the dried preparation to a powder. ally acceptable excipient. 14. The method of claim 1, further comprising the addition 27. The composition of claim 24, wherein the composition of an agriculturally acceptable adjuvant or agriculturally further comprises an agriculturally acceptable adjuvant or acceptable excipient. agriculturally acceptable excipient. 15. The method of claim 1, further comprising the step of 28. The composition of claim 24, wherein the cryoprotec applying the dried preparation of viable Methylobacterium to tion agent is a disaccharide. a plant or plant part. 29. The composition of claim 28, wherein the disaccharide is Sucrose. 16. The method of claim 1, wherein at least about 50%, 30. A plant that is at least partially coated with the compo 60%, or 70% of Methylobacterium cells in the dried prepa sition of claim 24. ration are viable. 31. A plant part that is at least partially coated with the 17. A composition comprising the dried preparation of composition of claim 24. viable Methylobacterium made by the method of claim 1. 32. The plant part of claim 31, wherein the plant part is 18. A plant that is at least partially coated with the compo selected from the group consisting of a seed, stem, root, sition of claim 17. flower, cotyledon, a coleoptile, fruit, and a leaf. 19. A plant part that is at least partially coated with the 33. The plant part of claim 32, wherein the plant part is a composition of claim 17. seed. 20. The plant part of claim 19, wherein the plant part is 34. The plant part of claim 33, wherein the seed is a lettuce selected from the group consisting of a seed, stem, root, seed and is coated with about 7x10 CFU of Methylobacte flower, cotyledon, a coleoptile, fruit, and a leaf. rium per seed to about 4x10 CFU of the Methylobacterium 21. The plant part of claim 20, wherein the plant part is a per seed. seed.