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    • 专利摘要:
    MICROBIAL INOCUUM COMPOSITIONS AND USES THEREOF IN AGRICULTURE. The present disclosure provides innovative agricultural microbial inoculum compositions for uses in promoting plant growth, plant productivity and/or soil quality. Innovative microbial inoculum compositions comprise one or more microbial species, one or more urease inhibitors and/or one or more nitrification inhibitors. The present disclosure also provides fertilizer compositions comprising said microbial inoculum compositions.
    公开号:BR112017026113B1
    申请号:R112017026113-8
    申请日:2016-06-01
    公开日:2022-02-01
    发明作者:Drew Bobeck；Cedric Pearce
    申请人:Koch Agronomic Services, Llc；
    IPC主号:
    专利说明:

    REFERENCE TO RELATED REQUEST
    [001]This application claims priority to US Provisional Patent Application No. 62/169,942, filed on June 2, 2015, which is incorporated herein by reference in its entirety. TECHNICAL FIELD
    [002] The present disclosure relates to innovative agricultural microbial inoculum compositions for uses in promoting plant growth, plant productivity and/or soil quality. Innovative microbial inoculum compositions comprise one or more microbial species, one or more urease inhibitors and/or one or more nitrification inhibitors. The present disclosure also relates to fertilizer compositions comprising said microbial inoculum compositions, their formulations and the uses thereof. BACKGROUND
    [003] The use of fertilizers to improve the production of agricultural plants and crops and to overcome unsatisfactory soil quality is widely practiced. The most commonly used commercially available nitrogen-containing fertilizers are inorganic chemical fertilizers such as urea. Extended use of urea is often associated with negative environmental consequences, such as nitrate contamination in runoff and groundwater, as well as emission of ammonia and nitrous oxide to the atmosphere. Attention to the application of nitrogen-based fertilizers has shifted from the role of promoting crop production to alleviating environmental pollution. There are a variety of new management practices and technologies that can promote efficient nitrogen use and alleviate environmental pollution.
    [004]One of the widely used technologies is the application of a urease inhibitor in combination with urea treatment. The urea component of the fertilizer applied to the soil becomes a source of ammonia as a result of the hydrolysis of urea catalyzed by urease, an enzyme produced by numerous fungi and bacteria that is well known to those skilled in the art. Urease inhibitors can slow the rate of conversion of urea to ammonia, thereby significantly reducing the amount of urea that would otherwise have to be applied to the soil, by reducing the amount of ammonia volatilization. One of the most common urease inhibitors is N-(n-butyl)thiophosphoric triamide (NBPT) (see, for example, US Patent No. 5,698,003).
    [005] Another widely used technology is the application of nitrification inhibitors to significantly reduce nitrate leaching and nitrogen gas emissions. Most of the nitrogen supplied as a commercially available fertilizer is eventually transformed into a nitrate form of nitrogen. In the presence of adequate oxygen, high temperatures and some moisture, N-ammonium is converted to N-nitrate through a biochemical process known as nitrification, which requires two forms of soil bacteria. The first bacterium, Nitrosomonas, converts N-ammonium to N-nitrite. The second bacterium, Nitrobacter, converts N-nitrite to N-nitrate. Nitrification inhibitors have a primary way of slowing down the nitrification process by inhibiting Nitrosomonas bacteria in the area where ammonium will be present. Some widely used nitrification inhibitors that are commercially available include 2-chloro-6-(trichloromethyl)-pyridine (Nitrapyrine) and dicyanodiamide (DCD).
    [006] In addition to the application of chemical enzyme inhibitors such as the N-(n-butyl)thiophosphoric triamide urease inhibitor (NBPT) and nitrification inhibitors such as dicyanodiamide (DCD), fertilizer compositions comprising microorganisms (called "biofertilizers" or "biostimulants") are increasingly considered as alternatives to conventional chemical fertilizers. The ability of specific bacterial species to promote plant growth has long been recognized. For example, nitrogen-fixing bacteria such as the Rhizobium species provide plants with essential nitrogen compounds. It was also shown that Azotobacter and Azospirillum species promote plant growth and increase crop productivity, promoting the accumulation of nutrients in plants. However, bacteria from these genera are often unable to compete effectively with native soil and plant flora, thus requiring the application of impractically large volumes of inoculum. SUMMARY OF THE INVENTION
    [007] To date, urease inhibitors and nitrification inhibitors have been successful to varying degrees, while biofertilizers have typically had limited success. Thus, there is a need for improved fertilizers or fertilizer additives, and for methods that are effective in providing nutrients for plant growth and that are environmentally safe and non-hazardous. One solution is to provide a combination of urease inhibitors and/or nitrification inhibitors with biofertilizers. However, the combination of urease inhibitors and/or nitrification inhibitors with biofertilizers is not simple. First, urease inhibitors and/or nitrification inhibitors can weaken or kill biofertilizers when combined. Second, urease inhibitors and/or nitrification inhibitors are typically dispensed in a solvent system (eg glycol, complex amines, aryl alcohols), which can also weaken or kill biofertilizers.
    [008] In WO 2015/104698 A2 the combination of a urease inhibitor with many microbial pesticides was disclosed. However, the document did not disclose any solvent system that could provide reasonable viability for the mentioned microbial pesticides. Furthermore, the document did not identify any solvent-tolerant bacteria.
    [009] Solvent-tolerant bacteria are potentially useful in many applications from microbial transformation to environmental remediation, as well as in biotechnological processes. Organic solvent tolerance may be a species-specific property, and may not be readily predictable. See, for example, "Association of organic solvent tolerance and fluoroquinolone resistance in clinical isolates of Escherichia coli", Anbu, P., Journal of Antimicrobial Chemotherapy, (1998) 41, 111-114. Most of the reported and well-studied solvent-tolerant bacteria are gram-negative bacteria. Gram-negative bacteria have the advantage of having an additional outer membrane that protects the cytoplasmic membrane by reducing periplasmic concentrations of harmful solvents to acceptable levels. Due to the inherent disadvantage of not having an outer membrane, only a few gram-positive organisms have been reported to exhibit solvent tolerance. See, for example, "Isolation and characterization of a novel organic solvent-tolerant Anoxybacillus sp. PGDY12, a thermophilic Gram-positive bacterium", Gao, Y., Journal of Applied Microbiology, 110, 472-478.
    [010] Surprisingly, in extensive efforts to identify solvent-tolerant bacteria to promote plant health, plant nutrition, and/or soil health, some agriculturally beneficial gram-positive organisms have been identified as being viable in some selected organic solvents. , while some gram-negative organisms are not viable in the same organic solvents. For example, the tested species of gram-positive organisms Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis and Bacillus pumilis demonstrated a viability time range of at least 2 hours to at least 21 days in a solution with at least one of the organic solvents. propylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol and dimethyl sulfoxide (DMSO). However, none of the agriculturally beneficial gram-negative species of Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas chlororaphis tested demonstrated viability in the organic solvents tested.
    [011] In one embodiment, the present disclosure provides an agricultural microbial inoculum composition comprising at least one microbial strain originating from one or more microbial species, and at least one active agent, wherein the active agent is a urease inhibitor, or a nitrification inhibitor, or a combination thereof, and further being that the at least one microbial strain is present in an amount effective to promote plant health, plant nutrition and/or soil health in the presence of the active agent .
    [012] In one embodiment, the present disclosure provides an agricultural microbial inoculum composition comprising: i. at least one agriculturally beneficial species of bacillus selected from the group consisting of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis, Bacillus pumilis and any combination thereof; and ii. at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO) and any combination thereof.
    [013] In one embodiment, the present disclosure provides an agricultural microbial inoculum composition comprising: i. at least one agriculturally beneficial species of bacillus selected from the group consisting of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis, Bacillus pumilis and any combination thereof; ii. at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO) and any combination thereof; and iii. a urease inhibitor selected from the group consisting of N-(n-butyl)thiophosphoric triamide (NBPT), N-(n-butyl)phosphoric triamide, thiophosphoryl triamide, phenyl phosphorodiamidate, N-cyclohexyl phosphoric triamide, N-cyclohexyl triamide thiophosphoric, phosphoric triamide, hydroquinone, p-benzoquinone, hexamidocyclotriphosphazene, thiopyridines, thiopyrimidines, thiopyridine-N-oxides, N,N-dihalo-2-imidazolidinone, N-halo-2-oxazolidinone, N-(2-nitrophenyl triamide )thiophosphoric, N-(2-nitrophenyl)phosphoric triamide and any combination thereof.
    [014] In one embodiment, the present disclosure provides an agricultural microbial inoculum kit comprising: Part A, wherein Part A comprises a urease inhibitor and at least one organic solvent, said urease inhibitor being selected from group consisting of N-(n-butyl)thiophosphoric triamide (NBPT), N-(n-butyl)phosphoric triamide, thiophosphoryl triamide, phenyl phosphorodiamidate, N-cyclohexyl phosphoric triamide, N-cyclohexyl thiophosphoric triamide, phosphoric triamide, hydroquinone, p-benzoquinone, hexamidocyclotriphosphazene, thiopyridines, thiopyrimidines, thiopyridine-N-oxides, N,N-dihalo-2-imidazolidinone, N-halo-2-oxazolidinone, N-(2-nitrophenyl)thiophosphoric triamide, N-triamide -(2-nitrophenyl)phosphoric and any combination thereof; and wherein said organic solvent is selected from the group consisting of propylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO) and any combination thereof; and Part B, with Part B comprising at least one species of agriculturally beneficial bacillus selected from the group consisting of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis, Bacillus pumilis and any combination thereof, each Part A and each Part B is contained in a separate container.
    [015] In one embodiment, the present disclosure provides an agricultural microbial inoculum kit comprising: Part A, with Part A comprising at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methyl-2 -pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO) and any combination thereof; and Part B, with Part B comprising at least one species of agriculturally beneficial bacillus selected from the group consisting of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis, Bacillus pumilis and any combination thereof, each Part A and each Part B is contained in a separate container.
    [016] In another embodiment, the present disclosure provides a method for accentuating a yield trait in a plant of interest, as compared to the yield trait of a reference or control plant, the method comprising placing a plant , a plant part or a plant seed of interest, or the surrounding soil, in contact with an effective amount of a microbial inoculum composition of the present disclosure.
    [017] The urease inhibitor or the nitrification inhibitor can mitigate nitrate contamination in runoff water and groundwater, as well as the emission of a large amount of ammonia and nitrous oxide to the atmosphere. Microbial species can additionally promote plant health, plant nutrition and soil health. The combination of both chemical enzyme inhibitors and microbial species in suitable compositions and formulations can serve as a better approach to improve the efficiency of nitrogen-based fertilizer use by optimizing plant productivity, soil quality and overall environmental sustainability. DETAILED DESCRIPTION OF THE INVENTION
    [018] Except where otherwise defined, all technical and scientific terms used herein have the same meaning commonly understood by those skilled in the art.
    [019] In one embodiment, the present disclosure provides an agricultural microbial inoculum composition comprising at least one microbial strain from one or more microbial species, and at least one active agent, wherein the active agent is a urease inhibitor, or a nitrification inhibitor, or a combination thereof, and further being that the at least one microbial strain promotes plant health, plant nutrition and/or soil health in the presence of the active agent.
    [020] In another embodiment, the present disclosure provides an agricultural microbial inoculum composition comprising at least one microbial strain from one or more microbial species and at least one active agent, wherein the active agent is a urease inhibitor, or a nitrification inhibitor, or a combination thereof, additionally being that the at least one microbial strain promotes plant health, plant nutrition and/or soil health in the presence of the active agent, one or more microbial species are selected from the following groups: (1) Species of spore-forming bacteria; (2) Species of spore-forming fungi; (3) Mycorrhizal organisms, including species of Laccaria bicolor, Glomus intraradices and Amanita; (4) Actinomyces species and strains thereof, including Streptomyces lydicus, Streptomyces griseoviridis, Streptomyces griseoviridis K61 (Mycostop, AgBio Development) and Streptomyces microflavus AQ 6121; (5) Bacillus species and strains thereof, including: Bacillus itcheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus oleronius, Bacillus megaterium, Bacillus mojavensis, Bacillus pumilus, Bacillus subtilis, Bacillus circulans, Bacillus globisporus, Bacillus firmus , Bacillus thuringiensis, Bacillus cereus, Bacillus amyloliquefaciens strain D747 (Double Nickel, Certis), Bacillus firmus strain I-1582 (Votivo and Nortica, Bayer), Bacillus licheniformis, Bacillus licheniformis strain SB3086 (EcoGuard, Novozymes), Bacillus pumilus GB34 (YieldShield, Bayer), QST2808 (Sonata, Bayer), Bacillus subtilis strain GB03 (Kodiak, Bayer), MBI 600 (Subtilex, Becker Underwood) & QST 713 (Serenade, Bayer), Bacillus subtilis strain GB122 with Bacillus amyloliquefaciens strain GB99 (BioYield, Bayer), Bacillus pumilus strain BU F-33, Bacillus thuringiensis galleriae strain SDS-502, Bacillus thuringiensis kurstaki, VBTS 2546, Bacillus cereus BP01, Bacillus subtilis strain EB120, Bacillus subtilis strain J-P13, Bacillus subtilis FB17, Bacillus subtilis strains QST30002 and QST3004 (NRRL B-50421 and NRRLB-50455), Bacillus subtilis strains QST30002 and QST3004 (NRRL B-50421 and NRRLB-50455) "sandpaper" mutants, enterotoxin-deficient quadruple mutants of Bacillus thuringiensis subspecies kurstaki strain VBTS 2477, Bacillus simplex strains 03WN13, 03WN23 and 03WN25, Bacillus subtilis strain QST 713, Bacillus mycoides BmJ NRRL B strain -30890, Bacillus subtilis strain DSM 17231 and B. licheniformis strain DSM17236, Bacillus aryabhattai, B. flexus, B. nealsonii, Bacillus sphaericus, Bacillus megaterium, B. vallismortis, Bacillus amyloliquefaciens (ATCC 23842), Bacillus licheniformis (ATCC 14580) ), Bacillus licheniformis (B-642), Bacillus licheniformis (B-14368), Bacillus thuringiensis (ATCC 10792), Bacillus thuringiensis (HD-17), Bacillus thuringiensis (HD-1), and Bacillus pumilis (NRS-272); (6) plant growth promoting rhizobacteria species (RPCVs) and strains thereof, including species reported to be able to fix nitrogen, e.g. Gluconacetobacter species (e.g. Gluconacetobacter diazotrophicus aka Acetobacter diazotrophicus), Spirillum species (e.g. Spirillum lipoferum ), Azospirillum species, Herbaspirillum seropedicae, Azoarcus species, Azotobacter species, Burkholderia species, Burkholderia sp. A396 and Paenibacillus polymyxa; (7) nitrogen-fixing bacterial species and strains thereof, including Rhizobium species (eg Bradyrhizobium species such as Bradyrhizobium japonicum and Rhizobium meliloti); (8) microbial species and strains thereof that are known to improve nutrient use efficiency, including Penicillium species (eg Penicillium bilaii, Penicillium bilaji) and Mesorhizobium cicero; (9) Microbial species and strains thereof that are known to have insecticidal or insect repellent effects, including Telenomus podisi, Baculovirus anticarsia, Trichogramma pretiosum, Trichogramma galloi, Chromobacterium subtsugae, Trichoderma fertile JM41R, Beauveria bassiana, Beauveria bassiana strain NRRL 30976, strain of Beauveria bassiana ATP02, DSM 24665, Paecilomyces fumosoroseus, Trichoderma harzianum, Verticillium lecanii, Isaria Petição 870190134244, of 12/16/2019, p. 15/48 CCM 8367 (CCEFO.011.PFR), Lecanicillium muscarium, Streptomyces microflavus and Muscodor albus; (10) Microbial species and strains thereof that are known to have nematicidal effects, e.g. Myrothecium verrucaria, Pasteuria species and strains thereof, including Pasteuria nishizawae, Pasteuria strain Pasteuria reneformis Pr-3, Paecilomyces lilacinus, Chromobacterium subtsugae, Pasteuria strain ATCC SD-5832, Metarhizium species and Flavobacterium species; (11) Microbial species and strains thereof that are known to have antifungal, antimicrobial and/or plant growth promoting effects, e.g. Gliocladium species, Pseudomonas species (e.g. Pseudomonas fluorescens, Pseudomonas fluorescens D7, P. putida and P. chlororaphis), Pseudomonas fluorescens strain NRRL B-21133, NRRL B-21053 or NRRL B-21102, Pseudomonas fluorescens VP5, Pseudomonas synxantha, Pseudomonas diazotrophicus, Enterobacter cloacae strain NRRL B-21050, Trichoderma species, Trichoderma de virens, Trichoderma atroviride, Coniothyrium minitans, Gliocladium species, Gliocladium virens, Gliocladium roseum strain 321U, Trichoderma harzianum species, Trichoderma harzianum Rifai, Clonostachys rosea strain 88-710, Pseudomonas rhodesiae FERM BP-10912, Serratia muthica CCGG2742, ceply coccus lavescens OH 182.9, Serratia plymuthica, Cladosporium cladosporioides, Mitsuaria species, Coprinus curtus, Virgibacillus halophilus , Saccharomyces species, Metschnikovia fruticola, Candida oleophila, Acremonium species, Pseudozyma aphidis, Pythium oligandrum, Phoma spp I-4278 strain, Achromobacter species, Geomyces species, Pseudomonas azotoformans strain, F30A strain, Brevibacillus parabrevis strain No 4; ASPERGILLUS NRRL 50427, NRRL 50428, NRRL 50429, NRRL 50430 and NRRL 50431, MYCAPARASITICAL SPHAERODES IDAC 301008-01, 02 or 03, Muscodor Albus NRRL 30547 or NRRL30548, Serratia Plymuthica CCGG2742, CEPA of pseudomonas koreensis 10IL21, 13IL01 strain of P. lini, Pantoea agglomerans strain 10IL31, Streptomyces strain RB72 scopuliridis, as endophyte Acremonium spp strain of Streptomyces spp BG76, Paracoccus kondratievae, Enterobacter cloacae, Cryptococcus flavescens, parafarraginis Lactobacillus, Lactobacillus buchneri, Lactobacillus or RAPI Lactobacillus zeae, Paenibacillus polymyxa, Serratia plymuthica, Phoma species, Pythium oligandrum, Mycosphaerella species and Variovorax species; (12) bacterial species and their strains from the group called Pink Pigmented Facultative Methylotrophs (PPFMs, "Pink-Pigmented Facultative Methylotrophs") - Pink Pigmented Optional Methylotrophs) including Methylobacterium species; and (13) microbial species and strains thereof which are known to have a herbicidal effect, for example Pyrenophora semeniperda; wherein the urease inhibitor is selected from the group consisting of N-(n-butyl)thiophosphoric triamide (NBPT), N-(n-butyl)phosphoric triamide, thiophosphoryl triamide, phenyl phosphorodiamidate, N-cyclohexyl phosphoric triamide, triamide N-cyclohexyl thiophosphoric, phosphoric triamide, hydroquinone, p-benzoquinone, hexamidocyclotriphosphazene, thiopyridines, thiopyrimidines, thiopyridine-N-oxides, N,N-dihalo-2-imidazolidinone, N-halo-2-oxazolidinone, N-( 2-nitrophenyl)thiophosphoric, N-(2-nitrophenyl)phosphoric triamide, derivatives thereof and any combination thereof; and wherein the nitrification inhibitor is selected from the group consisting of 2-chloro-6-trichloromethylpyridine, 5-ethoxy-3-trichloromethyl-1,2,4-thiadiazole, dicyandiamide, 2-amino-4-chloro-6-methyl -pyrimidine, 1,3-benzothiazol-2-thiol, 4-amino-N-1,3-thiazol-2-ylbenzene sulfonamide, thiourea, guanidine, 3,4-dimethylpyrazole phosphate, 2,4-diamino-6- trichloromethyl-5-triazine, polyether ionophores, 4-amino-1,2,4-triazole, 3-mercapto-1,2,4-triazole, potassium azide, carbon disulfide, sodium trithiocarbonate, ammonium dithiocarbamate, methylcarbamate 2,3,-dihydro-2,2-dimethyl-7-benzofuranol, N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester, ammonium thiosulfate, 1-hydroxypyrazole, 3 -methylpyrazole-1-carboxamide, 3-methylpyrazole, 3,5-dimethylpyrazole, 1,2,4-triazole, nitrification inhibitor G77 (CAS Registry No. 1373256-33-7), derivatives thereof and any combination thereof.
    [021] In one embodiment, the present disclosure provides a solid carrier based formulation for any microbial inoculum composition of the present disclosure, wherein the solid carrier is selected from mineral earths, for example silicates, silica gels, talc, kaolins , limestone, lime, chalk, clays, dolomite, diatomaceous earth, bentonite, montmorillonites; inorganic salts, for example aluminum sulfate, calcium sulfate, copper sulfate, iron sulfate, magnesium sulfate, silicon sulfate, magnesium oxide; polysaccharides, for example cellulose, starch; fertilizers, for example ammonium sulfate, ammonium phosphate, ammonium nitrate; products of vegetable origin, for example cereal flour, tree bark flour, wood flour, nutshell flour; grain flours suitable for use in the present disclosure, for example flours derived from corn, rice, wheat, barley, sorghum, millet, oats, triticale, rye, buckwheat, fonio and quinoa, and mixtures thereof.
    [022] In one embodiment, the present disclosure provides a solvent-based formulation for any agricultural microbial inoculum composition of the present disclosure, wherein the solvent is selected from alkanolamines such as triethanolamine, diethanolamine, monoethanolamine; alkyl diethanolamines, dialkyl monoethanolamines, the alkyl group being a branched or unbranched C1-C24 alkyl chain; methyl sulfoxide (DMSO); alkyl sulfones such as sulfolane (2,3,4,5-tetrahydrothiophene-1,1-dioxide); alkylamides, such as N-methyl pyrrolidone, N-ethyl pyrrolidone, or dimethylformamide; monoalcohols such as methanol, ethanol, propanol, isopropanol or benzyl alcohol; glycols, such as ethylene glycol, propylene glycol, diethylene glycol, or dipropylene glycol; glycol derivatives and protected glycols such as triethylene glycol monobutyl ether; glycerol and glycerol derivatives (trialcohols) including protected glycerols such as isopropylidene glycerol; dibasic esters and derivatives thereof; alkylene carbonates such as ethylene carbonate or propylene carbonate; monobasic esters such as ethyl lactate or ethyl acetate; carboxylic acid polymers such as maleic acid, oleic acid, itaconic acid, acrylic acid or methacrylic acid; monoalkyl glycol ethers and dialkyl glycol ethers; glycol esters; surfactants such as alkylbenzenesulfonates, lignin sulfonates, alkylphenol ethoxylates or polyethoxylated amines.
    [023] In one embodiment, the present disclosure provides an encapsulated formulation for any agricultural microbial inoculum composition of the present disclosure. In the soil environment, inoculated microbial species may have difficulties surviving among competing organisms and naturally occurring predators. To aid in the survival of microorganisms present in microbial inocula and fertilizer compositions of the present disclosure after application to the environment, one or more of the strains of microbial species may be encapsulated in, for example, a suitable polymeric matrix. In one example, the encapsulation may comprise alginate microspheres, as described by Young et al, 2006, "Encapsulation of plant growth-promoting bacteria in alginate beads enriched with humid acid", Biotechnology and Bioengineering 95:76-83. Those skilled in the art will understand that any suitable encapsulating matrix or material may be used. Encapsulation can be accomplished using methods and techniques known to those skilled in the art. The encapsulation of the microorganisms may include nutrients or other components of the inoculum or fertilizer composition, in addition to the microorganisms.
    [024] In one embodiment, the present disclosure provides an agricultural microbial inoculum composition comprising: i. at least one agriculturally beneficial bacillus species selected from the group consisting of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis, Bacillus pumilis and any combination thereof; and ii. at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO) and any combination thereof.
    [025] In one embodiment, the present disclosure provides an agricultural microbial inoculum composition comprising: i. at least one agriculturally beneficial species of bacillus selected from the group consisting of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis, Bacillus pumilis and any combination thereof; ii. at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO) and any combination thereof; and iii. a urease inhibitor selected from the group consisting of N-(n-butyl)thiophosphoric triamide (NBPT), N-(n-butyl)phosphoric triamide, thiophosphoryl triamide, phenyl phosphorodiamidate, N-cyclohexyl phosphoric triamide, N-cyclohexyl triamide thiophosphoric, phosphoric triamide, hydroquinone, p-benzoquinone, hexamidocyclotriphosphazene, thiopyridines, thiopyrimidines, thiopyridine-N-oxides, N,N-dihalo-2-imidazolidinone, N-halo-2-oxazolidinone, N-(2-nitrophenyl triamide )thiophosphoric, N-(2-nitrophenyl)phosphoric triamide and any combination thereof.
    [026] In one embodiment, the present disclosure provides an agricultural microbial inoculum composition comprising: i. at least one agriculturally beneficial bacillus species selected from the group consisting of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis, Bacillus pumilis and any combination thereof; ii. at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO) and any combination thereof; and iii. a nitrification inhibitor selected from the group consisting of 2-chloro-6-trichloromethylpyridine, 5-ethoxy-3-trichloromethyl-1,2,4-thiadiazole, dicyandiamide, 2-amino-4-chloro-6-methyl-pyrimidine, 1 ,3-benzothiazol-2-thiol, 4-amino-N-1,3-thiazol-2-ylbenzene sulfonamide, thiourea, guanidine, 3,4-dimethylpyrazole phosphate, 2,4-diamino-6-trichloromethyl-5- triazine, polyether ionophores, 4-amino-1,2,4-triazole, 3-mercapto-1,2,4-triazole, potassium azide, carbon disulfide, sodium trithiocarbonate, ammonium dithiocarbamate, 2,3-methylcarbamate ,-dihydro-2,2-dimethyl-7-benzofuranol, N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester, ammonium thiosulfate, 1-hydroxypyrazole, 3-methylpyrazole-1 -carboxamide, 3-methylpyrazole, 3,5-dimethylpyrazole, 1,2,4-triazole, nitrification inhibitor G77 (CAS Registry No. 1373256-33-7) and any combination thereof.
    [027] In one embodiment, the present disclosure provides an agricultural microbial inoculum kit comprising: Part A, wherein Part A comprises a urease inhibitor and at least one organic solvent, said urease inhibitor being selected from group consisting of N-(n-butyl)thiophosphoric triamide (NBPT), N-(n-butyl)phosphoric triamide, thiophosphoryl triamide, phenyl phosphorodiamidate, N-cyclohexyl phosphoric triamide, N-cyclohexyl thiophosphoric triamide, phosphoric triamide, hydroquinone, p-benzoquinone, hexamidocyclotriphosphazene, thiopyridines, thiopyrimidines, thiopyridine-N-oxides, N,N-dihalo-2-imidazolidinone, N-halo-2-oxazolidinone, N-(2-nitrophenyl)thiophosphoric triamide, N-triamide -(2-nitrophenyl)phosphoric and any combination thereof; and wherein said organic solvent is selected from the group consisting of propylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO) and any combination thereof; and Part B, with Part B comprising at least one species of agriculturally beneficial bacillus selected from the group consisting of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis, Bacillus pumilis and any combination thereof, each Part A and each Part B is contained in a separate container.
    [028] In one embodiment, the present disclosure provides an agricultural microbial inoculum kit comprising: Part A, with Part A comprising at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methyl-2 -pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO) and any combination thereof; and Part B, with Part B comprising at least one species of agriculturally beneficial bacillus selected from the group consisting of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis, Bacillus pumilis and any combination thereof, each Part A and each Part B is contained in a separate container.
    [029] In one embodiment, at least one agriculturally beneficial species of bacillus, in an agricultural microbial inoculum composition or kit, is selected from the group consisting of Bacillus licheniformis, Bacillus thuringiensis and any combination thereof.
    [030] In one embodiment, the agriculturally beneficial bacillus species in an agricultural microbial inoculum composition or kit comprises a bacillus strain selected from the group consisting of Bacillus amyloliquefaciens (ATCC 23842), Bacillus licheniformis (ATCC 14580), Bacillus licheniformis (B-642), Bacillus licheniformis (B-14368), Bacillus thuringiensis (ATCC 10792), Bacillus thuringiensis (HD-17), Bacillus thuringiensis (HD-1), Bacillus pumilis (NRS-272) and any combination thereof.
    [031] In one embodiment, the urease inhibitor in an agricultural microbial inoculum composition or kit, when present, is N-(n-butyl)thiophosphoric triamide (NBPT).
    [032] In one embodiment, the nitrification inhibitor in an agricultural microbial inoculum composition or kit, when present, is dicyanodiamide, nitrification inhibitor G77 (CAS Registry No. 1373256-33-7), or a combination thereof.
    [033] In one embodiment, the solvent in an agricultural microbial inoculum composition or kit comprises propylene glycol and N-methyl-2-pyrrolidone.
    [034] In one embodiment, the solvent in an agricultural microbial inoculum composition or kit comprises propylene glycol, N-methyl-2-pyrrolidone and triethylene glycol monobutyl ether.
    [035] In one embodiment, an agricultural microbial inoculum composition additionally comprises water, glycerol or a combination thereof.
    [036] In one embodiment, Part B of an agricultural microbial inoculum kit additionally comprises water, glycerol or a combination thereof.
    [037] In one embodiment, the present disclosure provides an agricultural microbial inoculum composition comprising: i. at least one agriculturally beneficial species of bacillus selected from the group consisting of Bacillus licheniformis, Bacillus thuringiensis and any combination thereof; and ii. at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO) and any combination thereof.
    [038] In one embodiment, the present disclosure provides an agricultural microbial inoculum composition comprising: i. at least one agriculturally beneficial bacillus species selected from the group consisting of Bacillus licheniformis, Bacillus thuringiensis and any combination thereof; ii. at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO) and any combination thereof; and iii. N-(n-butyl)thiophosphoric triamide (NBPT).
    [039] In one embodiment, the present disclosure provides an agricultural microbial inoculum kit comprising: Part A, with Part A comprising N-(n-butyl)thiophosphoric triamide (NBPT) at least one organic solvent selected from the group that consists of propylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO) and any combination thereof; and Part B, with Part B comprising at least one species of agriculturally beneficial bacillus selected from the group consisting of Bacillus licheniformis, Bacillus thuringiensis and any combination thereof, each Part A and each Part B being contained in a separate container .
    [040] In one embodiment, the present disclosure provides an agricultural microbial inoculum composition comprising: i. at least one agriculturally beneficial strain of bacillus selected from the group consisting of Bacillus amyloliquefaciens (ATCC 23842), Bacillus licheniformis (ATCC 14580), Bacillus licheniformis (B-642), Bacillus licheniformis (B-14368), Bacillus thuringiensis (ATCC 10792 ), Bacillus thuringiensis (HD-17), Bacillus thuringiensis (HD-1), Bacillus pumilis (NRS-272) and any combination thereof; and ii. at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO) and any combination thereof.
    [041] In one embodiment, the present disclosure provides an agricultural microbial inoculum composition comprising: i. at least one agriculturally beneficial strain of bacillus selected from the group consisting of Bacillus amyloliquefaciens (ATCC 23842), Bacillus licheniformis (ATCC 14580), Bacillus licheniformis (B-642), Bacillus licheniformis (B-14368), Bacillus thuringiensis (ATCC 10792 ), Bacillus thuringiensis (HD-17), Bacillus thuringiensis (HD-1), Bacillus pumilis (NRS-272) and any combination thereof; ii. at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO) and any combination thereof; and iii. N-(n-butyl)thiophosphoric triamide (NBPT).
    [042] In one embodiment, the present disclosure provides an agricultural microbial inoculum composition comprising: i. at least one agriculturally beneficial strain of bacillus selected from the group consisting of Bacillus amyloliquefaciens (ATCC 23842), Bacillus licheniformis (ATCC 14580), Bacillus licheniformis (B-642), Bacillus licheniformis (B-14368), Bacillus thuringiensis (ATCC 10792 ), Bacillus thuringiensis (HD-17), Bacillus thuringiensis (HD-1), Bacillus pumilis (NRS-272) and any combination thereof; ii. at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO) and any combination thereof; and iii. dicyandiamide, nitrification inhibitor G77 (CAS Registry No. 1373256-33-7), or a combination thereof.
    [043] In one embodiment, the present disclosure provides an agricultural microbial inoculum kit comprising: Part A, wherein Part A comprises N-(n-butyl)thiophosphoric triamide (NBPT) at least one organic solvent selected from the group that consists of propylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO) and any combination thereof; and Part B, with Part B comprising at least one strain of agriculturally beneficial bacillus selected from the group consisting of Bacillus amyloliquefaciens (ATCC 23842), Bacillus licheniformis (ATCC 14580), Bacillus licheniformis (B-642), Bacillus licheniformis ( B-14368), Bacillus thuringiensis (ATCC 10792), Bacillus thuringiensis (HD-17), Bacillus thuringiensis (HD-1), Bacillus pumilis (NRS-272) and any combination thereof; each Part A and each Part B is contained in a separate container.
    [044] In one embodiment, the present disclosure provides an agricultural microbial inoculum kit comprising: Part A, with Part A comprising at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methyl-2 -pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO) and any combination thereof; and Part B, with Part B comprising at least one strain of agriculturally beneficial bacillus selected from the group consisting of Bacillus amyloliquefaciens (ATCC 23842), Bacillus licheniformis (ATCC 14580), Bacillus licheniformis (B-642), Bacillus licheniformis ( B-14368), Bacillus thuringiensis (ATCC 10792), Bacillus thuringiensis (HD-17), Bacillus thuringiensis (HD-1), Bacillus pumilis (NRS-272) and any combination thereof; each Part A and each Part B is contained in a separate container.
    [045] The weight percentage of a urease inhibitor such as NBPT in any embodiment of an agricultural microbial inoculum composition or kit of the present disclosure, when present, is in the range of 0.02% to 80%. In one embodiment, the weight percentage is in the range of 0.02% to 70%. In one embodiment, the weight percentage is in the range of 0.02% to 60%. In one embodiment, the weight percentage is in the range of 0.02% to 50%. In one embodiment, the weight percentage is in the range of 0.02% to 40%. In one embodiment, the weight percentage is in the range of 0.02% to 30%. In one embodiment, the weight percentage is in the range of 0.02% to 20%. In one embodiment, the weight percentage is in the range of 0.02% to 10%. In one embodiment, the weight percentage is in the range of 0.02% to 5%. In one embodiment, the weight percentage is in the range of 5% to 60%. In one embodiment, the weight percentage is in the range of 5% to 50%. In one embodiment, the weight percentage is in the range of 5% to 40%. In one embodiment, the weight percentage is in the range of 5% to 30%. In one embodiment, the weight percentage is in the range of 10% to 60%. In one embodiment, the weight percentage is in the range of 10% to 50%. In one embodiment, the weight percentage is in the range of 10% to 40%. In one embodiment, the weight percentage is in the range of 10% to 30%. In one embodiment, the weight percentage is in the range of 15% to 60%. In one embodiment, the weight percentage is in the range of 15% to 50%. In one embodiment, the weight percentage is in the range of 15% to 40%. In one embodiment, the weight percentage is in the range of 15% to 30%. In one embodiment, the weight percentage is in the range of 30% to 60%. In one embodiment, the weight percentage is in the range of 30% to 50%. In one embodiment, the weight percentage is in the range of 40% to 60%. The weight percentage is based on the entire microbial inoculum composition.
    [046] The weight percentage of a nitrification inhibitor such as DCD, when present, in any embodiment of an agricultural microbial inoculum composition or kit of the present disclosure, is in the range of 1% to 80%. In one embodiment, the weight percentage is in the range of 1% to 70%. In one embodiment, the weight percentage is in the range of 1% to 60%. In one embodiment, the weight percentage is in the range of 1% to 50%. In one embodiment, the weight percentage is in the range of 1% to 40%. In one embodiment, the weight percentage is in the range of 1% to 30%. In one embodiment, the weight percentage is in the range of 1% to 20%. In one embodiment, the weight percentage is in the range of 1% to 10%. In one embodiment, the weight percentage is in the range of 1% to 5%. In one embodiment, the weight percentage is in the range of 10% to 80%. In one embodiment, the weight percentage is in the range of 10% to 70%. In one embodiment, the weight percentage is in the range of 10% to 60%. In one embodiment, the weight percentage is in the range of 10% to 50%. In one embodiment, the weight percentage is in the range of 10% to 40%. In one embodiment, the weight percentage is in the range of 10% to 30%. In one embodiment, the weight percentage is in the range of 20% to 80%. In one embodiment, the weight percentage is in the range of 20% to 70%. In one embodiment, the weight percentage is in the range of 20% to 60%. In one embodiment, the weight percentage is in the range of 20% to 50%. In one embodiment, the weight percentage is in the range of 20% to 40%. In one embodiment, the weight percentage is in the range of 20% to 30%. The weight percentage is based on the entire microbial inoculum composition.
    [047] The weight percentage of an organic solvent such as propylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO) or any combination thereof in any embodiment of an agricultural microbial inoculum composition or kit of the present disclosure is in the range of 20% to 99%. In one embodiment, the weight percentage is in the range of 20% to 90%. In one embodiment, the weight percentage is in the range of 20% to 80%. In one embodiment, the weight percentage is in the range of 20% to 70%. In one embodiment, the weight percentage is in the range of 20% to 60%. In one embodiment, the weight percentage is in the range of 20% to 50%. In one embodiment, the weight percentage is in the range of 30% to 99%. In one embodiment, the weight percentage is in the range of 30% to 90%. In one embodiment, the weight percentage is in the range of 30% to 80%. In one embodiment, the weight percentage is in the range of 30% to 70%. In one embodiment, the weight percentage is in the range of 30% to 60%. In one embodiment, the weight percentage is in the range of 30% to 50%. In one embodiment, the weight percentage is in the range of 40% to 99%. In one embodiment, the weight percentage is in the range of 40% to 80%. In one embodiment, the weight percentage is in the range of 40% to 70%. In one embodiment, the weight percentage is in the range of 40% to 60%. The weight percentage is based on the entire microbial inoculum composition.
    [048] In one embodiment, an agricultural microbial inoculum composition or kit of the present disclosure comprises propylene glycol with a weight percentage in the range of 40% to 70%, N-methyl-2-pyrrolidone with a weight percentage in the range of 15% to 40%, NBPT with a weight percentage in the range of 10% to 30% and, optionally, a colorant with a weight percentage in the range of 0.1% to 5%. The weight percentage is based on the entire microbial inoculum composition.
    [049] In one embodiment, an agricultural microbial inoculum composition or kit of the present disclosure comprises propylene glycol with a weight percentage in the range of 10% to 30%, N-methyl-2-pyrrolidone with a weight percentage in the range of 30% to 60%, NBPT with weight percentage in the range of 15% to 40%, triethylene glycol monobutyl ether with weight percentage in the range of 1% to 5%, and optionally a colorant with weight percentage in the range of 0% to 1%. The weight percentage is based on the entire microbial inoculum composition.
    [050] In one embodiment, the concentration of the bacillus species in the microbial inoculum composition of the present disclosure is at least 1.0 x 10 2 spores/ml. In one embodiment, the concentration is at least 1.0 x 10 3 spores/ml. In one embodiment, the concentration is at least 1.0 x 10 4 spores/ml. In one embodiment, the concentration is at least 1.0 x 10 5 spores/ml. In one embodiment, the concentration is at least 1.0 x 10 6 spores/ml. In one embodiment, the concentration is at least 1.0 x 10 7 spores/ml. In one embodiment, the concentration is at least 1.0 x 10 8 spores/ml. In one embodiment, the concentration is at least 1.0 x 109 spores/ml. In one embodiment, the concentration is at least 1.0 x 10 10 spores/ml. In one embodiment, the concentration is at least 1.0 x 10 11 spores/ml. In one embodiment, the concentration is at least 1.0 x 10 12 spores/ml. In one embodiment, the concentration is in the range of 1.0 x 10 2 to 1.0 x 10 12 spores/ml. In one embodiment, the concentration is in the range of 1.0 x 10 3 to 1.0 x 10 12 spores/ml. In one embodiment, the concentration is in the range of 1.0 x 10 4 to 1.0 x 10 12 spores/ml. In one embodiment, the concentration is in the range of 1.0 x 10 5 to 1.0 x 10 12 spores/ml. In one embodiment, the concentration is in the range of 1.0 x 10 6 to 1.0 x 10 12 spores/ml. In one embodiment, the concentration is in the range of 1.0 x 10 7 to 1.0 x 10 12 spores/ml. In one embodiment, the concentration is in the range of 1.0 x 10 8 to 1.0 x 10 12 spores/ml. In one embodiment, the concentration is in the range of 1.0 x 109 to 1.0 x 1012 spores/ml.
    [051] In one embodiment of the present disclosure, the agricultural microbial inoculum composition may serve as a fertilizer in its own right.
    [052] A dye may also be included in the agricultural microbial inoculum composition of the present disclosure. Any commonly used dye, including food dyes, can be used to provide visual evidence of the uniformity of the microbial inoculum composition. The weight percentage of a dye in the total microbial inoculum composition is 0 to 10%. In one embodiment, the weight percentage is from 0.1% to 5%.
    [053] Examples of suitable dyes in the present disclosure include, but are not limited to, FD&C Blue #1, FD&C Green #3, FD&C Yellow #5, FD&C Red #3, FD&C Red #40, FD&C Yellow No. 6 and AGROTAIN® ULTRA green dye or a combination thereof.
    [054] In another embodiment, the present disclosure provides a fertilizer composition comprising any agricultural microbial inoculum composition in any embodiment of the present disclosure, wherein the fertilizer may be a granulated fertilizer, such as granulated urea, a liquid fertilizer, such as urea- ammonium nitrate (UAN), an aqueous solution of urea and ammonium nitrate, or anhydrous ammonia (NH3).
    [055] In another embodiment, the present disclosure provides a method for enhancing a yield trait in a plant of interest, as compared to the yield trait of a reference or control plant, the method comprising placing a plant , a plant part or a plant seed of interest, or the surrounding soil, in contact with an effective amount of an agricultural microbial inoculum composition of the present disclosure, wherein the microbial inoculum composition comprises: . i. at least one agricultural microbial strain from one or more microbial species, and ii. at least one active agent, wherein the active agent is a urease inhibitor, a nitrification inhibitor, or a combination thereof, and the agricultural microbial inoculum composition in the effective amount is effective in enhancing the yield trait in the plant. interest, in relation to the yield trait in the reference or control plant, when the plant of interest is brought into contact with the effective amount.
    [056] In another embodiment, the present disclosure provides a method for enhancing a productivity trait in the plant, such as increasing plant growth and/or productivity, the method comprising applying to the plant, plant part, seeds or to the soil in which the plant or plant seed is grown, an effective amount of an agricultural microbial inoculum composition of any embodiment of the present disclosure.
    [057] In another embodiment, the present disclosure provides a method of improving soil quality, the method comprising applying to the soil, or plants or plant seeds in said soil, an effective amount of a microbial inoculum composition. agriculture as disclosed in any embodiment of the present disclosure.
    [058] In any embodiment of the disclosure, the concentrations of each agricultural microbial strain to be added to the microbial inoculum and fertilizer compositions as disclosed herein will depend on a variety of factors, including the identity and number of individual strains employed, the species of plant being treated, the nature and conditions of the soil to be treated, the exact nature of the microbial inoculum or fertilizer composition to be applied, the type and form of the active agent, the form in which the inoculum or fertilizer is applied, and the means by which it is applied, and the stage of the plant's growing season during which the application takes place. In any of these cases, the appropriate concentrations must be effective in enhancing the productivity trait in the presence of the active agent and can be determined by one skilled in the art using only routine experiments. By way of example only, the concentration of each strain present in the inoculum or fertilizer composition can be from about 1.0 x 102 colony forming units (CFU)/ml to about 5.0 x 1012 CFU/ml per acre. , from about 1.0 x 102 CFU/ml to about 5.0 x 1010 CFU/ml per acre, from about 1.0 x 102 CFU/ml to about 5.0 x 108 CFU/ml per acre , from about 1.0 x 102 CFU/ml to about 5.0 x 106 CFU/ml per acre, or from about 1.0 x 102 CFU/ml to about 5.0 x 104 CFU/ml per acre.
    [059] In one embodiment of the present disclosure, a microbial food source, such as algae ash or glycerol, may be included in any embodiment of the present disclosure.
    [060] The term "microbial species" refers to naturally occurring or specifically evolved variants or mutants of microbial species such as bacteria and fungi, as disclosed herein. Variants or mutants may or may not have the same identifying biological characteristics as the specific strains exemplified here, as long as they share similar advantageous properties in terms of promoting plant growth and providing nutrients for plant growth in the soil. Variants of certain microbial strains may include, but are not limited to, those developed through gene integration techniques, such as those mediated by insertional or transposon elements, or by homologous recombination, other recombinant DNA techniques to modify, insert, delete , activating or silencing genes, intraspecific protoplast fusion, mutagenesis by irradiation with ultraviolet light or X-rays, or by treatment with a chemical mutagen such as nitrosoguanidine, methylmethane sulfonate, nitrogen mustard and the like, as well as bacteriophage-mediated transduction. Suitable and applicable methods are well known in the art and are described, for example, in J.H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (1972); J.H. Miller, A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (1992); and J. Sambrook, D. Russell, Molecular Cloning: A Laboratory Manual, 3rd. Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (200l).
    [061] The term "plant productivity" or "productivity trait", as used here, refers to any aspect of a plant's growth or development that is a reason the plant is cultivated. Thus, for the purposes of the present description, improved or increased "plant productivity" or "accentuated productivity trait" broadly refers to improvements in the biomass or productivity of leaves, stems, grains, fruits, vegetables, flowers or other plant parts that are harvested or used for various purposes, and enhancements to the growth of plant parts, including stems, leaves, and roots.
    [062] The term "improving soil quality" refers to increasing the quantity and/or availability of nutrients required by, or beneficial to, plants for growth. Just as an example, these nutrients include nitrogen, phosphorus, potassium, copper, zinc, boron and molybdenum. Also encompassed by the term "improving soil quality" is the reduction or minimization of the amount of an element that can be harmful to plant growth or development, such as iron and manganese. Thus, improving soil quality through the use of microbial inoculum and fertilizer compositions of the present disclosure thus aid and promote the growth of plants in the soil.
    [063] The term "effective amount" refers to an amount of microbial inoculum composition or fertilizer applied to a given area of soil or vegetation that is sufficient to promote one or more beneficial or desired outcomes, for example in terms of rates of plant growth, crop yield, or soil nutrient availability. An "effective amount" may be provided in one or more administrations. The exact amount needed will vary depending on factors such as the identity and number of individual strains employed, the species of plant being treated, the nature and conditions of the soil being treated, the exact nature of the microbial inoculum or fertilizer composition being treated. applied, the form in which the inoculum or fertilizer is applied and the means by which it is applied, and the stage of the plant's growing season during which the application occurs. For any of these cases, a suitable "effective amount" can be determined by one skilled in the art, using only routine experiments.
    [064] The term "viability" in the present disclosure refers to the ability of a microbial cell to repeatedly divide on an agar surface to produce a visible colony. The temperature for the viability assessment is about 37°C in the present disclosure. The viability time is counted from the moment a freshly prepared microbial culture in a nutrient broth is added to an organic solvent, or to a mixture of more than one organic solvent. After the microbial culture in a nutrient broth is added to the organic solvent, or to a mixture of more than one solvent, a small amount of the mixture is removed at a time for incubation at about 37 °C. The period of time between the time the microbial culture in a nutrient broth is added to an organic solvent and the time the mixture is removed for incubation is defined as the viability time for the purpose of viability assessment. Bacteria viability test in solutions with organic solvents
    [065] The purpose of bacterial viability testing is to assess the viability of agriculturally beneficial bacterial species in solutions with organic solvents.
    [066] Agriculturally beneficial bacterial species, both gram-negative and gram-positive, are used for the test.
    [067] Agriculturally beneficial bacterial species were obtained from the American Type Culture Collection (ATCC), or the Agricultural Research Service Culture Collection. Bacteria species were cultured in suitable medium, LB broth, nutrient broth, and microscopically checked for purity with Gram stain and in Petri dishes. The bacteria species are pure, that is, no unusual colonies were observed.
    [068] All test samples are prepared using a two-step method.
    [069] First, selected agriculturally beneficial bacteria were cultured overnight in Luria-Bertani (LB) medium at 37 °C with agitation. Proliferation was measured with a Bausch and Lomb Spectronic spectrophotometer at 600 nm (OD600) to provide samples with cell optical density at 600 nm (OD600) between 1.2 and 1.5. Overnight cultures typically contain from 1.6 x 108 to 3.4 x 108 colony forming units per ml (CFU/ml).
    [070] Second, 0.5 ml of the prepared sample of the bacteria species in LB medium was added to an organic solvent or a mixture of organic solvents (4.5 ml).
    [071] A 10 μl sample of the organic solvent solution with the species of bacteria is removed immediately to assess the viability of the bacteria (T = 0).
    [072] The remaining part of the organic solvent solution with the bacteria species is incubated at 37 °C for future testing. A volume of 10 μl of sample was removed from the organic solvent solution incubated with the bacteria species at T=2 hours, 4 hours, 1 day, 2 days, 5 days, 7 days, 9 days, 12 days, 15 days, 18 days and 21 days to evaluate the viability of the bacteria.
    [073] Each time, the sample (10 μl) that was removed for viability assessment was placed on agar plates, which were incubated overnight at 37 °C. The colonies of bacteria present on the plates indicate that the bacteria have tolerated the solvent and are therefore viable. If no bacterial colonies proliferate, the bacteria have not demonstrated solvent tolerance.
    [074] Agricultural beneficial bacteria that are viable for at least two hours from the time the bacteria sample is added to an organic solvent are considered to be viable in the organic solvent solution of the present invention.
    [075] Bacillus amyloliquefaciens (ATCC 23842) provided at least two hours of viability in NMP and at least 5 days of viability in glycerol.
    [076] Bacillus licheniformis (ATCC 14580) provided at least one day of viability in triethylene glycol monobutyl ether, at least 5 days of viability in PG, at least 5 days of viability in NMP, at least 21 days of viability in glycerol and at least 21 days of viability in DMSO.
    [077] Each of Bacillus licheniformis (B-642) and Bacillus licheniformis (B-14368) provided at least 21 days of viability in PG, at least 21 days of viability in NMP, at least 21 days of viability in ether monobutyl triethylene glycol, at least 21 days of viability in glycerol and at least 21 days of viability in DMSO.
    [078] Bacillus thuringiensis (ATCC 10792) provided at least 2 hours of viability in PG, at least 2 hours of viability in NMP, at least 2 hours of viability in triethylene glycol monobutyl ether, at least 21 days of viability in glycerol and at least 21 days of viability in DMSO.
    [079] Each of Bacillus thuringiensis (HD-17) and Bacillus thuringiensis (HD-1) provided at least 21 days of viability in PG, at least 21 days of viability in NMP, at least 21 days of viability in ether monobutyl triethylene glycol, at least 21 days of viability in glycerol and at least 21 days of viability in DMSO.
    [080] Bacillus pumilis (NRS-272) provided at least 21 days of viability in PG, at least 21 days of viability in triethylene glycol monobutyl ether, at least 21 days of viability in glycerol and at least 21 days of viability in DMSO
    [081] Surprisingly, all three selected agriculturally beneficial Gram-negative species of Pseudomonas fluorescens (ATCC 53958), Pseudomonas putida (ATCC 49128), and Pseudomonas chlororaphis (ATCC 55670) selected, which were expected to have better solvent tolerance, lost nearly viability was instantaneously when bacteria samples prepared with OD600 between 1.3 and 1.5 in nutrient broth were added to all organic solvents tested except glycerol.
    [082] The viability test of bacteria in solutions with organic solvents in the present disclosure demonstrated that all examples of Bacillus licheniformis and Bacillus licheniformis showed viability with a time interval of at least 2 hours to at least 21 days in a solution with the at least one of the organic solvents PG, NMP, triethylene glycol monobutyl ether, glycerol and DMSO.
    [083] The viability test of bacteria in solutions with organic solvents in the present disclosure showed that each of the bacilli of the strains Bacillus amyloliquefaciens (ATCC 23842), Bacillus licheniformis (ATCC 14580), Bacillus licheniformis (B-642), Bacillus licheniformis (B-14368), Bacillus thuringiensis (ATCC 10792), Bacillus thuringiensis (HD-17), Bacillus thuringiensis (HD-1) and Bacillus pumilis (NRS-272) showed viability with a time interval of at least 2 hours to at least least 21 days in a solution with at least one of the organic solvents PG, NMP, triethylene glycol monobutyl ether, glycerol and DMSO. Examples Table 1: Examples with viability of at least 2 hours in at least one of the organic solvents PG, NMP, triethylene glycol monobutyl ether, glycerol or DMSO

    权利要求:
    Claims (28)
    [0001]
    1. Vehicle-based agricultural microbial inoculum composition, CHARACTERIZED by the fact that it comprises: i. at least one agriculturally beneficial Bacillus species selected from the group consisting of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis, Bacillus pumilis, and any combination thereof; ii. at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO), and any combination thereof, wherein the at least one organic solvent is present in an amount of 20 to 99 weight percent, based on the total weight of the microbial inoculum composition; and iii. a urease inhibitor selected from the group consisting of N-(n-butyl)thiophosphoric triamide (NBPT), N-(n-butyl)phosphoric triamide, thiophosphoryl triamide, phenyl phosphorodiamidate, N-cyclohexyl phosphoric triamide, N-cyclohexyl thiophosphoric triamide , phosphoric triamide, hydroquinone, p-benzoquinone, hexamidocyclotriphosphazene, thiopyridines, thiopyrimidines, thiopyridine-N-oxides, N,N-dihalo-2-imidazolidinone, N-halo-2-oxazolidinone, N-(2-nitrophenyl) triamide thiophosphoric, N-(2-nitrophenyl)phosphoric triamide, and any combination thereof.
    [0002]
    2. Agricultural microbial inoculum composition according to claim 1, CHARACTERIZED in that the agriculturally beneficial Bacillus species is selected from the group consisting of Bacillus licheniformis, Bacillus thuringiensis, and any combination thereof;
    [0003]
    3. Agricultural microbial inoculum composition, according to claim 1, CHARACTERIZED by the fact that the agriculturally beneficial Bacillus species is selected from the group of strains consisting of Bacillus amyloliquefaciens (ATCC 23842), Bacillus licheniformis (ATCC 14580), Bacillus licheniformis (B-642), Bacillus licheniformis (B-14368), Bacillus thuringiensis (ATCC 10792), Bacillus thuringiensis (HD-17), Bacillus thuringiensis (HD-1), Bacillus pumilis (NRS-272), and any combination of the same.
    [0004]
    4. Agricultural microbial inoculum composition, according to claim 1, CHARACTERIZED by the fact that the urease inhibitor is N-(n-butyl)thiophosphoric triamide (NBPT).
    [0005]
    5. Agricultural microbial inoculum composition, according to claim 1, CHARACTERIZED in that it additionally comprises a nitrification inhibitor selected from the group consisting of 2-chloro-6-trichloromethylpyridine, 5-ethoxy-3-trichloromethyl-1,2 ,4-thiadiazole, dicyandiamide, 2-amino-4-chloro-6-methyl-pyrimidine, 1,3-benzothiazol-2-thiol, 4-amino-N-1,3-thiazol-2-ylbenzene sulfonamide, thiourea, guanidine, 3,4-dimethylpyrazole phosphate, 2,4-diamino-6-trichloromethyl-5-triazine, polyether ionophores, 4-amino-1,2,4-triazole, 3-mercapto-1,2,4-triazole , potassium azide, carbon disulfide, sodium trithiocarbonate, ammonium dithiocarbamate, 2,3,-dihydro-2,2-dimethyl-7-benzofuranol methylcarbamate, N-(2,6-dimethylphenyl) methyl ester -N-(methoxyacetyl)-alanine, ammonium thiosulfate, 1-hydroxypyrazole, 3-methylpyrazole-1-carboxamide, 3-methylpyrazole, 3,5-dimethylpyrazole, 1,2,4-triazole, nitrification inhibitor G77 (CAS registration no. 1373256-33-7), and any combination thereof.
    [0006]
    6. Agricultural microbial inoculum composition, according to claim 5, CHARACTERIZED by the fact that the nitrification inhibitor is dicyandiamide, nitrification inhibitor G77 (CAS registration No. 1373256-33-7) or a combination thereof.
    [0007]
    7. Agricultural microbial inoculum composition, according to claim 1, CHARACTERIZED by the fact that the percentage by weight of the urease inhibitor is in the range of 0.02% to 80%, based on the total weight of the microbial inoculum composition agricultural.
    [0008]
    8. Agricultural microbial inoculum composition, according to claim 7, CHARACTERIZED by the fact that the percentage by weight of the urease inhibitor is in the range of 10% to 60%.
    [0009]
    9. Agricultural microbial inoculum composition, according to claim 1, CHARACTERIZED by the fact that the agriculturally beneficial Bacillus species has at least 2 hours of viability from the moment said agriculturally beneficial Bacillus species comes into contact with said organic solvent.
    [0010]
    10. Agricultural microbial inoculum composition, according to claim 1, CHARACTERIZED by the fact that the concentration of the agriculturally beneficial Bacillus species is in the range of 1.0 x 102 to 1.0 x 1012 spores/ml.
    [0011]
    11. Agricultural microbial inoculum composition, according to claim 1, CHARACTERIZED in that it additionally comprises a dye.
    [0012]
    12. Agricultural microbial inoculum composition, according to claim 1, CHARACTERIZED in that it comprises propylene glycol and N-methyl-2-pyrrolidone.
    [0013]
    13. Agricultural microbial inoculum composition, according to claim 12, CHARACTERIZED by the fact that the percentage by weight of propylene glycol is in the range of 40% to 70%, based on the total weight of the agricultural microbial inoculum composition, the percentage by weight of N-methyl-2-pyrrolidone is in the range of 15% to 40%, based on the total weight of the agricultural microbial inoculum composition, the urease inhibitor is NBPT, and the weight percentage of NBPT is in the range of 10% to 30%, based on the total weight of the microbial inoculum composition.
    [0014]
    14. Agricultural microbial inoculum composition, according to claim 1, CHARACTERIZED in that it comprises propylene glycol, N-methyl-2-pyrrolidone, and triethylene glycol monobutyl ether.
    [0015]
    15. Agricultural microbial inoculum composition, according to claim 14, CHARACTERIZED by the fact that the percentage by weight of propylene glycol is in the range of 10% to 30%, based on the total weight of the agricultural microbial inoculum composition, the percentage by weight of N-methyl-2-pyrrolidone is in the range of 30% to 60%, based on the total weight of the agricultural microbial inoculum composition, the weight percentage of triethylene glycol monobutyl ether is in the range of 1% to 5 %, based on the total weight of the agricultural microbial inoculum composition, the urease inhibitor is NBPT, and the weight percentage of NBPT is in the range of 15% to 40%, based on the total weight of the agricultural microbial inoculum composition.
    [0016]
    16. Fertilizer composition, CHARACTERIZED by the fact that it comprises the composition of agricultural microbial inoculum as defined in claim 1.
    [0017]
    17. Method for promoting plant growth, plant productivity and/or soil quality, CHARACTERIZED in that it comprises the application of an effective amount, to a plant, to a plant part, to a plant seed or soil, of the agricultural microbial inoculum composition as defined in claim 1
    [0018]
    18. Agricultural microbial inoculum kit, CHARACTERIZED in that it comprises: Part A, wherein Part A comprises a urease inhibitor and at least one organic solvent, wherein said urease inhibitor is selected from the group consisting of triamide N -(n-butyl)thiophosphoric triamide (NBPT), N-(n-butyl)phosphoric triamide, thiophosphoryl triamide, phenyl phosphorodiamidate, N-cyclohexyl phosphoric triamide, N-cyclohexyl thiophosphoric triamide, phosphoric triamide, hydroquinone, p-benzoquinone, hexamidocyclotriphosphazene , thiopyridines, thiopyrimidines, thiopyridine-N-oxides, N,N-dihalo-2-imidazolidinone, N-halo-2-oxazolidinone, N-(2-nitrophenyl)thiophosphoric triamide, N-(2-nitrophenyl)phosphoric triamide , and any combination thereof; and wherein said organic solvent is selected from the group consisting of propylene glycol (PG), N-methyl-2-pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO), and any combination thereof ; and Part B, wherein Part B comprises at least one agriculturally beneficial Bacillus species selected from the group consisting of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis, Bacillus pumilis, and any combination thereof, wherein each of Part A and Part B is contained in a separate container, and wherein the at least one organic solvent is present in an amount from 20 to 99 percent by weight, based on the combined weight of Part A and Part B.
    [0019]
    19. Agricultural microbial inoculum kit according to claim 18, CHARACTERIZED in that Part B comprises at least one Bacillus species selected from the group consisting of Bacillus licheniformis, Bacillus thuringiensis, and any combination thereof.
    [0020]
    20. Agricultural microbial inoculum kit, according to claim 18, CHARACTERIZED by the fact that Part B comprises at least one strain of agriculturally beneficial Bacillus, selected from the group consisting of Bacillus amyloliquefaciens (ATCC 23842), Bacillus licheniformis (ATCC 14580), Bacillus licheniformis (B-642), Bacillus licheniformis (B-14368), Bacillus thuringiensis (ATCC 10792), Bacillus thuringiensis (HD-17), Bacillus thuringiensis (HD-1), Bacillus pumilis (NRS-272), and any combination thereof.
    [0021]
    21. Agricultural microbial inoculum kit, according to claim 18, CHARACTERIZED by the fact that the urease inhibitor is N-(n-butyl)thiophosphoric triamide (NBPT).
    [0022]
    22. Agricultural microbial inoculum kit, according to claim 21, CHARACTERIZED by the fact that Part A comprises propylene glycol and N-methyl-2-pyrrolidone.
    [0023]
    23. Agricultural microbial inoculum kit, according to claim 22, CHARACTERIZED by the fact that the percentage by weight of propylene glycol is in the range of 40% to 70%, based on the total weight of Part A, the percentage by weight of N-methyl-2-pyrrolidone is in the range of 15% to 40%, based on the total weight of Part A, and the weight percentage of NBPT is in the range of 10% to 30%, based on the total weight of Part A THE.
    [0024]
    24. Agricultural microbial inoculum kit, according to claim 21, CHARACTERIZED by the fact that Part A comprises propylene glycol, N-methyl-2-pyrrolidone, and triethylene glycol monobutyl ether.
    [0025]
    25. Agricultural microbial inoculum kit, according to claim 24, CHARACTERIZED by the fact that the percentage by weight of propylene glycol is in the range of 10% to 30%, based on the total weight of Part A, the percentage by weight of N-methyl-2-pyrrolidone is in the range of 30% to 60% based on the total weight of Part A, the weight percentage of triethylene glycol monobutyl ether is in the range of 1% to 5% based on the weight of Part A, and the weight percentage of NBPT is in the range of 15% to 40%, based on the total weight of Part A.
    [0026]
    26. Agricultural microbial inoculum kit, according to claim 18, CHARACTERIZED by the fact that the agriculturally beneficial Bacillus species has at least 2 hours of viability from the moment said agriculturally beneficial Bacillus species in Part A enters in contact with said organic solvent in Part B.
    [0027]
    27. Agricultural microbial inoculum kit, according to claim 18, CHARACTERIZED by the fact that the percentage by weight of the urease inhibitor is in the range of 0.02% to 80%, based on the total weight of Part A.
    [0028]
    28. Agricultural microbial inoculum kit, according to claim 18, CHARACTERIZED by the fact that the concentration of the agriculturally beneficial Bacillus species is in the range of 1.0 x 102 to 1.0 x 1012 spores/ml, after Part To be mixed with Part B.
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    同族专利:
    公开号 | 公开日
    AR113623A2|2020-05-27|
    CL2017002928A1|2018-03-09|
    CA2932067C|2019-02-26|
    EP3288385A1|2018-03-07|
    US9790134B2|2017-10-17|
    US20160355445A1|2016-12-08|
    AR104896A1|2017-08-23|
    CN107709275A|2018-02-16|
    AU2016270813A1|2017-11-30|
    BR112017026113A2|2018-08-14|
    US20180002244A1|2018-01-04|
    US20200123076A1|2020-04-23|
    CA2932067A1|2016-12-02|
    US10513467B2|2019-12-24|
    AU2016270813B2|2018-02-15|
    CN107709275B|2019-01-11|
    ZA201707751B|2019-03-27|
    WO2016196681A1|2016-12-08|
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    法律状态:
    2019-09-17| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2021-08-24| B350| Update of information on the portal [chapter 15.35 patent gazette]|
    2021-12-07| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2022-02-01| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 01/06/2016, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US201562169942P| true| 2015-06-02|2015-06-02|
    US62/169,942|2015-06-02|
    PCT/US2016/035344|WO2016196681A1|2015-06-02|2016-06-01|Microbial inoculant compositions and uses thereof in agriculture|
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