NZ750053A - Microbial inoculant compositions and uses thereof in agriculture - Google Patents

Abstract

The present disclosure provides novel agricultural microbial inoculant compositions for uses in promoting plant growth, plant productivity and/or soil quality. The novel microbial inoculant 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 inoculant compositions.

Description

MICROBIAL INOCULANT COMPOSITIONS AND USES THEREOF IN AGRICULTURE CROSS-REFERENCE TO RELATED APPLICATION This application is a onal of New Zealand patent application , which is the al phase entry in New d of PCT international application (published as WO 96681), and claims priority to U.S. Provisional Patent Application No. 62/169,942, filed on June 2, 2015, which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD The present disclosure relates to novel agricultural microbial inoculant compositions for uses in promoting plant growth, plant productivity and/or soil quality. The novel microbial inoculant 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 sing said microbial inoculant compositions, formulations and the uses thereof.

BACKGROUND The use of fertilizers to enhance plant and crop production and overcome poor soil quality is widespread. Most commonly ed commercially available nitrogen ning fertilizers are inorganic chemical fertilizers such as urea. The extended use of urea is often associated with negative environmental consequences, such as e contamination in run off and ground water, and emission of ammonia and s oxide to the atmosphere. Attention to nitrogen fertilizer application 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 nitrogen use efficiency and alleviate environmental pollution.

One of the widely used technologies is the application of a urease inhibitor in combination with the urea treatment. The urea component of fertilizer applied to the soil becomes a source of ammonia as a result of urease catalyzed ysis of urea, an enzyme produced by numerous fungi and bacteria that is well known to d artisans. Urease inhibitors can slow down the conversion rate of urea to ammonia, thereby icantly reducing the quantity of urea that otherwise has to be applied on the soil by reducing the amount of ammonia volatilization. One of the most common urease inhibitors is N-(n-butyl) thiophosphoric triamide (NBPT) (See e.g. U.S. Patent No. 5,698,003). r widely used technology is the application of nitrification inhibitors to significantly reduce nitrate ng and gaseous nitrogen emissions. Most nitrogen supplied as a commercial fertilizer is ultimately transformed to a nitrate form of nitrogen. In the ce of adequate oxygen, warm temperatures, and some re, ammonium-N is converted to nitrate- N through a biochemical process known as nitrification that requires two forms of soil bacteria.

The first bacterium Nitrosomonas converts ammonium-N to nitrite-N. The second bacterium Nitrobacter converts nitrite-N to nitrate-N. Nitrification inhibitors have one primary way of ng the nitrification process by inhibiting the bacteria Nitrosomonas in the area where ammonium is to be present. Some widely used nitrification inhibitors that are commercially available include 2-chloro(trichloromethyl)-pyridine (Nitrapyrin) and dicyandiamide (DCD).

In addition to the application of al enzyme inhibitors such as urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) and ication inhibitors such as dicyandiamide (DCD), fertilizer compositions comprising microorganisms (so-called "bio-fertilizers" or “biostimulants” ) are increasingly considered as alternatives to conventional chemical fertilizers. The ability of ic bacterial species to promote plant growth has long been recognized. For example, nitrogen-fixing bacteria such as Rhizobium species e plants with essential nitrogenous nds. Species of Azotobacter and rillum have also been shown to promote plant growth and increase crop yield, promoting the accumulation of nutrients in plants.

However bacteria of these genera are often unable to compete effectively with native soil and plant flora, thereby requiring the application of impractically large volumes of inocula.

SUMMARY OF THE INVENTION To date, urease tors and nitrification inhibitors have met with varied success, while bio-fertilizers have typically met with limited success. Thus, there remains a need for improved fertilizers or fertilizer additives and methods that are effective in ing nutrients for plant growth and are environmentally safe and non-hazardous. One solution is to provide a combination of urease tors and/or nitrification inhibitors with bio-fertilizers. Nevertheless, the combination of urease inhibitors and/or nitrification inhibitors with bio-fertilizers is not ht forward. First, urease tors and/or nitrification inhibitors can weaken or kill the biofertilizers when combined. Second, urease inhibitors and/or nitrification inhibitors are typically dispensed in a solvent system (e.g. glycol, complex amines, aryl alcohols), which can also weaken or kill the bio-fertilizers. pesticides. However, it did not disclose any solvent system that could provide reasonable viability for the listed microbial ides. In addition, it did not identify any solvent-tolerant Solvent-tolerant bacteria are potentially useful in many applications of microbial transformation for environmental remediation as well as in biotechnological processes. Organic solvent tolerance may be a species-specific property and may not be easily 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 ed and well-studied solvent-tolerant ia are Gram-negative bacteria.

Gram-negative bacteria have the advantage of having an additional outer membrane that protects the cytoplasmic membrane by reducing the asmic concentrations of harmful ts to acceptable levels. Owing to the inherent disadvantage of lacking an outer membrane, only a few Gram-positive organisms have been ed 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.

Surprisingly, in extensive efforts to identify solvent-tolerant bacteria to e plant health, plant nutrition, and/or soil , a few agriculturally beneficial Gram-positive organisms are identified to be viable in some ed organic solvents while some Gram-negative organisms are not viable in the same organic solvents. For e, tested Gram-positive organisms species Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis and Bacillus pumilus have demonstrated viability time range from at least 2 hours to at least 21 days in a solution with at least one of the c solvents propylene glycol (PG), N-methyl pyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, and dimethyl sulfoxide (DMSO). However, none of the tested agriculturally beneficial Gram-negative species Pseudomonas fluorescens, Pseudomonas putida, and Pseudomonas chlororaphis demonstrated viability in the tested organic solvents. It is an object of the present invention to go some way towards overcoming these problems and/or to at least provide the public with a useful choice. [00010a] In a first aspect the present invention provides a ition comprising: i. a us species selected from the group ting of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis, Bacillus pumilus, and mixtures thereof; ii. an organic solvent selected from the group consisting of propylene glycol (PG), N- methylpyrrolidone (NMP), ylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO), and mixtures thereof, wherein the organic solvent is t in an amount of 20 weight percent to 99 weight percent, based on the total weight of the composition; and iii. a urease tor selected from the group consisting of N-(n-butyl)thiophosphoric triamide (NBPT), utyl)phosphoric triamide, thiophosphoryl triamide, phenyl phosphorodiamidate, N-cyclohexyl phosphoric triamide, N-cyclohexyl thiophosphoric triamide, oric triamide, hydroquinone, oquinone, hexamidocyclotriphosphazene, ridines, thiopyrimidines, thiopyridine-N-oxides, N,N-dihaloimidazolidinone, N-halooxazolidinone, N-(2-nitrophenyl)thiophosphoric triamide, N-(2-nitrophenyl)phosphoric triamide, and mixtures thereof. b] In a second aspect the present invention provides a fertilizer composition comprising the composition according to the first aspect, and a nitrogen source. [00010c] In a third aspect the present invention provides a method for promoting plant , plant productivity and/or soil quality, wherein the method comprises applying an effective amount of the composition according to the first aspect to a plant, plant part, plant seed or soil. d] In a fourth aspect the present invention provides a kit sing: Part A, wherein Part A comprises a urease inhibitor and an orga nic solvent, wherein said 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, N-cyclohexyl thiophosphoric triamide, phosphoric triamide, hydroquinone, p-benzoquinone, hexamidocyclotriphosphazene, thiopyridines, thiopyrimidines, thiopyridine-N-oxides, N,N-dihaloimidazolidinone, N-halooxazolidinone, N-(2-nitrophenyl)thiophosphoric triamide, N-(2-nitrophenyl)phosphoric triamide, and mixtures thereof; and wherein said organic solvent is selected from the group consisting of propylene glycol (PG), N- methylpyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO), and mixtures thereof; and Part B, n Part B ses a Bacillus s selected from the group ting of us amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis , Bacillus pumilus, and mixtures thereof, n each of Part A and Part B is contained in a separate co ntainer and wherein the organic solvent is present in an amount g from 20 weight % to 99 weight %, based on the combined weight of Part A and Part B.

Also described is an agricultural microbial ant composition comprising at least one microbial strain from one or more microbial species, and at least one active agent, n the active agent is a urease inhibitor or a nitrification inhibitor or a combination thereof, and r wherein the at least one microbial strain is present at an effective amount to promote plant health, plant nutrition, and/or soil health in the presence of the active agent.

In one embodiment, the present disclosure describes an agricultural microbial inoculant ition comprising: i. at least one agriculturally beneficial Bacillus species selected from the group consisting of Bacillus amyloliquefaciens, us licheniformis, Bacillus thuringiensis , Bacillus pumilus, and any combination thereof; and ii. at least one organic t selected from the group consisting of propylene glycol (PG), N-methylpyrrolidone (NMP), triethylene glycol monobuty l ether, glycerol, dimethyl sulfoxide (DMSO), and any ation thereof.

In one embodiment, the present disclosure describes an agricultural microbial inoculant composition comprising: i. at least one agriculturally beneficial Bacillus species selected from the group consisting of Bacillus amyloliquefaciens, Bacillus iformis, Bacillus thuringiensis , Bacillus pumilus, and any combination thereof; ii. at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methylpyrrolidone (NMP), triethylene glycol monobuty l ether, glycerol, dimethyl sulfoxide , and any combination thereof; and iii. a urease inhibitor ed 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-dihaloimidazolidinone, N-halo oxazolidinone, N-(2-nitrophenyl)thiophosphoric triamide, N-(2-nitrophenyl)phosphoric triamide, and any combination thereof.

In one ment, the present disclosure describes an agricultural microbial inoculant kit comprising: Part A, wherein Part A comprises a urease inhibitor and at leas t one organic solvent, wherein said urease inhibitor is ed from the group consisted of N-(n-butyl)thiophosphoric triamide (NBPT), utyl)phosphoric triamide, thiophosphoryl triamide, phenyl phosphorodiamidate, N-cyclohexyl phosphoric triamide, N-cyclohexyl thiophosphoric de, phosphoric triamide, hydroquinone, p-benzoquinone, hexamidocyclotriphosphazene,thiopyridines, thiopyrimidines, ridine-N-oxides, N,N- dihaloimidazolidinone, N-halooxazolidinone, 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-methylpyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl s ulfoxide (DMSO), and any combination thereof; and Part B, wherein Part B ses at least one Bacillus species selected from the group consisting of Bacillus amyloliquefaciens, Bacillus iformis, us thuringiensis , Bacillus pumilus, and any combination thereof, wherein each Part A and Part B is contained in a te conta iner.

In one ment, the present disclosure describes an agricultural microbial inoculant kit comprising: Part A, wherein Part A comprises at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methylpyrrolidone (NM P), triethylene glycol monobutyl ether, glycerol, yl sulfoxide (DMSO), and any combination thereof; and Part B, wherein Part B comprises at least one Bacillus species selected from the group consisting of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis , Bacillus pumilus, and any combination thereof, wherein each Part A and Part B is contained in a separate conta iner.

In another embodiment, the present disclosure describes a method of enhancing a yield trait in a subject plant as compared to the yield trait of a reference or control plant, the method comprising contacting a subject plant, plant part, plant seed, or surrounding soil with an effective amount of a microbial inoculant composition of the present disclosure .

The urease inhibitor or nitrification inhibitor can mitigate e contamination in run off and ground water, and the emission of a large amount of ammonia and s oxide to the atmosphere. The ial species can further e plant health, plant nutrition, and soil health. The combination of both chemical enzyme inhibitors and microbial species in suitable compositions and formulations may serve as a better approach to improve the efficiency of nitrogen-based fertilizer usage by improving plant productivity, soil quality, and the overall environmental sustainability.

DETAILED DESCRIPTION OF THE INVENTION Unless defined otherwise, all technical and scientific terms used herein have the same meaning as ly understood by those of ordinary skill in the art.

In one embodiment, the t disclosure bes an ltural microbial inoculant 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 n the at least one microbial strain promotes plant health, plant nutrition, and/or soil health in the presence of the active agent.

In another embodiment, the present disclosure describes an ltural microbial inoculant 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 ation thereof, further wherein the at least one microbial strain es plant health, plant nutrition, and/or soil health in the presence of the active agent, wherein one or more microbial species are selected from the following group: (1) Spore forming species of bacteria; ] (2) Spore forming species of fungi; (3) Mycorrhizal organisms including Laccaria bicolor, Glomus intraradices, and Amanita species; (4) myces species and strains thereof, ing Streptomyces lydicus, Streptomyces griseoviridis, Streptomyces griseoviridis K61 (Mycostop; AgBio development), and Streptomyces microflavus AQ 6121; (5) Bacillus s and strains thereof, including: Bacillus itcheniformis; Bacillus megaterium; Bacillus pumilus, Bacillus amyloliquefaciens, Bacillus licheniformis; Bacillus oleronius; Bacillus megaterium; Bacillus nsis; us pumilus; Bacillus subtilis; Bacillus circulans; Bacillus globisporus; Bacillus firmus, Bacillus thuringiensis, Bacillus cereus, Bacillus amyloliquefaciens strain D747 (Double ; ), Bacillus firmus strain I-1582 (Votivo and Nortica; Bayer), Bacillus licheniformis, Bacillus licheniformis strain SB3086 (EcoGuard; Novozymes), Bacillus s strain GB34 (YieldShield; Bayer), QST2808 (Sonata; Bayer), us subtilis strains GB03 (Kodiak; Bayer), MBI 600 (Subtilex; Becker Underwood) & QST 713 (Serenade; Bayer), Bacillus subtilis strain GB122 plus Bacillus amyloliquefaciens strain GB99 eld; 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 s QST30002 and QST3004 (NRRL B-50421 and NRRLB-50455), us subtilis strains QST30002 and QST3004 (NRRL B-50421 and 50455) sandpaper mutants, us thuringiensis subsp kurstaki strain VBTS 2477 quadruple enterotoxindeficient s, Bacillus x s 03WN13, 03WN23 and 03WN25, Bacillus subtilis strain QST 713, Bacillus mycoides isolate BmJ NRRL B-30890, Bacillus subtilis strain DSM 17231 and B iformis strain DSM17236, Bacillus aryabhattai, B. flexus, B. nealsonii, Bacillus sphaericus, us megaterium, B. mortis, 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 pumilus (NRS-272); (6) Species of “Plant Growth Promoting Rhizobacteria” (PGPRs) and strains f, including species ed to be capable of nitrogen fixation, for example Gluconacetobacter species (e.g. Gluconacetobacter rophicus a.k.a. Acetobacter diazotrophicus), Spirillum species (e.g. Spirillum lipoferum), Azospirillum species, Herbaspirillum seropedicae, Azoarcus species, Azotobacter species, lderia species, Burkholderia sp. A396, and Paenibacillus polymyxa; (7) N-fixing bacterial species and strains thereof, including Rhizobium species (e.g.

Bradyrhizobium species such as Bradyrhizobium japonicum, and Rhizobium ti); (8) Microbial species and strains thereof that are known to improve nutrient use efficiency, including Penicillium species (e.g. Penicillium bilaii, Penicillium bilaji), and Mesorhizobium cicero; (9) Microbial species and s 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, ria bassiana strain ATP02, DSM 24665, Paecilomyces fumosoroseus, Trichoderma num, Verticillium lecanii, Isaria fumosorosea CCM 8367 (CCEFO.011.PFR), Lecanicillium ium, 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 ria nishizawae, Pasteuria Pasteuria reneformis strain Pr-3, Paecilomyces lilacinus, Chromobacterium subtsugae, Pasteuria strain ATCC SD-5832, Metarhizium species, and Flavobacterium s; (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 3 or NRRL B- 21102, Pseudomonas fluorescens VP5, Pseudomonas synxantha, Pseudomonas diazotrophicus, bacter cloacae strain NRRL B-21050, Trichoderma species, Trichoderma virens, Trichoderma atroviride strains, 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 plymuthica CCGG2742, Cryptococcus lavescens strain OH 182.9, Serratia hica, Cladosporium cladosporioides, Mitsuaria s, Coprinus curtus, acillus halophilus, Saccharomyces species, Metschnikovia ola, a ila, Acremonium species, Pseudozyma aphidis, Pythium oligandrum, Phoma spp strain I-4278, Achromobacter species, Geomyces species, Pseudomonas azotoformans, strain F30A, Brevibacillus evis strain No 4; non-toxigenic Aspergillus strains NRRL 50427, NRRL 50428, NRRL 50429, NRRL 50430 and NRRL 50431, Sphaerodes mycoparasitica strains IDAC 301008-01, -02, or -03, Muscodor albus strain NRRL 30547 or 548, Serratia plymuthica 42, Pseudomonas koreensis strain 10IL21, P lini strain 13IL01, Pantoea agglomerans strain , Streptomyces scopuliridis strain RB72, Acremonium spp endophytes, Streptomyces spp BG76 strain, Paracoccus kondratievae, Enterobacter cloacae, Cryptococcus cens, Lactobacillus parafarraginis, Lactobacillus buchneri, Lactobacillus rapi or Lactobacillus zeae, Paenibacillus polymyxa, Serratia plymuthica, Phoma species, m oligandrum, Mycosphaerella species, and Variovorax species; (12) Bacterial species and strains thereof from the group termed Pink-Pigmented Facultative Methylotrophs (PPFMs) including Methylobacterium species; and ] (13) Microbial s and strains thereof that are known to have herbicidal effect e.g., Pyrenophora semeniperda; wherein the urease inhibitor is selected from the group consisting of N-(nbutyl )thiophosphoric de (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-dihaloimidazolidinone, N-halo oxazolidinone, N-(2-nitrophenyl)thiophosphoric triamide, N-(2-nitrophenyl)phosphoric de, derivatives thereof, and any combination thereof; and wherein the nitrification inhibitor is selected from the group consisting of 2-chloro trichloromethylpyridine, xytrichloromethyl-1,2,4-thiadiazol, dicyandiamide, 2-amino chloromethyl-pyrimidine, 1,3-benzothiazolethiol, 4-amino-N-1,3-thiazolylbenzene sulfonamide, thiourea, guanidine, 3,4-dimethylpyrazole phosphate, 2,4-diamino oromethyltriazine, polyetherionophores, 4-amino-1,2,4-triazole, 3-mercapto-1,2,4- triazole, potassium azide, carbon bisulfide, sodium trithiocarbonate, um dithiocarbamate, 2,3,-dihydro-2,2-dimethylbenzofuranol methylcarbamate, N-(2,6-dimethylphenyl)-N- (methoxyacetyl)-alanine methyl ester, ammonium thiosulfate, 1-hydroxypyrazole, 3- methylpyrazolecarboxamide, ylpyrazole, 3,5-dimethylpyrazole, 1,2,4-triazole, G77 Nitrification Inhibitor (CAS Registration No. 13732567), de rivatives thereof, and any combination thereof.

] In one embodiment, the present sure describes a solid carrier-based formulation for any microbial inoculant composition of the present disclosure, wherein the solid carrier is selected from mineral earths, e.g. silicates, silica gels, talc, kaolins, limestone, lime, chalk, clays, dolomite, diatomaceous earth, bentonite, montmorillonites; inorganic salts, e.g. aluminum e, calcium e, copper sulfate, iron sulfate, magnesium sulfate, silicon sulfate, ium oxide; polysaccharides, e.g. cellulose, starch; fertilizers, e.g., um sulfate, ammonium phosphate, ammonium nitrate; products of ble origin, e.g. cereal meal, tree bark meal, wood meal, nutshell meal; grain flours suitable for the use in the present disclosure, e.g. flours from corn, rice, wheat, barley, sorghum, millet, oat, ale, rye, buckwheat, fonio and quinoa, and mixtures thereof, In one embodiment, the present disclosure describes a solvent-based formulation for any agricultural microbial inoculant composition of the present disclosure, wherein the solvent is selected from alkanolamines such as anolamine, diethanolamine, monoethanolamine; alkyldiethanolamines, dialkylmonoethanolamines, wherein the alkyl group is C1-C24 branched or unbranched alkyl chain; dimethylsulfoxide (DMSO); ulfones such as ane (2,3,4,5- tetrahydrothiophene-1,1-dioxide); alkyl amides such as N-methylpyrrolidone, N- ethylpyrrolidone, or dimethylformamide; monoalcohols such as methanol, ethanol, propanol, isopropanol, or benzyl alcohol; glycols such as ethylene glycol, propylene glycol, lene glycol, or dipropylene glycol; glycol derivatives and protected glycols such as triethylene glycol monobutyl ether; glycerol and glycerol derivatives (trialcohols) ing protected glycerols such as isopropylidine glycerol; dibasic esters and derivatives thereof; alkylene carbonates such as ethylene carbonate or propylene carbonate; monobasic esters such as ethyl lactate or ethyl acetate; polymers of carboxylic acids such as maleic acid, oleic acid, ic acid, acrylic acid, or methacrylic acid; monoalkyl glycol ethers and dialkyl glycol ethers; glycol esters; surfactants such as enzenesulfonates, lignin sulfonates, alkylphenol ethoxylates, or polyethoxylated amines.

In one embodiment, the present disclosure describes an encapsulated formulation for any agricultural microbial inoculant composition of the present disclosure. In the soil environment, inoculated microbial species can find survival difficult among naturally ing competitor and predator organisms. To aid in survival of microorganisms present in microbial inoculants and fertilizer compositions of the present disclosure upon application in the environment, one or more of the microbial species strains may be encapsulated in, for example, a suitable polymeric . In one example, encapsulation may comprise alginate beads such as has been described by Young et al, 2006, Encapsulation of plant growth-promoting bacteria in alginate beads enriched with humid acid, hnology and Bioengineering 83. Those skilled in the art will appreciate that any suitable ulation material or matrix may be used.

Encapsulation may be ed using methods and techniques known to those skilled in the art.

Encapsulated microorganisms can include nutrients or other components of the inoculant or fertilizer composition in addition to the microorganisms.

In one embodiment, the present disclosure describes an agricultural ial inoculant composition sing: i. at least one agriculturally beneficial Bacillus species selected from the group consisting of Bacillus amyloliquefaciens, Bacillus licheniformis, us thuringiensis , Bacillus pumilus, and any combination thereof; and ii. at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methylpyrrolidone (NMP), triethylene glycol monobuty l ether, glycerol, dimethyl sulfoxide (DMSO), and any ation thereof.

] In one embodiment, the present disclosure describes an ltural microbial inoculant composition comprising: i. at least one agriculturally beneficial us species selected from the group consisting of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus giensis , Bacillus pumilus, and any combination thereof; ii. at least one organic t selected from the group consisting of propylene glycol (PG), N-methylpyrrolidone (NMP), triethylene glycol monobuty l 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 thiophosphoric triamide, phosphoric triamide, hydroquinone, p-benzoquinone, hexamidocyclotriphosphazene, ridines, thiopyrimidines, thiopyridine-N-oxides, N,N-dihaloimidazolidinone, N-halo oxazolidinone, N-(2-nitrophenyl)thiophosphoric triamide, N-(2-nitrophenyl)phosphoric triamide, and any combination thereof.

In one embodiment, the present disclosure describes an ltural microbial ant composition sing: i. at least one agriculturally beneficial Bacillus species selected from the group consisting of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis , us s, and any combination f; ii. at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methylpyrrolidone (NMP), triethylene glycol monobuty l ether, glycerol, dimethyl sulfoxide , and any combination thereof; and iii. a nitrification tor selected from the group consisting of 2-chloro trichloromethylpyridine, 5-ethoxytrichloromethyl-1,2,4-thiadiazol, dicyandiamide, 2-amino chloromethyl-pyrimidine, 1,3-benzothiazolethiol, 4-amino-N-1,3-thiazolylbenzene sulfonamide, thiourea, guanidine, methylpyrazole phosphate, 2,4-diamino trichloromethyltriazine, poly etherionophores, 4-amino-1,2,4-triazole, 3-mercapto-1,2,4- triazole, potassium azide, carbon bisulfide, sodium trithiocarbonate, ammonium dithiocarbamate, 2,3,-dihydro-2,2-dimethylbenzofuranol methylcarbamate, N-(2,6-dimethylphenyl)-N- (methoxyacetyl)-alanine methyl ester, ammonium thiosulfate, 1-hydroxypyrazole, 3- methylpyrazolecarboxamide, ylpyrazole, 3,5-dimethylpyrazole, 1,2,4-triazole, G77 Nitrification Inhibitor (CAS Registration No. 13732567), an d any combination thereof.

In one embodiment, the present sure bes an agricultural microbial inoculant kit comprising: Part A, wherein Part A comprises a urease inhibitor and at leas t one organic solvent, wherein said urease inhibitor is selected from the group consisted of N-(n-butyl)thiophosphoric triamide (NBPT), N-(n-butyl)phosphoric triamide, thiophosphoryl triamide, phenyl phosphorodiamidate, N-cyclohexyl phosphoric triamide, ohexyl thiophosphoric triamide, phosphoric triamide, hydroquinone, p-benzoquinone, hexamidocyclotriphosphazene,thiopyridines, rimidines, thiopyridine-N-oxides, N,N- dihaloimidazolidinone, N-halooxazolidinone, N-(2-nitrophenyl)thiophosphoric triamide, itrophenyl)phosphoric triamide, and any combination thereof; and wherein said organic solvent is selected from the group consisting of propylene glycol (PG), N-methylpyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl s ulfoxide (DMSO), and any combination f; and Part B, wherein Part B comprises at least one Bacillus species ed from the group consisting of us amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis , Bacillus pumilus, and any combination thereof, wherein each Part A and Part B is contained in a separate conta iner.

In one embodiment, the t disclosure describes an ltural microbial inoculant kit comprising: Part A, wherein Part A comprises at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methylpyrrolidone (NM P), triethylene glycol monobutyl ether, glycerol, dimethyl ide (DMSO), and any combination f; and Part B, wherein Part B comprises at least one Bacillus species selected from the group consisting of Bacillus amyloliquefaciens, Bacillus iformis, Bacillus thuringiensis , Bacillus pumilus, and any ation thereof, wherein each Part A and Part B is contained in a separate conta iner.

In one embodiment, the at least one agriculturally beneficial Bacillus species in an agricultural microbial inoculant composition or a kit is selected from the group consisting of Bacillus licheniformis, Bacillus giensis, and any combination thereof.

In one embodiment, the agriculturally beneficial Bacillus species in an agricultural microbial inoculant composition or a kit comprises 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) , us thuringiensis (HD-17), Bacillus thuringiensis , us pumilus (NRS- 272), and any combination f.

In one ment, the urease inhibitor in an agricultural microbial inoculant composition or a kit, where present, is N-(n-butyl)thiophosphoric triamide (NBPT).

In one embodiment, the nitrification inhibitor in an agricultural microbial inoculant composition or a kit, where present, is dicyandiamide, G77 Nitr ification Inhibitor (CAS Registration No. 67), or a combination thereof.

In one embodiment, the solvent in an agricultural microbial inoculant composition or a kit ses propylene glycol and N-methylpyrrolidone.

In one embodiment, the solvent in an agricultural microbial inoculant composition or a kit comprises propylene glycol, N-methylpyrrolidone, and ylene glycol monobutyl ether.

In one embodiment, an agricultural ial inoculant composition further comprises water, glycerol or a combination thereof.

In one embodiment, Part B of an agricultural microbial inoculant kit r comprises water, glycerol or a combination thereof.

In one embodiment, the present disclosure describes an agricultural microbial inoculant composition comprising: i. at least one agriculturally cial Bacillus species 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 ting of propylene glycol (PG), N-methylpyrrolidone (NMP), triethylene glycol monobuty l ether, glycerol, dimethyl sulfoxide (DMSO), and any combination thereof.

In one embodiment, the present disclosure bes an agricultural microbial inoculant composition comprising: i. at least one lturally beneficial Bacillus species ed from the group consisting of Bacillus licheniformis, us thuringiensis , and any combination thereof; ii. at least one c t ed from the group consisting of propylene glycol (PG), ylpyrrolidone (NMP), triethylene glycol monobuty l ether, glycerol, dimethyl sulfoxide (DMSO), and any combination thereof; and iii. N-(n-butyl)thiophosphoric triamide (NBPT).

] In one embodiment, the present disclosure describes an agricultural microbial inoculant kit comprising: Part A, wherein Part A comprises N-(n-butyl)thiophosphoric tria mide (NBPT) and at least one organic solvent selected from the group consisting of propylene glycol (PG), yl- 2-pyrrolidone (NMP), triethylene glycol monobutyl ether, glycer ol, dimethyl sulfoxide (DMSO), and any combination thereof; and Part B, wherein Part B ses at least one Bacillus species selected from the group consisting of Bacillus licheniformis, Bacillus thuringiensis , and any combination f, wherein each Part A and Part B is contained in a separate conta iner.

In one embodiment, the present disclosure describes an agricultural microbial inoculant composition comprising: i. at least one agriculturally beneficial Bacillus strain selected from the group consisting of Bacillus amyloliquefaciens (ATCC 23842), Bacillus iformis (ATCC 14580), Bacillus licheniformis (B-642), Bacillus licheniformis 68), Bacillus thuringiensis (ATCC 10792) , Bacillus thuringiensis (HD-17), Bacillus thuringiensis (HD-1), Bacillus pumilus (NRS- 272), and any combination thereof; and ii. at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methylpyrrolidone (NMP), triethylene glycol monobuty l ether, glycerol, dimethyl sulfoxide (DMSO), and any combination f.

In one embodiment, the present sure describes an agricultural microbial inoculant composition comprising: i. at least one agriculturally beneficial 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 giensis (HD-17), Bacillus thuringiensis (HD-1), us pumilus (NRS- 272), and any combination thereof; ii. at least one organic solvent selected from the group consisting of ene glycol (PG), N-methylpyrrolidone (NMP), triethylene glycol monobuty l ether, ol, dimethyl ide (DMSO), and any combination thereof; and iii. N-(n-butyl)thiophosphoric triamide (NBPT).

In one embodiment, the t disclosure describes an agricultural microbial inoculant composition comprising: i. at least one agriculturally beneficial us strain selected from the group consisting of Bacillus amyloliquefaciens (ATCC 23842), Bacillus iformis (ATCC 14580), Bacillus licheniformis (B-642), Bacillus licheniformis (B-14368), Bacillus thuringiensis (ATCC 10792) , Bacillus thuringiensis (HD-17), Bacillus thuringiensis (HD-1), Bacillus pumilus (NRS- 272), and any combination thereof; ii. at least one organic solvent selected from the group consisting of propylene glycol (PG), N-methylpyrrolidone (NMP), triethylene glycol monobuty l ether, glycerol, dimethyl ide (DMSO), and any combination thereof; and ] iii. dicyandiamide, G77 Nitrification Inhibitor (CAS Registrati on No. 13732567), or a combination thereof.

In one embodiment, the present disclosure describes an agricultural microbial inoculant kit comprising: Part A, n Part A comprises utyl)thiophosphoric tria mide (NBPT) and at least one organic solvent ed from the group consisting of propylene glycol (PG), N-methyl- 2-pyrrolidone (NMP), triethylene glycol monobutyl ether, glycer ol, dimethyl sulfoxide (DMSO), and any combination thereof; and Part B, wherein Part B comprises at least one agriculturally be neficial Bacillus strain selected from the group consisting of Bacillus iquefaciens (ATCC 23 842), Bacillus licheniformis (ATCC 14580), Bacillus licheniformis (B-642), Bacillus licheniformis (B-14368), us thuringiensis (ATCC 10792) , Bacillus thuringiensis (HD-17), Bacillus thuringiensis (HD-1), us pumilus (NRS-272), and any combination thereof, wherein each Part A and Part B is contained in a separate conta iner. 3] In one ment, the present disclosure describes an agricultural microbial inoculant kit comprising: Part A, wherein Part A comprises at least one organic solvent s elected from the group consisting of propylene glycol (PG), N-methylpyrrolidone (NM P), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO), and any combination thereof; and ] Part B, wherein Part B ses at least one agriculturally be neficial Bacillus strain selected from the group ting of Bacillus amyloliquefaciens (ATCC 23 842), Bacillus licheniformis (ATCC 14580), Bacillus licheniformis (B-642), Bacillus licheniformis (B-14368), Bacillus thuringiensis (ATCC 10792) , Bacillus thuringiensis (HD-17), Bacillus thuringiensis , Bacillus s (NRS-272), and any combination thereof, wherein each Part A and Part B is contained in a separate conta iner.

The weight percentage of a urease inhibitor such as NBPT in any embodiment of an agricultural microbial inoculant composition or a kit of the present disclosure, where presents, is in the range of 0.02-80%. In one embodiment, the weight percentage is in the range of 0.02- 70%. In one embodiment, the weight percentage is in the range of 0.02-60%. In one embodiment, the weight percentage is in the range of 0%. In one embodiment, the weight percentage is in the range of 0.02-40%. In one embodiment, the weight percentage is in the range of 0.02-30%. In one embodiment, the weight percentage is in the range of 0.02-20%. In one embodiment, the weight percentage is in the range of 0.02-10%. In one embodiment, the weight percentage is in the range of 0.02-5%. In one embodiment, the weight tage is in the range of 5-60%. In one ment, the weight percentage is in the range of 5-50%. In one embodiment, the weight percentage is in the range of 5-40%. In one embodiment, the weight percentage is in the range of 5-30 %. In one embodiment, the weight percentage is in the range of -60%. In one embodiment, the weight percentage is in the range of 10-50%. In one embodiment, the weight percentage is in the range of 10-40%. In one embodiment, the weight percentage is in the range of 10-30%. In one ment, the weight percentage is in the range of 15-60%. In one embodiment, the weight percentage is in the range of 15-50%. In one embodiment, the weight percentage is in the range of 15-40%. In one embodiment, the weight percentage is in the range of 15-30%. In one embodiment, the weight percentage is in the range of 30-60%. In one embodiment, the weight percentage is in the range of . In one embodiment, the weight percentage is in the range of 40-60%. The weight percentage is based on the entirety of the microbial inoculant composition.

The weight percentage of a nitrification inhibitor such as DCD, where present, in any embodiment of an agricultural microbial inoculant composition or a kit of the present disclosure is in the range of 1-80%. In one embodiment, the weight percentage is in the range of 1-70%. In one embodiment, the weight percentage is in the range of 1-60%. In one embodiment, the weight percentage is in the range of 1-50%. In one embodiment, the weight tage is in the range of 1-40%. In one embodiment, the weight percentage is in the range of 1-30%. In one embodiment, the weight percentage is in the range of 1-20%. In one embodiment, the weight percentage is in the range of 1-10%. In one embodiment, the weight percentage is in the range of 1-5%. In one embodiment, the weight percentage is in the range of 10-80%. In one embodiment, the weight percentage is in the range of 10-70%. In one embodiment, the weight percentage is in the range of . In one embodiment, the weight percentage is in the range of 10-50%. In one embodiment, the weight percentage is in the range of 10-40%. In one embodiment, the weight percentage is in the range of 10-30%. In one embodiment, the weight percentage is in the range of 20-80%. In one embodiment, the weight percentage is in the range of 20-70%. In one embodiment, the weight percentage is in the range of 20-60%. In one embodiment, the weight tage is in the range of 20-50%. In one embodiment, the weight percentage is in the range of 20-40%. In one embodiment, the weight percentage is in the range of 20-30%. The weight percentage is based on the entirety of the microbial inoculant composition.

The weight percentage of an organic solvent such as propylene g lycol (PG), N- methylpyrrolidone (NMP), triethylene glycol monobutyl ether, ol, dimethyl ide , or any ation f in any embodiment of an agricultural microbial inoculant composition or a kit of the present disclosure is in the range of . In one embodiment, the weight percentage is in the range of 20- 90%. In one embodiment, the weight percentage is in the range of 20-80%. In one embodiment, the weight tage is in the range of 20-70%. In one embodiment, the weight percentage is in the range of 20-60%. In one embodiment, the weight percentage is in the range of 20-50%. In one ment, the weight percentage is in the range of 30-99%. In one embodiment, the weight percentage is in the range of 30-90%. In one embodiment, the weight percentage is in the range of 30-80%. In one embodiment, the weight percentage is in the range of 30-70%. In one embodiment, the weight percentage is in the range of 30-60%. In one embodiment, the weight percentage is in the range of . In one embodiment, the weight percentage is in the range of 40-99%. In one embodiment, the weight percentage is in the range of 40-80%. In one embodiment, the weight percentage is in the range of . In one ment, the weight percentage is in the range of 40-60%. The weight percentage is based on the ty of the microbial inoculant composition.

In one embodiment, an agricultural microbial inoculant composition or a kit of the present disclosure comprises propylene glycol with the weight percentage in the range of 40- 70%, N-methylpyrrolidone with the weight percentage in the range of 15-40%, NBPT with the weight percentage in the range of 10-30%, and optionally a dye with the weight tage in the range of 0.1-5%. The weight tage is based on the entirety of the microbial inoculant composition. 1] In one embodiment, an agricultural microbial inoculant composition or an kit of the present disclosure comprises propylene glycol with the weight percentage in the range of 10- %, N-methylpyrrolidone with the weight percentage in the range of 30-60%, NBPT with the weight percentage in the range of , triethylene glycol monobutyl ether with the weight percentage in the range of 1-5%, and optionally a dye with the weight percentage in the range of 0-1%. The weight percentage is based on the entirety of the microbial inoculant composition.

In one embodiment, the concentration of the us species in the microbial inoculant composition of the present sure is at least 1.0 x 102 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 105 spores/mL. In one embodiment, the concentration is at least 1 .0 x 106 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 /mL. In one embodiment, the concentration is at least 1 .0 x 1010 spores/mL. In one embodiment, the concentration is at least 1.0 x 10 11 spores/mL. In one embodiment, the tration is at least 1.0 x 10 12 /mL. In one embodiment, the concentration is in the range of 1.0 x 10 2–1.0 x 1012 spores/mL. In one embodiment, the concentration is in the range of 1.0 x 103–1.0 x 1012 spores/mL. In one embodiment, the tration is in the range of 1.0 x 104–1.0 x 1012 spores/mL. In one embodiment, the concentration is in the range of 1.0 x 10 5–1.0 x 1012 spores/mL. In one embodiment, the concentration is in the range of 1.0 x 10 6–1.0 x 1012 spores/mL. In one embodiment, the concentration is in the range of 1.0 x 10 7–1.0 x 1012 spores/mL. In one embodiment, the concentration is in the range of 1.0 x 10 8–1.0 x 1012 spores/mL. In one embodiment, the concentration is in the range of 1.0 x 10 9–1.0 x 1012 spores/mL.

In one embodiment of the present disclosure, the agricultural microbial ant composition may serve as a fertilizer by itself.

A dye may also be included in the agricultural microbial inoculant composition in the present disclosure. Any commonly used dye including food dyes may be used to provide visual evidence of the uniformity of the ial inoculant composition. The weight percentage of a dye in the total microbial inoculant composition is 0-10%. In one ment, the weight percentage is 0.1-5%. es of dyes le in the present disclosure include but are not limited to FD&C Blue No. 1, FD&C Blue No. 1, FD&C Green No. 3, FD&C Yellow No. 5, FD&C Red No. 3, FD&C Red No. 40, FD&C Yellow No. 6, and AGROTAIN® ULTRA green dye, or a combination thereof.

In another embodiment, the present disclosure describes a fertilizer composition comprising any agricultural ial inoculant composition in any embodiment of the present disclosure, wherein the fertilize can be a granular fertilizer such as urea ar, a liquid fertilizer such as urea ammonium nitrate (UAN), an aqueous urea and ammonia nitrate aqueous solution, or anhydrous ammonia (NH3).

In another embodiment, the t disclosure describes a method of enhancing a yield trait in a subject plant as compared to the yield trait of a reference or control plant, the method sing contacting a subject plant, plant part, plant seed, or surrounding soil with an effective amount of an agricultural microbial inoculant composition of the present disclosure, wherein the microbial inoculant composition comprises: 8] 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, nitrification tor, or a combination thereof, wherein the agricultural microbial inoculant composition at the effective amount is effective in enhancing the yield trait in the subject plant relative to the yield trait in the reference or control plant when the subject plant is ted with the ive amount.

In another ment, the present disclosure bes a method for enhancing a yield trait in the plant, such as increasing plant growth and/or productivity, wherein the method comprises applying to the plant, plant part, plant seeds or to the soil in which the plant or plant seeds are grown an effective amount of an agricultural microbial inoculant composition of any embodiment of the present disclosure.

In another embodiment, the present disclosure describes a method for improving soil quality, wherein the method comprises ng to soil or to the plants or plant seeds in said soil an effective amount of an agricultural microbial inoculant composition as disclosed in any embodiment of the present disclosure.

In any embodiment of the disclosure, the concentrations of each agricultural microbial strain to be added to microbial inoculants and fertilizer compositions as disclosed herein will depend on a variety of factors including the identity and number of dual strains ed, the plant s being treated, the nature and condition of the soil to be d, the exact nature of the microbial inoculant or fertilizer composition to be applied, the type and form of active agent, the form in which the inoculant or fertilizer is applied and the means by which it is d, and the stage of the plant growing season during which application takes place. For any given case, appropriate concentrations should be effective in enhancing the yield trait in the presence of the active agent, and may be determined by one of ordinary skill in the art using only routine experimentation. By way of e only, the tration of each strain present in the inoculant or fertilizer composition may 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.

In one embodiment of the present disclosure, a microbial food source such as kelp or ol may be included in any embodiment of the present disclosure.

The term “microbial species” refers to either naturally occurring or specifically developed variants or mutants of microbial species such as bacteria and fungi as disclosed herein. Variants or mutants may or may not have the same fying biological characteristics of the specific strains exemplified herein, provided they share similar advantageous properties in terms of promoting plant growth and ing nutrients for plant growth in the soil. Variants of certain microbial strains may include but not limited to those developed by gene ation techniques such as those mediated by insertional elements or transposons or by homologous recombination, other recombinant DNA ques for modifying, inserting, deleting, 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, en mustard and the like, and 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, N.Y. (1972); J. H. Miller, A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1992); and J. Sambrook, D.

Russell, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (200l). 6] The term “plant productivity” or “yield trait” as used herein refers to any aspect of growth or development of a plant that is a reason for which the plant is grown. Thus, for purposes of the present disclosure, improved or sed “plant productivity” or “enhanced yield trait” refers broadly to improvements in biomass or yield of leaves, stems, grain, fruit, vegetables, s, or other plant parts ted or used for various purposes, and improvements in growth of plant parts, including stems, leaves and roots.

The term “improving soil quality” refers to the increasing the amount and/or availability of nutrients required by, or beneficial to plants, for growth. For example only, such nutrients include nitrogen, phosphorous, potassium, copper, zinc, boron and molybdenum. Also encompassed by the term “improving soil quality” is reducing or minimizing the amount of an element that may be detrimental to plant growth or development such as, for example iron and manganese. Thus, improving soil quality by use of microbial inoculants and fertilizer compositions of the present disclosure y assists and promotes the growth of plants in the soil.

The term “effective amount” refers to an amount of microbial ant or fertilizer ition applied to a given area of soil or vegetation that is sufficient to promote one or more cial or desired es, for example, in terms of plant growth rates, crop yields, or nutrient availability in the soil. An “effective amount” can be provided in one or more administrations. The exact amount required will vary depending on factors such as the identity and number of individual strains employed, the plant s being treated, the nature and condition of the soil to be d, the exact nature of the microbial inoculant or fertilizer composition to be applied, the form in which the inoculant or fertilizer is applied and the means by which it is applied, and the stage of the plant growing season during which application takes place. For any given case, an appropriate “effective amount” may be ined by one with ordinary skill in the art using only routine experimentation.

The term “viability” in the present disclosure refers to the capability of repeated on of a microbial cell on an agar surface to produce a e colony. The temperature for the viability evaluation is about 37 oC 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 a mixture of more than one organic solvent. After the microbial culture in a nutrient broth is added to the organic solvent or the mixture of more than one solvent, a small amount of mixture is taken out at certain time for incubation at about 37 oC. The time period between the moment the microbial culture in a nt broth is added to an c solvent and the moment that the mixture is taken out for incubation is defined as the viability time for the viability evaluation e.

Bacteria Viability Test in Solutions with Organic Solvents 0] The purpose of the bacteria viability test is to evaluate the v iability of agriculturally beneficial bacteria species in solutions with organic solvents. 1] Both agriculturally beneficial Gram-positive and Gram-negative bacteria species are used for the test.

Agriculturally beneficial bacteria species were obtained from t he American Type Culture Collection (ATCC), or the Agricultural Research Service Culture Collection. The bacteria s were grown on appropriate media, LB broth, nutrient broth, and checked microscopically with Gram staining and on Petri plates for purity. The bacteria species are pure, i.e., no unusual colonies were ed.

All the samples for testing are prepared by a two-step method.

First, selected agriculturally beneficial bacteria were grown o vernight in Luria- Bertani (LB) medium at 37 oC with ion. Growth was measured with a Bausch and Lomb Spectronic Spectrophotometer at at 600 nm (OD600) to provide s with cell optical density at 600 nm (OD600) between 1.2 and 1.5. The overnight cultures typically n 1.6x108 to 3.4 x 108 colony forming units per mL (CFU/mL).

] Second, 0.5 mL of the prepared bacteria s sample in LB me dium was added to an organic solvent or a mixture of organic solvents (4.5 mL).

A sample of 10 µL of the organic solvent solution with bacteria species is removed immediately for bacteria viability evaluation (T=0).

The remaining organic solvent solution with the bacteria species is ted at 37 oC for future test. A volume of 10 µL of sample was taken out from the incubated organic solvent on 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 for ia viability evaluation.

Each time the sample (10 µL) that was taken out for viability evaluation was placed onto agar , which was ted overnight at 37 o C. Colonies of bacteria present on the plates indicates that the bacteria have tolerated the solvent and was therefore viable. If there is no growth of colonies of bacteria, the bacteria have demonstrated no tolerance in the solvent.

The agriculturally beneficial bacteria that are viable for at l east two hours from the moment that the bacteria sample is added to an organic solvent are considered to be viable in the organic solvent solution described herein.

Bacillus amyloliquefaciens (ATCC 23842) provided at least two hours of viability in NMP, and at least 5 days of viability in glycerol.

Bacillus licheniformis (ATCC 14580) provided at least one day of viability in triethylene glycol monobutyl ether, at least 5 days of viab ility 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.

Bacillus licheniformis (B-642) and Bacillus licheniformis (B-14368) each provided at least 21 days of viability in PG, at least 21 days of viability in NMP, at least 21 days of viability in triethylene glycol tyl ether, at least 21 days of viab ility in glycerol, and at least 21 days of viability in DMSO.

Bacillus giensis (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 ylene glycol monobutyl ether, at least 21 days of viability in glycerol, and at least 21 days of viability in DMSO.

Bacillus thuringiensis (HD-17) and Bacillus thuringiensis (HD-1) each provided at least 21 days of viability in PG, at least 21 days of viability in NMP, at least 21 days of viability in triethylene glycol tyl ether, at least 21 days of viab ility in glycerol, and at least 21 days of viability in DMSO.

Bacillus pumilus (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 viab ility in glycerol, and at least 21 days of ity in DMSO.

Surprisingly, all three selected agriculturally beneficial Gram-negative species Pseudomonas fluorescens (ATCC 53958), Pseudomonas putida (ATCC , and Pseudomonas chlororaphis (ATCC 55670), which were expected to have better t- nce, lost viability almost instantly when the prepared ba cteria samples with OD600 between 1.3 and 1.5 in nutrient broth were added to all tested organic solvent except glycerol.

The bacteria viability test in solutions with c solvents in the present disclosure demonstrated that all the examples of Bacillus licheniformis and Bacillus licheniformis provided viability with time range from at least 2 hours to at least 21 days in a solution with at least one of the c solvents PG, NMP, triethylene glycol tyl ether, glycerol and DMSO.

The bacteria viability test in solutions with organic solvents in the present disclosure demonstrated that the Bacillus strains Bacillus amyloliquefaciens (ATCC , Bac illus 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 pumilus 72) each provided ity with time range from at least 2 hours to at least 21 days in a solution with at least one of the organic ts 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 tyl ether, glycerol or DMSO Example No. Bacillus Species Bacillus Strains Solvent 1 amyloliquefaciens ATCC 23842 NMP 2 amyloliquefaciens ATCC 23842 Glycerol 3 licheniformis ATCC 14580 Triethylene glycol monobutyl ether 4 licheniformis ATCC 14580 PG licheniformis ATCC 14580 NMP 6 licheniformis ATCC 14580 ol 7 licheniformis ATCC 14580 DMSO 8 licheniformis B-642 PG 9 licheniformis B-642 NMP licheniformis B-642 Triethylene glycol monobutyl ether 11 licheniformis B-642 Glycerol 12 licheniformis B-642 DMSO 13 iformis B-14368 PG 14 licheniformis B-14368 NMP licheniformis B-14368 Triethylene glycol monobutyl ether 16 licheniformis B-14368 Glycerol 17 licheniformis B-14368 DMSO 18 thuringiensis ATCC 10792 PG 19 thuringiensis ATCC 10792 NMP thuringiensis ATCC 10792 Triethylene glycol monobutyl ether 21 thuringiensis ATCC 10792 Glycerol 22 thuringiensis ATCC 10792 DMSO 23 thuringiensis HD-17 PG 24 thuringiensis HD-17 NMP thuringiensis HD-17 Triethylene glycol monobutyl ether 26 thuringiensis HD-17 ol 27 thuringiensis HD-17 DMSO 28 thuringiensis HD-1 PG 29 thuringiensis HD-1 NMP thuringiensis HD-1 Triethylene glycol monobutyl ether 31 thuringiensis HD-1 Glycerol 32 giensis HD-1 DMSO 33 pumilus NRS-272 PG 34 pumilus NRS-272 Triethylene glycol monobutyl ether pumilus NRS-272 Glycerol 36 pumilus NRS-272 DMSO The following numbered paragraphs define particular aspects of the present invention: 1. An agricultural microbial inoculant composition comprising: i. at least one agriculturally beneficial Bacillus species selected from the group consisting of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis , us pumilus, and any ation thereof; and ii. at least one organic t selected from the group consisting of propylene glycol (PG), N-methylpyrrolidone (NMP), triethylene glycol monobuty l ether, glycerol, dimethyl sulfoxide , and any combination thereof. 2. The agricultural microbial inoculant composition of paragraph 1, wherein the agriculturally beneficial us species is selected from the group consisting of us licheniformis, Bacillus thuringiensis, and any combination thereof. 3. The agricultural microbial inoculant composition of paragraph 1, wherein 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), us thuringiensis (ATCC 10792) , Bacillus thuringiensis (HD-17), Bacillus thuringiensis (HD-1), us pumilus (NRS-272), and any combination thereof. 4. The agricultural microbial inoculant composition of paragraph 1, further comprising a urease tor 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, uinone, oquinone, hexamidocyclotriphosphazene, ridines, thiopyrimidines, thiopyridine-N-oxides, N,N-dihaloimidazolidinone, N-halooxazolidinone, N-(2- nitrophenyl)thiophosphoric triamide, N-(2-nitrophenyl)phosphoric triamide, and any ation thereof.

. The agricultural microbial inoculant composition of paragraph 4, wherein the urease inhibitor is N-(n-butyl)thiophosphoric triamide (NBPT). 6. The agricultural microbial inoculant composition of paragraph 1, further comprising a nitrification inhibitor selected from the group consisting of 2-chlorotrichloromethylpyridine, -ethoxytrichloromethyl-1,2,4-thiadiazol, dicyandiamide, 2-aminochloromethylpyrimidine , 1,3-benzothiazolethiol, 4-amino-N-1,3-thiazolylbenzene sulfonamide, thiourea, guanidine, 3,4-dimethylpyrazole phosphate, 2,4-diaminotrichloromethyltriazine, poly etherionophores, 4-amino-1,2,4-triazole, 3-mercapto-1,2,4-triazole, ium azide, carbon bisulfide, sodium trithiocarbonate, ammonium dithiocarbamate, 2,3,-dihydro-2,2-dimethyl benzofuranol methylcarbamate, N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester, ammonium thiosulfate, 1-hydroxypyrazole, 3-methylpyrazolecarboxamide, 3- pyrazole, methylpyrazole, 1,2,4-triazole, G77 Nitri fication Inhibitor (CAS Registration No. 13732567), and any combination thereof. 7. The agricultural microbial inoculant composition of paragraph 6, wherein the nitrification inhibitor is dicyandiamide, G77 ication Inhibitor (CAS Re tion No. 13732567) or a combination thereof. 8. The agricultural microbial ant composition of paragraph 4, wherein the weight percentage of the urease inhibitor is in the range of 0.02-80% based on the total weight of the agricultural microbial inoculant composition. 9. The agricultural microbial inoculant composition of paragraph 8, wherein the weight percentage of the urease inhibitor is in the range of 10-60%.

. The agricultural microbial ant composition of paragraph 1, wherein the agriculturally beneficial Bacillus species has at least 2 hours of viability from the time when said agriculturally beneficial Bacillus species contacts said organic solvent. 11. The agricultural ial inoculant composition of paragraph 1, wherein the agriculturally cial us species tration is in the range of 1.0 x 10 2 1.0 x 1012 spores/mL. 12. The agricultural microbial inoculant composition of paragraph 1, further comprising a dye. 13. The agricultural ial inoculant composition of paragraph 4, comprising propylene glycol and N-methylpyrrolidone. 14. The agricultural microbial inoculant composition of paragraph 13, wherein the weight percentage of propylene glycol is in the range of 40-70% based on the total weight of the agricultural microbial ant composition, the weight percentage of ylpyrrolidone is in the range of 15-40% based on the total weight of the agricultural microbial inoculant composition, and the weight percentage of NBPT is in the range of 10-30% based on the total weight of the microbial inoculant composition.

. The agricultural ial inoculant composition of paragraph 4 comprising propylene glycol, N-methylpyrrolidone, and triethylene glycol monobutyl ether. 16. The agricultural microbial inoculant composition of paragraph 15, wherein the weight percentage of propylene glycol is in the range of 10-30% based on the total weight of the agricultural microbial inoculant composition, the weight percentage of N-methylpyrrolidone is in the range of 30-60% based on the total weight of the agricultural microbial inoculant composition, the weight percentage of triethylene glycol monobutyl ether is in the range of 1-5% based on the total weight of the agricultural microbial inoculant composition, and the weight percentage of NBPT is in the range of 15-40% based on the total weight of the agricultural microbial ant composition. 17. An agricultural microbial inoculant kit comprising: Part A, wherein Part A comprises a urease inhibitor and at leas t one organic solvent, wherein said urease inhibitor is selected from the group ted of N-(nbutyl )thiophosphoric triamide (NBPT), N-(n-butyl)phosphoric triamide, thiophosphoryl triamide, phenyl phosphorodiamidate, N-cyclohexyl phosphoric triamide, N-cyclohexyl osphoric triamide, phosphoric triamide, hydroquinone, p-benzoquinone, hexamidocyclotriphosphazene,thiopyridines, thiopyrimidines, ridine-N-oxides, N,N-dihaloimidazolidinone, N-halooxazolidinone, N-(2-nitrophenyl)thiophosphoric triamide, itrophenyl)phosphoric triamide, and any combination thereof; and wherein said organic solvent is selected from the group consisting of propylene glycol (PG), ylpyrrolidone (NMP), triethylene glycol ty l ether, glycerol, dimethyl sulfoxide (DMSO), and any combination thereof; and Part B, n Part B comprises at least one Bacillus species selected from the group ting of Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis, Bacillus pumilus, and any combination thereof. wherein each Part A and Part B is contained in a separate conta iner. 18. The agricultural microbial inoculant kit of paragraph 17, n Part B comprises at least one Bacillus species selected from the group consisting of Bacillus iformis, us thuringiensis, and any combination thereof. 19. The agricultural microbial inoculant kit of paragraph 17, P art B comprises at least one agriculturally beneficial 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) , us thuringiensis (HD-17), Bacillus thuringiensis (HD-1), Bacillus pumilus (NRS-272), and any combination thereof.

. The agricultural microbial inoculant kit of paragraph 17, wherein the urease inhibitor is N-(nbutyl )thiophosphoric triamide (NBPT). 21. The agricultural microbial inoculant kit of paragraph 20, wherein Part A comprises ene glycol and N-methylpyrrolidone. 22. The agricultural microbial inoculant kit of paragraph 21, wherein the weight percentage of propylene glycol is in the range of 40-70% based on the total weight Part A, the weight percentage of N-methylpyrrolidone is in the range of 15-40% based on the total weight of Part A, and the weight percentage of NBPT is in the range of 10-30% based on the total weight of Part A. 23. The agricultural microbial inoculant kit of paragraph 20, wherein Part A comprises propylene glycol, ylpyrrolidone, and triethylene glycol monobutyl ether. 24. The ltural microbial inoculant kit of paragraph 23, wherein the weight percentage of propylene glycol is in the range of 10-30% based on the total weight of Part A, the weight percentage of N-methylpyrrolidone is in the range of 30-60% based on the total weight of Part A, the weight percentage of triethylene glycol monobutyl ether is in the range of 1-5% based on the total weight of Part A, and the weight percentage of NBPT is in the range of 15-40% based on the total weight of A.

. The agricultural microbial inoculant kit of paragraph 17, wherein the agriculturally beneficial Bacillus species has at least 2 hours of viability from the time when said lturally beneficial Bacillus species in Part A contacts said organic solvent in Part B. 26. The agricultural microbial inoculant kit of aph 17, wherein the weight percentage of the urease inhibitor is in the range of 0.02-80% based on the total weight of Part A. 27. The agricultural microbial inoculant kit of paragraph 17, wherein the agriculturally beneficial Bacillus species concentration is in the range of 1.0 x 10 2 to 1.0 x 1012 spores/mL after Part A is mixed with Part B. 28. A method for promoting plant growth, plant productivity and/or soil quality, wherein the method comprises applying an effective amount of the agricultural ial inoculant ition according to paragraph 1 to a plant, plant part, plant seed or soil. 29. A fertilizer composition comprising the agricultural microbial inoculant composition according to paragraph 1.

The term “comprising” as used in this specification and claims means “consisting at least in part of”. When interpreting statements in this specification, and claims which e the term “comprising”, it is to be understood that other features that are onal to the features prefaced by this term in each statement or claim may also be present. Related terms such as “comprise” and “comprised” are to be interpreted in similar manner.

In this ication where reference has been made to patent ications, other external documents, or other sources of information, this is generally for the e of ing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

In the description in this specification reference may be made to subject matter that is not within the scope of the claims of the current application. That subject matter should be readily fiable by a person skilled in the art and may assist in putting into practice the invention as d in the claims of this application.

Claims (25)

1. A composition comprising: i. a Bacillus species selected from the group consisting of us amyloliquefaciens, Bacillus licheniformis, Bacillus thuringiensis, Bacillus pumilus, and mixtures thereof; ii. an organic solvent selected from the group consisting of ene glycol (PG), N- methylpyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO), and mixtures f, wherein the organic solvent is present in an amount of 20 weight percent to 99 weight percent, based on the total weight of the composition; and iii. a urease inhibitor selected from the group consisting of N-(n-butyl)thiophosphoric triamide (NBPT), utyl)phosphoric triamide, thiophosphoryl triamide, phenyl phosphorodiamidate, N-cyclohexyl phosphoric triamide, N-cyclohexyl thiophosphoric de, phosphoric triamide, hydroquinone, p-benzoquinone, hexamidocyclotriphosphazene, ridines, thiopyrimidines, thiopyridine-N-oxides, N,N-dihaloimidazolidinone, N-halooxazolidinone, N-(2-nitrophenyl)thiophosphoric triamide, N-(2-nitrophenyl)phosphoric triamide, and mixtures thereof.

2. The ition of claim 1, wherein the Bacillus species is Bacillus licheniformis, Bacillus thuringiensis, or a ation thereof.

3. The composition of claim 1, wherein the Bacillus species is selected from the group of strains consisting of Bacillus amyloliquefaciens (ATCC 23842), Bacillus licheniformis (ATCC 14580), us licheniformis (B-642), Bacillus licheniformis (B-14368), us thuringiensis (ATCC 10792) , Bacillus thuringiensis (HD-17), Bacillus thuringiensis (HD-1), us pumilus (NRS- 272).

4. The composition of claim 1, wherein the urease inhibitor is N-(n-butyl)thiophosphoric triamide (NBPT).

5. The composition of claim 1, further comprising a nitrification inhibitor selected from the group consisting of 2-chlorotrichloromethylpyridine, 5-ethoxytrichloromethyl-1,2,4- thiadiazol, dicyandiamide, 2-aminochloromethyl-pyrimidine, 1,3-benzothiazolethiol, 4- amino-N-1,3-thiazolylbenzene sulfonamide, thiourea, guanidine, methylpyrazole phosphate, 2,4-diaminotrichloromethyltriazine, poly etherionophores, 4-amino-1,2,4- triazole, 3-mercapto-1,2,4-triazole, potassium azide, carbon ide, sodium trithiocarbonate, ammonium dithiocarbamate, 2,3,-dihydro-2,2-dimethylbenzofuranol methylcarbamate, N- (2,6-dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester, ammonium thiosulfate, 1- hydroxypyrazole, 3-methylpyrazolecarboxamide, 3-methylpyrazole, 3,5-dimethylpyrazole, 1,2,4-triazole, G77 Nitrification Inhibitor (CAS Registration N o. 13732567), and es thereof.

6. The composition of claim 5, wherein the nitrification inhibitor is dicyandiamide, G77 Nitrification Inhibitor (CAS Registration No. 13732567) or a combination thereof.

7. The composition of claim 1, wherein the urease inhibitor is t in an amount ranging from 10 weight % to 80 weight % based on the total weight of the ition.

8. The composition of claim 1, wherein the Bacillus s is viable for at least 2 hours from the time when saidl Bacillus species contacts said organic solvent.

9. The composition of claim 1, wherein the Bacillus species concentration is in the range of 1.0 x 102 spores/mL to 1.0 x 1012 spores/mL.

10. The ition of claim 1, further comprising a dye.

11. The ition of claim 1, comprising propylene glycol and N-methylpyrrolidone.

12. The composition of claim 11, wherein the propylene glycol is present in an amount ranging from 40 weight % to 70 weight %, the N-methylpyrrolidone is present in an amount ranging from 15 weight % to 40 weight %, the urease tor is NBPT, and the NBPT is present in an amount ranging from10 weight % to 30 weight % based on the total weight of the composition.

13. A fertilizer ition comprising the composition ing to claim 1, and a nitrogen source.

14. A method for promoting plant growth, plant productivity and/or soil quality, wherein the method comprises applying an effective amount of the composition according to claim 1 to a plant, plant part, plant seed or soil.

15. A kit comprising: Part A, wherein Part A comprises a urease inhibitor and an orga nic solvent, wherein said urease inhibitor is selected from the group consisting of N-(n-butyl)thiophosphoric triamide , N-(n-butyl)phosphoric triamide, osphoryl triamide, phenyl phosphorodiamidate, N-cyclohexyl phosphoric triamide, N-cyclohexyl thiophosphoric triamide, phosphoric triamide, hydroquinone, p-benzoquinone, hexamidocyclotriphosphazene, thiopyridines, thiopyrimidines, ridine-N-oxides, N,N-dihaloimidazolidinone, N-halooxazolidinone, N-(2-nitrophenyl)thiophosphoric triamide, N-(2-nitrophenyl)phosphoric triamide, and mixtures thereof; and wherein said organic solvent is selected from the group consisting of propylene glycol (PG), N- methylpyrrolidone (NMP), triethylene glycol monobutyl ether, glycerol, dimethyl sulfoxide (DMSO), and mixtures thereof; and Part B, n Part B comprises a Bacillus s selected from the group consisting of Bacillus iquefaciens, us licheniformis, us thuringiensis , Bacillus pumilus, and mixtures thereof, wherein each of Part A and Part B is contained in a separate co ntainer and wherein the organic solvent is present in an amount ranging from 20 weight % to 99 weight %, based on the combined weight of Part A and Part B.

16. The kit of claim 15, wherein Part B comprises a Bacillus species selected from the group consisting of Bacillus licheniformis, Bacillus thuringiensis .

17. The kit of claim 15, wherein Part B comprises a Bacillus strain selected from the group consisting of Bacillus amyloliquefaciens (ATCC 23842), Bacillus licheniformis (ATCC 14580), Bacillus licheniformis ), Bacillus licheniformis (B-14368), Bacillus thuringiensis (ATCC 10792) , Bacillus thuringiensis (HD-17), Bacillus thuringiensis (HD-1), Bacillus pumilus (NRS- 272).

18. The kit of claim 15, n the urease inhibitor is N-(n-butyl)thiophosphoric triamide (NBPT).

19. The kit of claim 18, wherein Part A ses propylene glycol and N-methylpyrrolidone.

20. The kit of claim 19, wherein the propylene glycol is present in an amount ranging from 40 weight % to 70 weight % based on the total weight of Part A, the N-methylpyrrolidone is present in an amount ranging from 15 weight % to 40 weight % based on the total weight of Part A, and the weight percentage of NBPT is present in an amount ranging from 10 weight % to 30 weight % based on the total weight of Part A.

21. The kit of claim 15, wherein the Bacillus species is viable for at least 2 hours from the time when said Bacillus species in Part A contacts said organic solvent in Part B.

22. A composition of any one of claims 1 to 12 substantially as herein described or exemplified.

23. A fertilizer composition of claim 13 substantially as herein described or ified.

24. A method of claim 14 ntially as herein described or exemplified.

25. A kit of any one of claims 15 to 21 substantially as herein described or exemplified.