Native diazotroph recruitment

Abstract

A soil treatment composition capable of recruiting native diazotrophs or other nitrogen fixing organisms to a desired soil site. In one example, a soil treatment composition includes a combination of plant growth promoting rhizobacteria (PGPR) and a nutrient enhancer for attracting native diazotrophs to a desired soil site. The treatment compositions may be mixed or otherwise blended in a tank prior to, or during, a spraying operation. Once mixed, the treatment compositions may be applied to a desired soil site via numerous applications, e.g., in-furrow, 2x2, sidedress, broadcast, or through fertigation. After being applied, such compositions may effectively recruit native diazotrophs which, in turn, may increase nutrient availability, uptake, and utilization.

Description

Attorney Docket No. 40014-1001W001NATIVE DIAZOTROPH RECRUITMENTRELATED APPLICATION

[0001] The present disclosure claims the benefit of U.S. Provisional Application 63 / 725,902, filed November 27, 2024, the entirety of which is incorporated herein by reference.TECHNICAL FIELD

[0002] This disclosure relates to the recruitment of diazotrophs or other nitrogen fixing organisms. More specifically, this disclosure relates to a composition and method for recruiting native diazotrophs or other nitrogen fixing organisms to a rhizosphere or desired soil site.BACKGROUND

[0003] Nitrogen is the plant nutrient needed in the largest amount to support healthy plants and most likely to be limiting to plant growth. To aid in the production of nitrogen (N), microbes known as diazotrophs, e.g., rhizobacteria, may reduce atmospheric dinitrogen gas (N2) to active, biologically available forms, e g., NH3, in a process called nitrogen fixation. (Pajares & Bohannan, 2016). For example, rhizobacteria, a type of diazotroph, are commonly found in soil, especially on the root nodules of leguminous plants, and depending on plant density and cropping system, can contribute 27-92% of total plant nitrogen uptake in legumes (van Kessel and Hartley, 2000; Peoples et al., 2012; Allito et al., 2020) and 57-600 kg of N / ha per annum (Sindhu et al., 2019).

[0004] Generally, nitrogen fixation is a putative mode of action postulated for plant growth promoting rhizobacteria (PGPR)-induced soil health and yield-related improvements. To detect a presence of nitrogen fixation in soil microbial communities, the nifH gene— encoding a key component of the nitrogenase enzyme — has been used as a reliable biomarker.

[0005] Beyond providing nitrogen, nitrogen supplied via nitrogen fixation has been shown to aid with the breakdown of crop residues to support a subsequent crop rotation; inducement ofAttorney Docket No. 40014-1001W001 nutrient, e.g., P, K, Ca, and Mg, uptake; and resistance to biotic and abiotic stresses. While advantageous to plant growth and vigor, many agricultural soils do not have sufficient diazotrophs in terms of number, quality, and effectiveness to generate sufficient nitrogen. Thus, over time, rhizobial strains have been isolated and incorporated into powdered, granular and liquid substrates to be applied by farmers to soils annually or as seed coatings.

[0006] However, the conditions required to keep these bacteria viable are not straight forward. For example, inoculant products require moist substrates (e.g., up to 62% w / w of dry weight basis) (Roughley and Vincent, 1967; Albareda et al., 2008; Denton et al., 2009) otherwise their viability quickly declines during transportation, storage, sowing, or as coatings on seeds as rhizobia are susceptible to desiccation (dehydration) (Stout et al., 1993; Mary et al., 1994; Deaker et al., 2012). In addition to the handling issues, and despite the uptake of these biological inoculants increasing, the high cost has prevented widespread uptake of these technologies globally. Therefore, what is needed are different treatment compositions and methods for recruiting native diazotrophs to a desired soil site.SUMMARY

[0007] The present disclosure sets forth a soil treatment composition capable of recruiting native diazotrophs or other nitrogen fixing organisms to a desired soil site. In one example, a soil treatment composition includes a combination of plant growth promoting rhizobacteria (PGPR) and a nutrient enhancer for attracting native diazotrophs to a desired soil site. Alternatively, in other examples, a soil treatment composition may include PGPR as a soluble concentrate in a water-based or oil -based solution. The treatment compositions may be mixed or otherwise blended in a tank prior to, or during, a spraying operation. Once mixed, the treatment compositions may be applied to a desired soil site via numerous applications, e.g., in-furrow, 2x2, sidedress, broadcast, or through fertigation. After being applied, such compositions may effectively recruit native diazotrophs which, in turn, may increase nutrient availability, uptake, and utilization. Further, it is to be understood that the disclosed treatment composition may be applied to any number of soil sites at numerous times during a growing season.Attorney Docket No. 40014-1001W001

[0008] The above summary is not intended to describe each illustrated embodiment or every implementation of the subject matter hereof. The figures and the detailed description that follow more particularly exemplify various examples.BRIEF DESCRIPTION OF THE DRAWINGSubject matter hereof may be more completely understood in consideration of the following detailed description of various examples in connection with the accompanying figures, in which:

[0009] Fig. 1 illustratively shows a treatment composition and soil site in accordance with one example;

[0010] Fig. 2 illustratively shows a method for recruiting native diazotrophs in accordance with one example;

[0011] Fig. 3 illustratively shows enrichment data of bacteria with nifH homologs after applying a treatment composition; and

[0012] Fig. 4 illustratively shows colony plate counts after applying a treatment composition. While various examples are amenable to various modifications and alternative forms, specifics thereof have been shown by way of example in the drawings and will be described in detail. It should be understood, however, that the intention is not to limit the claimed inventions to the particular examples described. On the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the subject matter as defined by the claims.DETAILED DESCRIPTION

[0013] FIG. 1 illustratively shows a treatment composition and soil site in accordance with one example. As shown, a treatment composition 102 may be applied to a soil site 124 with one or more plant(s) 118 to recruit native diazotroph(s) 122 to a rhizosphere of plant 118 or soil 120. While site 124 illustratively includes a singular plant 118 within soil 120, it is to be understood that composition 102 may be applied during various growing periods or cycles, e.g., VI, V2, V3,Attorney Docket No. 40014-1001W001V4.. . etc., with or without plant(s) 118. For example, composition 102 may be applied in-furrow along with plant seedlings in a furrow or trench, in a 2x2 ft-sq garden bed, as sidedressing to actively growing plants or crops, during broadcasting of seeds throughout a soil surface, or during fertigation to a root zone of plants. Plant 102 may take the form of vegetables, fruits, herbs, flowers, grains, or any other agricultural product that may benefit from native diazotroph recruitment and nitrogen fixation. Depending on a type of treatment site 124, it is to be understood that soil 120 may include any number of soils, e.g., loamy soils, clay loams, sandy loams, silt loams, silty clay loams, peat and muck soil, etc.

[0014] As shown, soil treatment composition 102 may include plant growth promoting rhizobacteria (PGPR) 104, a nutrient enhancer 112, and other components 116 for recruiting native diazotrophs or as further described infra. In an examples, beginning with PGPR 104, PGPR 104 may include a plurality of strains of bacteria, such as, for example, three different strains of bacteria in a water-based or oil-based solution, such as one strain of Bacillus licheniformis (e.g. 1 x 108cfu / mL) 106 and two or more strains of any combination of Bacillus velezensis / subtilis / methylotrophicus ( e.g. 2 x 108cfu / mL) 108. However, other Bacillus strains 110 may be added or substituted as well, e.g., Bacillus megaterium (1 x 108cfu / mL), Bacillus amyloliquefaciens (1 x 108cfu / mL), etc. In one example, PGPR 104 may be commercially available under the brand name BIOPATH and POWERCOAT. However, other commercial suppliers may be used as well.

[0015] By themselves, rhizobacteria 106 and 108 may serve to increase nutrient availability, uptake, and utilization to improve early season plant growth and vigor. Specifically, rhizobacteria 106 and 108 may include bioactives that produce enzymes and organic acids that improve a solubilization of liquid fertilizer nutrients into plant available forms and release bound nutrients, e.g., K, Ca, Mg, etc. More nutrient availability may lead to increased nutrient uptake, provide greater root volume, and improve overall plant growth and vigor. In turn, yield potential and harvest quality may be optimized. For example, a meta-analysis of over 119 field trials in the United States revealed an average yield increase of 4.22 bu / acre in maize treated with rhizobacteria 106 and 108 as compared to untreated controls.Attorney Docket No. 40014-1001W001 field-studies / ). Additionally, as noted above, inaddition to supplying a greater amount of nutrients, rhizobacteria 106 and 108 by themselves, or in combination with nutrient enhancer 112, may recruit native diazotrophs 122 to a plant rhizosphere or treatment site as discussed below.

[0016] Nutrient enhancer 112 may include food-derived carbon and plant-available nutrients. For example, nutrient enhancer 112 may be available under the brand name SYNERGY and include any number of nutrients. For example, nutrient enhancer 112 may include macronutrients nitrogen, phosphorus, potassium, magnesium, sulfur, and calcium and the micronutrients boron, chlorine, manganese, iron, nickel, copper, zinc, molybdenum, or any combination thereof. In one specific non-limiting example, enhancer 112 may include (all at example weight percent) secondary metabolites, e.g., betaine (0.23%) and polyphenols (0.5%); organic acids, e.g., lactic acid (0.4%) and amino acids (7.7%); carbon, e.g., soluble carbon (6%) and insoluble carbon (2%); micronutrients, e.g., calcium (650ppm), magnesium (2700 ppm), boron (4 ppm), iron (60 ppm), manganese (12 ppm), copper (2 ppm), and zinc (35 ppm), along with a nitrogen, phosphorus, and potassium (NPK) ratio of 1-1-1. Enhancer 112 may be derived from food-waste, e.g., uneaten or spoiled food such as pumpkins, bread, potatoes, fruits, vegetables, etc., and supplemented with potash, nitrogen, and phosphate during manufacture to obtain a NPK ratio of 1-1-1. In accordance with the present disclosure, it is to be understood that any type and combination of food-waste may be used to produce enhancer 112.

[0017] Moreover, treatment composition 102 may include other ingredients 116, e.g., fertilizers, complements, enhancers, etc., as well. For example, liquid urea-ammonium nitrate (UAN) or other liquid fertilizers may be added to composition 102 during, or prior to, a spraying operation. However, other nitrogen-based fertilizers, phosphorus-based fertilizers, potassium- based fertilizers, complete NPK fertilizers, etc. may be used as well.

[0018] To mix or otherwise blend PGPR 104 with nutrient enhancer 112, any number of mixing or blending protocols may be followed. In one example, PGPR 104 and enhancer 112 application rates may be 16 oz / acre and 32 oz / acre, respectively, yielding a blending ratio of 1 :2. However, other application rates and ratios may be used as well including 1 : 1, 1 :2, 1 :3, 1 :4; 1:5Attorney Docket No. 40014-1001W001 or more; 5: 1; 4: 1; 3: 1; 2: 1 or less, and anything therebetween. Blending or mixing may occur in any number of tanks, containers, or vessels prior to, or during, a desired spraying operation to produce treatment composition 102. For example, PGPR 104 and enhancer 112 may be blended in a 2 x 2.5-gal case, 275-gal tote. Alternatively, such components may be blended or mixed in a spraying device, e.g., boom sprayer, handheld sprayer, aerial sprayer, self-propelled sprayer, tractor sprayer, etc., storage container or tank.

[0019] Once blended or mixed, composition 102 may be added to treatment site 124 in accordance with any of the methods set forth above, e.g., in-furrow, sidedressing, broadcasting, etc. Given the vast array of options, any number of application protocols may be followed. For example, for starter / at plant applications, 16 to 32 fl oz of composition 102 may be applied per acre. As sidedress or topdress, 16 to 32 fl oz of composition 102 may be applied per acre and, for broadcast or manure, 64 fl oz of composition 102 may be applied per acre. However, depending on a desired application, additional amounts may be applied as well. Once applied, composition 102 may operate to recruit native diazotrophs to a desired soil site.

[0020] Fig. 2 illustratively shows a method for recruiting native diazotrophs to a desired soil site in accordance with one example. As shown, method 200 begins at block 202 where ingredients are gathered for producing a native diazotroph recruitment composition. Ingredients may include PGPR 204, nutrient enhancer(s) 206, and other ingredients 208. PGPR 204 may include one strain of Bacillus licheniformis (1 x 108cfu / mL) and two strains of Bacillus velezensis (2 x 108cfu / mL). Nutrient enhancer 206 may include a variety of food-derived carbon and plant-available nutrients such as secondary metabolites, betaine (0.23%) and polyphenols (0.5%); organic acids, lactic acid (0.4%) and amino acids (7.7%); carbon, soluble carbon (6%) and insoluble carbon (2%); micronutrients, calcium (650 ppm), magnesium (2700 ppm), boron (4 ppm), iron (60 ppm), manganese (12ppm), copper (2 ppm), and zinc (35 ppm), with a NPK ratio of 1-1-1. Finally, additional components 208, e.g., liquid fertilizer(s), enhancers, complements, etc., may be included as well. Liquid fertilizers 208 may include liquid ureaammonium nitrate (UAN) or other nitrogen-based fertilizers, phosphorus-based fertilizers, potassium-based fertilizers, complete NPK fertilizers, etc.Attorney Docket No. 40014-1001W001

[0021] Next, method 200 proceeds to block 210 where the gathered ingredients are blended or otherwise mixed to form a native diazotroph recruitment composition. Depending on a desired quantity, any sized or shaped tank 212, container 214, or other vessel 216 may be used. In one example, a nutrient enhancer may be blended with a PGPR and a liquid fertilizer during, or prior to, a spraying operation. Once blended or mixed, the diazotroph recruitment composition may be stored, e.g., in a 2 x 2.5-gal case, 275-gal tote, etc., transported, or immediately applied, e g., via in-furrow 220, 2x2 222, sidedress 224, broadcast 226, fertigation 228, etc. 230, to any number of treatment sites. In turn, native diazotrophs may be recruited to a rhizosphere or other soil site

[0022] In some embodiments according to the disclosure, a composition may comprise a combination of PGPR 104 and enhancer 112, a combinations of PGPR 104, or each strain of PGPR 104 and enhancer 112 each in a range from about 0.01 mg per liter (L) to about 100 g per liter of the composition. Thus, in some embodiments, a composition of the disclosure may comprise the above alternatives (a combination of PGPR 104 and enhancer 112, a combinations of PGPR 104, or each strain of PGPR 104 and enhancer 112 individually) in a range from about 0.01 mg / L to about 1 mg / L, about 0.01 mg / L to about 10 mg / L, about 0.01 mg / L to about 100 mg / L, about 0.01 mg / L to about 1 g / L, about 0.01 mg / L to about lOg / L, about 0.01 mg / L to about 100 g / L, about 0.01 g / L to about 1 g / L, about 0.01 g / L to about 5 g / L, about 0.01 g / L to about 10 g / L, about 0.1 g / L to about 15 g / L, about 0.01 g / L to about 20 g / L, about 0.01 g / L to about 30 g / L, about 0.01 g / L to about 40 g / L, about 0.01 g / L to about 100 g / L, about 0.5 g / L to about 1 g / L, about 0.5 g / L to about 5 g / L, about 0.5 g / L to about 10 g / L, about 0.5 g / L to about 20 g / L, about 0.5 g / L to about 30 g / L, about 0.5 g / L to about 40 g / L, about 0.5 g / L to about 50 g / L, about 0.5 g / L to about 100 g / , about 1 g / L to about 5 g / L, about 1 g / L to about 10 g / L, about 1 g / L to about 15 g / L, about 1 g / L to about 20 g / L, about 1 g / L to about 30 g / L, about 1 g / L to about 40 g / L, about 1 g / L to about 50 g / L, about 1 g / L to about 100 g / L, about 5 g / L to about 10 g / L, about 5 g / L to about 15 g / L, about 5 g / L to about 20 g / L, about 5 g / L to about 30 g / L, about 5 g / L to about 40 g / L, 1, about 5 g / L to about 50 g / L, about 5 g / L to about 100 g / L, about 10 g / L to about 15 g / L, about 10 g / L to about 20 g / L, about 10 g / L to about 30 g / L, about 10 g / L to about 40 g / L, about 10 g / L to about 50 g / L, about 10 g / L to about 100 g / L, about 15 g / L to aboutAttorney Docket No. 40014-1001W00120 g / L, about 15 g / L to about 30 g / L, about 15 g / L to about 40 g / L, about 15 g / L to about 50 g / L, about 15 g / L to about 100 g / L, about 20 g / L to about 30 g / L, or about 20 g / L to about 40 g / L, about 20 g / L to about 50 g / L, about 20 g / L to about 100 g / L, about 30 g / L to about 40 g / L, about 30 g / L to about 50 g / L, , about 30 g / L to about 100 g / L, about 40 g / L to about 50 g / L or about 40 g / L to about 100 g / L of the composition, or any value or range therein.

[0023] Other components 116 may be included, such as, for example, including, but not limited to, a surfactant, a humectant, an adjuvant, an antioxidant, a preservative, a plant macronutrient, a plant micronutrient, a plant growth regulator, a pesticide, a fungicide, an antiviral, an anti-bacterial, an herbicide, or any combination thereof.

[0024] Example surfactants can include, but are not limited to, alkali metal, alkaline earth metal and ammonium salts of ligno-sulfonic acid, naphthalenesulfonic acid, phenolsulfonic acid, dibutylnaphthalenesulfonic acid, alkylarylsulfonates, sodium dodecyl sulfate, alkyl sulfates, alkyl sulfonates, fatty alcohol sulfates, fatty acids and sulfated fatty alcohol glycol ethers, of sulfonated condensates naphthalene and naphthalene derivatives with formaldehyde, condensates of naphthalene or of naphthalenesulfonic acid with phenol and formaldehyde, polyoxyethylene octyl-phenyl ether, ethoxylated isooctylphenol, octylphenol, nonylphenol, alkylphenyl polyglycol ethers, tributylphenyl polyglycol ether, tristearylphenyl polyglycol ether, alkylaryl polyether alcohols, alcohol and fatty alcohol / ethylene oxide condensates, ethoxylated castor oil, polyoxyethylene alkyl ethers, ethoxylated polyoxypropylene, lauryl alcohol, polyglycol ether acetal, sorbitol esters, lignin-sulfite waste liquors and / or methylcellulose.

[0025] In some embodiments, a surfactant may be present in an amount from about 0.1% to about 40% w / w of the composition. In some embodiments, a surfactant may be present in an amount from about 0.1% to about 1%, about 1% to about 10%, about 10% to about 20%, about 10% to about 25%, about 10% to about 30%, about 10% to about 35%, about 15% to about 20%, about 15% to about 25%, about 15% to about 30%, about 15% to about 35%, about 15% to about 40%, about 20% to about 25%, about 20% to about 30%, about 20% to about 35%, about 20% to about 40%, about 25% to about 30%, about 25% to about 35%, about 25% to about 40%, about 30% to about 35%, about 30% to about 40%, about 35% to about 40% w / w of the composition orAttorney Docket No. 40014-1001W001 any range or value therein). Thus, in some embodiments, the surfactant may be present in a composition in an amount of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% w / w of the composition or any range or value therein.

[0026] Example humectants can include, but are not limited to, glycerol, sorbitol, xylitol, maltitol, glyceryl triacetate, sodium lactate, urea formaldehyde, propylene glycol, ethylene glycol and / or fatty acids. An example antioxidant can include, but is not limited to, ascorbic acid, tocopherols, propyl gallate, tertiary butylhydroquinone, butylated hydroxyanisole, and / or butylated hydroxytoluene.

[0027] An example preservative can include, but is not limited to, sorbic acid, sodium sorbate, sorbates, benzoic acid, sodium benzoate, benzoates, hydroxybenzoate and derivatives, sulfur dioxide and sulphites, nitrite, nitrate, lactic acid, propionic acid and sodium propionate, tocopherol, plant extract, hops, salt, sugar, vinegar, alcohol (e.g. methanol and ethanol), diatomaceous earth and castor oil, citric acid, ascorbic acid, sodium ascorbate, phenol derivatives (butylated hydroxytoluene, butylated hydroxyanisole, BHA, BHT, TBHQ and propyl gallate), gallic acid, sodium gallate, sulfur dioxide, sulphites, tocopherols, and / or methylchloroisothiazolinone, l,2-Benzisothiazolin-3-one (BIT), Hexahydro- 1,3, 5 -tri s- hydroxyethyl -s-triazine (HTHT), 5-chloro-2-methyl-2H-isothiazol-3-one (CMIT), 2-methyl-2H- isothiazol-3-one (MIT), Zinc pyrithione (ZPT), 2-Bromo-2-nitropropane- 1,3 -diol (Bronopol), Formaldehyde, l,3-Dimethylol-5,5-dimethylhydantoin (DMDMH), 2,2-Dibromo-3- nitrilopropionamide (DBNPA), and / or Poly (hexamethylene biguanide) hydrochloride (PHMB).

[0028] In some embodiments, a preservative may be present in an amount ranging from about 0.001% to about 5% w / w or any range or value therein. In some embodiments, a composition may comprise a preservative in an amount ranging from about 0.001% to about 0.1%, about 0.001% to about 0.5%, about 0.001% to about 1%, about 0.001% to about 2%, aboutAttorney Docket No. 40014-1001W0010.001% to about 3%, about 0.001% to about 4%, about 0.01% to about 0.1%, about 0.01% to about 0.5%, about 0.01% to about 1%, about 0.01% to about 2%, about 0.01% to about 3%, about 0.01% to about 4%, about 0.01% to about 5%, about 0.05% to about 0.1%, about 0.05% to about 0.5%, about 0.05% to about 1%, about 0.05% to about 2%, about 0.05% to about 3%, about 0.05% to about 4%, about 0.05% to about 5%, about 0.1% to about 0.5%, about 0.1% to about 1%, about 0.1% to about 2%, about 0.1% to about 3%, about 0.1% to about 4%, about 0.1% to about 5%, about 0.5% to about 1%, about 0.5% to about 2%, about 0.5% to about 3%, about 0.5% to about 4%, about 0.5% to about 5%, about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 2% to about 3%, about 2% to about 4%, about 4% to about 5% , about 3% to about 4%, about 3% to about 5%, about 4% to about 5% w / w of the composition, or any range or value therein. Thus, in some embodiments, the preservative may be present in a composition comprising the microorganism C. tofieldiae and / or extracts from said microorganism and / or filtrates from said microorganism in an amount of about. 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% w / w of the composition or any range or value therein.

[0029] Example plant growth regulators include, but are not limited to, auxin (including but not limited to naphthalene acetic acid (NAA) and / or indole-3 -butyric acid (IBA) and / or indole-3- acetic acid (IAA, 3-IAA)), cytokinin, abscisic acid, gibberellin, ethylene, salicylic acid, jasmonic acid, brassinosteriod (e g., brassinolide), or any combination thereof. Example pesticides include, but are not limited to, malathion, parathion, methyl parathion, chlorpyrifos, diazinon, dichlorvos, phosmet, fenitrothion, tetrachlorvinphos, azamethiphos, fenvalerate, cyfluthrin, lambda- cyhalothrin, zeta-cypermethrin, permethrin, piperonyl butoxide, imidacloprid, acetamiprid, clothianidin, nitenpyram, nithiazine, thiacl oprid, thiamethoxam, ryanodol, 9,21- didehydroryanodol, chlorantraniliprole, flubendiamide, and / or cyantraniliprole. Example fungicides include, but are not limited to, prothioconazole, trifloxystrobin, azoxystrobin, propiconazole, and / or pyraclostrobin. Example anti-bacterials (bactericides) include, but are notAttorney Docket No. 40014-1001W001 limited to, methylisothiazolinone, chloromethylisothiazolinone, benzisothiazolinone, octylisothiazolinone, dichlorooctylisothiazolinone, and / or butylbenzisothiazolinone. Example herbicides can include, but are not limited to, glyphosate, 2,4-dichlorophenoxyacetic acid, atrazine, S-metolachlor, and / or 3, 6-dichloro-2-methoxybenzoic acid.

[0030] In some embodiments, a composition may further comprise an antifoaming agent. Any antifoaming agent for use with agricultural and / or food products may be used. Example antifoaming agents include, but are not limited to, long chain unsaturated fatty acids including, but not limited to Cl 2 to Cl 4, Cl 8.1 and Cl 8'2 unsaturated fatty acids, and / or synthetic polysiloxanes (silicones) including, but not limited to, polydimethylsiloxane, and / or hydrophobic silica. In some embodiments, a composition may comprise an amount of antifoaming agent in a range from about 0.0001% to about 0.05% w / w of the composition or any range or value therein. Thus, in some embodiments, the antifoaming agent may be present in the composition in an amount of about 0.0001%, 0.0002%, 0.0003%, 0.0004%, 0.0005%, 0.0006%, 0.0007%, 0.0008%, 0.0009%, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, or 0.05% w / w of the composition or any range or value therein.

[0031] In some embodiments, a composition may further comprise a biocide. Any biocide for use with agricultural and / or food products may be used. When included in the composition, a biocide may be present at about 0.1 g L'1to about 20 g L'1of the composition, or any range or value therein. Thus, in some embodiments, the biocide may be present in the composition in an amount of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 g per liter of the composition, or any range or value therein.Experimental Data

[0032] To demonstrate native diazotroph recruitment and the functional impact of the observed soil microbiome changes, the presence of nifH homologs, encoding a key component of the nitrogenase enzyme responsible for nitrogen fixation, was measured in bacteria significantlyAttorney Docket No. 40014-1001W001 induced within the microbiome of the treated soils compared to untreated soils (see Fig. 3). Treated soils were obtained from Deuel, South Dakota; Hamilton, Indiana; and York, Nebraska. In these trials, PGPR (BioPath®) and nutrient enhancer (Synergy®) were mixed with UAN and immediately applied as a knifed-in side dress to soils during a V6 spraying operation. In turn, the presence of nifH homologs was measured and compared for treated and untreated soils. As shown in the middle column, there were notable increases in the relative abundance of bacteria enriched with nifH homologs for treatment compositions including PGPR and a nutrient enhancer. Namely, soil treated with the composition of PGPR and a nutrient enhancer showed a relative abundance of bacteria with nifH homologs 10-15% greater than untreated soil and sequence read counts of 163736 x 106.

[0033] Alternatively, a treatment composition of PGPR, by itself, showed effective as well. Specifically, as shown in the left column, the relative abundance of bacteria with nifH homologs was over 30% greater as compared to untreated soil with sequence read counts of 38 x 106. For comparison sake, a treatment composition of a nutrient enhancer, by itself, did not show effective with a relative abundance between 0 and 5% (see right column).

[0034] This trend is further evidenced in Fig. 4 which illustratively shows colony plate counts on Burk’s growth medium for nitrogen fixing microbes. Generally, higher colony counts are correlated to an abundance of culturable nitrogen fixing microbes in a soil sample. In this study, maize was treated with low- and medium-concentration fertilizer in combination with PGPR and a nutrient enhancer in agricultural soil grown in a greenhouse. As shown in columns b and c, a nutrient enhancer and PGPR significantly increased the number of nitrogen fixing microbes recovered from soil around the rhizosphere compared to the control at both low and medium fertilizer levels.

[0035] Various examples of assemblies, devices, and methods have been described herein. These examples are given only by way of example and are not intended to limit the scope of the claimed inventions. It should be appreciated, moreover, that the various features of the examples that have been described may be combined in various ways to produce numerous additional examples. Moreover, while various materials, dimensions, shapes, configurations and locations,Attorney Docket No. 40014-1001W001 etc. have been described for use with disclosed examples, others besides those disclosed may be utilized without exceeding the scope of the claimed inventions.

[0036] Persons of ordinary skill in the relevant arts will recognize that the subject matter hereof may comprise fewer features than illustrated in any individual embodiment described above. The examples described herein are not meant to be an exhaustive presentation of the ways in which the various features of the subject matter hereof may be combined. Accordingly, the examples are not mutually exclusive combinations of features; rather, the various examples can comprise a combination of different individual features selected from different individual examples, as understood by persons of ordinary skill in the art. Moreover, elements described with respect to one embodiment can be implemented in other examples even when not described in such examples unless otherwise noted.

[0037] Although a dependent claim may refer in the claims to a specific combination with one or more other claims, other examples can also include a combination of the dependent claim with the subject matter of each other dependent claim or a combination of one or more features with other dependent or independent claims. Such combinations are proposed herein unless it is stated that a specific combination is not intended.

[0038] Any incorporation by reference of documents above is limited such that no subject matter is incorporated that is contrary to the explicit disclosure herein. Any incorporation by reference of documents above is further limited such that no claims included in the documents are incorporated by reference herein. Any incorporation by reference of documents above is yet further limited such that any definitions provided in the documents are not incorporated by reference herein unless expressly included herein.

[0039] For purposes of interpreting the claims, it is expressly intended that the provisions of 35 U.S.C. § 112(f) are not to be invoked unless the specific terms “means for” or “step for” are recited in a claim.

Claims

Attorney Docket No. 40014-1001W001CLAIMSWhat is claimed is:

1. A soil treatment composition for recruiting native diazotrophs comprising: a plurality of strains of plant growth promoting rhizobacteria; and a nutrient enhancer comprising food-derived carbon and plant-available nutrients.

2. The composition of claim 1, wherein the plurality of strains plant growth promoting rhizobacteria comprises a water-based or oil-based solution.

3. The composition of claim 1, wherein the plurality of strains of plant growth promoting rhizobacteria are from the genus Bacillus.

4. The composition of claim 3, wherein the plurality of strains comprises at least two strains selected from the group consisting of: Bacillus licheniformis, Bacillus velezensis, Bacillus subtilis, Bacillus methylotrophicus, Bacillus megaterium, and Bacillus amyloliquefaciens.

5. The composition of claim 4, wherein the plurality of strains comprises one strain of Bacillus licheniformis and two or more strains of any combination of Bacillus velezensis, Bacillus subtilis, and Bacillus methylotrophicus .

6. The composition of claim any of the preceding claims, wherein plant available nutrients of the nutrient enhancer comprises nitrogen, phosphorus, potassium, magnesium, sulfur, calcium, boron, chlorine, manganese, iron, nickel, copper, zinc, molybdenum, or any combination thereof.

7. The composition of claim 7, wherein the food-derived carbon and plant available nutrients comprise secondary metabolites, organic acids, soluble carbon sources, insoluble carbon sources, or combinations thereof.Attorney Docket No. 40014-1001W0018. The composition of claim 7, wherein the secondary metabolites are selected from betaine, polyphenols, and combinations thereof.

9. The composition of claim 7, wherein the organic acids are selected from lactic acid and amino acids, and combinations thereof.

10. The composition of claim 6, wherein the plant available nutrients include nitrogen, phosphorus, and potassium in an NPK ratio of 1-1-1.11 . The composition of any of the preceding claims, wherein the food-derived carbon comprises food-waste from uneaten or spoiled food including one or more of pumpkins, bread, potatoes, fruits, and vegetables.

12. The composition of claim any of the preceding claims, further comprising a surfactant, a humectant, an adjuvant, an antioxidant, a preservative, a plant macronutrient, a plant micronutrient, a plant growth regulator, a pesticide, a fungicide, an antiviral, an anti-bacterial, an herbicide, an anti-foaming agent, or combinations thereof.

13. A method for recruiting native diazotrophs to a desired soil site comprising: providing a composition comprising a plurality of strains of plant growth promoting rhizobacteria, and a nutrient enhancer comprising food-derived carbon and plant-available nutrients; and applying or providing instructions to apply the composition to the desired soil site for recruiting native diazotrophs and increasing yield of a crop or plant planted in the desired soil site.

14. The method of claim 13, wherein application of the composition to the desired soil site comprises in-furrow application along with plant seedlings in a furrow or trench, in a gardenAttorney Docket No. 40014-1001W001 bed, as sidedressing to actively growing plants or crops, during broadcasting of seeds throughout a soil surface, or during fertigation to a root zone of plants.

15. The method of claim 13 or 14, wherein the soil site comprises loamy soils, clay loams, sandy loams, silt loams, silty clay loams, peat and muck soil, or combinations thereof.

16. The method of claim 13 or 14, wherein the plurality of strains of plant growth promoting rhizobacteria are from the genus Bacillus.

17. The method of claim 16, wherein the plurality of strains comprises at least two strains selected from the group consisting of: Bacillus licheniformis, Bacillus velezensis, Bacillus subtilis, Bacillus methylotrophicus, Bacillus megaterium, and Bacillus amyloliquefaciens.

18. The method of claim 17, wherein the plurality of strains comprises one strain of Bacillus licheniformis and two or more strains of any combination of Bacillus velezensis, Bacillus subtilis, and Bacillus methylotrophicus.

19. The method of any of claims 13-18, wherein the plant available nutrients of the nutrient enhancer comprises nitrogen, phosphorus, potassium, magnesium, sulfur, calcium, boron, chlorine, manganese, iron, nickel, copper, zinc, molybdenum, or any combination thereof.

20. The method of claim 19, wherein the food-derived carbon and plant available nutrients comprise secondary metabolites, organic acids, soluble carbon sources, insoluble carbon sources, or combinations thereof.

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