AU2015205819B2 - Bradyrhizobium strains - Google Patents

Abstract

According to the present invention new isolates of Bradyrhizobium japonicum have been isolated and possess unique properties. These Bradyrhizobia are plant growth promoting rhizobacterium (PGPR), possess superior tolerance/resistance to 5 desiccation, and enhance the overall performance of leguminous plant growth.

Description

BRAD YRHIZOBIUM STRAINS

The present application is a divisional application of Australian Application No. 2012351899, which is incorporated in its entirety herein by reference.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

This application contains a reference to a deposit of biological material, which deposit is incorporated herein by reference. For complete information see Table 1.

FIELD OF THE INVENTION

The present invention relates to isolated Bradyrhizobium bacterium having enhanced characteristics, including but not limited to, enhanced desiccation resistance.

BACKGROUND OF THE INVENTION

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

In order to maintain healthy growth, plants must extract a variety of elements from the soil in which they grow. These elements include nitrogen and the so-called micro-nutrients {e.g., copper, iron and zinc), but many soils are deficient in such elements or they contain them only in forms which cannot be readily taken up by plants (it is generally believed that essential elements cannot be readily taken up by plants unless they are present in dissolved form in the soil). Nitrogen is an essential element for most plants as it plays a role in the synthesis of amino acids, proteins, nucleotides, nucleic acids, chlorophyll, co-enzymes and in the overall growth and health of the plant. To counteract such deficiencies, sources of the deficient elements are commonly applied to soils in order to improve growth rates and yields obtained from crop plants. For example, nitrate and/or ammonium are often added to soil to counteract a lack of available nitrogen.

In the field of crop science, it is well known that many cultivated crops require that the soil provide relatively large amounts of nitrogen to the plant. The notable exceptions to those plants requiring nitrogen via the soil are plants from the legume family.

Specifically, leguminous plants are unique from non-leguminous plants in their ability to fix atmospheric nitrogen into ammonia. The ability to fix atmospheric nitrogen into a useable nitrogen source for the plant obviates the need for the plant to obtain nitrogen from the soil. Nitrogen fixation, however, requires a symbiotic relationship between the leguminous plant and native bacterial within the soil. One of the most extensively studied partners in this symbiotic relationship is bacteria belonging to the genus Bradyrhizobium or Rhizobium. Gresshoff, P. (1999). Identification of Plant Genes Involved in Plant-Microbe Interactions. Stacey, G. & Keen, T. (Ed.), Plant-Microbe Interactions (4th ed.) (Ch. 6). St. Paul: APS Press.

Symbiosis is generally achieved through an exchange of complex bidirectional signaling between the plant and the microbe and the microbe and the plant. Typically, plant factors, such as flavonoids and flavonoid like substances, induce colonization of the bacteria into the root nodule of the leguminous plant (Gresshoff, 1999). Once the bacteria have colonized the root nodule, the bacteria effect morphological changes in the plant, namely root hair curling and the development of a new root organ - the nodule (Gresshoff, 1999). The nodule permits the establishment of a new physiological environment for the nodule inducing bacteria to differentiate into a nitrogen-fixing endosymbiont, or bacteriod, for the colonized plant (Gresshoff, 1999).

In order to assist with the symbiotic exchange of bi-directional signaling between the plant and microbe, bacteria, such as Bradyrhizobia sp., are often coated on a seed. To prolong the viability of the microbe on the seed, it is desirable that the microbe be tolerant to desiccation and dry environmental conditions generally.

There remains a need for microbes with enhanced desiccation resistance.

Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

SUMMARY OF THE INVENTION

According to a first aspect, the invention provides a method of treating a seed, comprising applying to said seed an inoculum that comprises at least one mutagenized Bradyrhizobium japonicum strain having the deposit accession number NRRL B-50612.

Described herein are novel bacterial strains having enhanced desiccation resistance, especially when the novel strains are compared to its parental strain, e.g., Bradyrhizobium sp., parental strain USDA 532C. The inventors have isolated and tested a significant number of bacterial strains for their desiccation resistance properties.

As disclosed throughout, the isolated strains are strains of the genus Bradyrhizobium spp. In particular, the isolated strains are strains of Bradyrhizobium japonicum. Even more particularly, the isolated strains are isolated Bradyrhizobium japonicum strains selected from the group consisting of: the strain having the deposit accession number NRRL B-50608; the strain having the deposit accession number NRRL B-50609; the strain having the deposit accession number NRRL B-50610; the strain having the deposit accession number NRRL B-50611; the strain having the deposit accession number NRRL B-50612, or a combination of at least two or more of the above deposited strains.

Also described herein are compositions comprising a carrier and one or more of the bacterial strains described herein. In an embodiment, the composition comprises one or more plant signal molecules. In one embodiment, the composition comprises at least one lipo-chitooligosaccharide (LCO). In another embodiment the composition comprises at least one chitooligosaccharide (CO). In still another embodiment, the composition comprises at least one flavonoid. In still yet another embodiment, the composition comprises jasmonic acid or a derivative thereof. In another embodiment, the composition comprises linoleic acid or a

'derivative thereof. In yet another embodiment,/the'- oi^pesiitoGCQffipdses iinolenioadd or a derivative thereof. In still yet another embodiment:, the 'composition' comprises a karnkin.

Farther described herein is a: method for enhancing the growth of a plant or plant part composing contacting a plant or plant: part; with one; or mom with one or more of the bacterial strains described herein,: The method comprises introducing: into the soil an inpouium of one or more of the bacterial strains described herein. In another embodiment, the method comprises introducing Into the soil an inoculum of one or more of the bacteria! strains as a seed coating,

Aisp described herein is a method for enhancing nitrogen fixation in; a plant(s) com prising growing a piantfs)' in a soil that contains a one or more of the bacteria! strai ns: described herein. In one embodiment,: the plant(s) is a leguminous ptantis), nomieguminbus p!ahf(s)j dr combinations thereof, in another embodiment, the plant le a plant selected from the group;consisting of soybean, been:,: alfalfa, dover, com, lettuce tomatoes, potatoes, cucumbers, and combinations thereof,

Further described herein are seeds coated with the bacteria! strains described herein, BRIEF DESCRIPTION OF DRAWINGS

Fig, 1 is a graphical represenfation of the preliminary screening of desiccation resistant mutants compared when compared to the desiccation Fesisianee of parental strain USDAS32G.

Fig, 2 is a graphical representation of the second screening of desiccation resistant mutants compared when compared to the desiccation resistance of parental strain USDA S32C,

Fig. 3 is a graphical representation of the third screening of desiccation resistant mutants compared when compared to the desiccation resistance of parental strain USDA 532C.

Fig, 4 is a graphicai representation of the fourth screening of desiccation resistant mutants compared when compared to the desiccation resistance of parental strain USDA S32C.

Fig, 5 is a bar graph representation of the desiccation resistance of the selected desiccation resistant mutants compared when compared to the desiccation resistance of parental strain USDA 532C at zero (0} and fourteen (14) days.

Fig, 6 Is a bar graph representation of the desiccation resistance of the seiected desiccation resistant mutants compared when compared to the desiccation resistance of parental strain USDA 532C at seven (7) and fourteen (14} days.

DETAILED DESCRIPTION OF THE INVENTION

The disclosed bacterial strains have been Isolated and tested for their ability to solubilize phosphorous. This is described in detail in the "Examples'' section provided below. The disclosed embodiments further relate to compositions, seed coatings, methods for Increasing the availability of phosphorus for plant uptake from soil, and methods for increasing the phosphorus uptake In plants comprising growing the plants in a soil oontaining a phosphoms source.

Definitions:

As used herein, the singular forms "a”, "an* and "the” are intended to include the plural forms as well unless the context dearly indicates otherwise.

As used herein, the term "bioiegicaily pure culture* Is intended to mean a culture essentially free from biological Contamination and hiving a genetic uniformity such that different subcultures taken therefrom will display substantially identical genotypes and phenotypes (e.g,, cultures have a purity of at least 60%, of at least 65%, at least 70%, at least 75%, at least 80%, at ieast 85%, at least 50%, at feast 91%,-at 1088192%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%. at least 99%, up to 100% pure).

As used herein, the term “isolate, isolates, isoiating, and/or isolated, etc.'- is intended to mean that the referenced material is removed from the environment in which St is normally found.

As used herein, the term "inoculum* Is intended to mean any form of bacterial cells, or spores, which is capable of propagating on or In the soli when the conditions of temperature, moisture, etc., are favorable for bacterial growth. : As used herein, the terms “spore" has: Its normal meaning which is well known and understood by those of skill in the art and generally refers to a microorganism in its dormant, protected state.

As used herein, the term *source” of a particular element is intended to: mean a: compound of that element which, at least in the soil conditions under consideration, does nof make the element fully available for plant uptake.

As used herein, the terms “effective amount*, “effective concentration8, or “effective dosage" is intended to mean the amount, concentration, or dosage of the one or more baeterlai isolates sufficient to cause an increase in the growth of a giant or plant part or an increase in nitrogen fixation. The actual effective dosage in absolute value depends on factors including, but not limited to, the sice (e.g., the area, the total acreage, etc,) of the land for application with the bacterial isolates. Synergistic or antagonistic interactions between the other active or inert ingredients which may Increase or reduce activity of the bacterial isolates, and the stability of the baoferiai isolates in compositions and/or as seed treatments. The “effective amounf, 'effective concentration*., or ''effective dosage” of the bacterial composition may be determined, eg., by a routine dose response experiment.

As used herein, the terms ''carrier* or"agronomfcaily acceptable carrier" are intended to refer to any material' which can be used to deliver the actives (e,g., a bacteria! strain) to a seed, soil, plant or plant pad.

As used herein, the term "soiTcompatihle carrier” js: intended: to refer to any material which can be added to a soli without: eausing/bavlog an, adverse effect on plant growth, soil structure, soil drainage,, Or the Ike,

As used herein, the is intended ;tp refer to any material which can be added to a seiad'Vi^^ ah adverse effect on the seed, the plant that grows from the seed, seed germination, or the like.

As used herein, the term “agriculturally beneficial ingredients)" is intended ίο mean any agent or combination of agents capable of causing or providing a beneficial and/or useful effect in agriculture.

As used herein, “at least one bioiogicaiiy active ingredient” is intended to mean bioiogicaily active ingredients (e.g., signal molecules. other microorganisms, etc, ) other than-the·' one or more bacteriai isolates described herein.

As used herein, the toon “desiccation" is intended to mean a state of extreme dryness, conditions without moisture and/or water. The terms “desiccation resistance" and/or “desiccation tolerance" are intended to encompass the ability of an organism to withstand and/or survive and/or endure conditions of extreme dryness.

As used herein, terms "nitrogen fixation", “fixation of atmospheric nitrogen", or "nitrogen fixing";, etc, are: intended to encompass biological processes in which molecular nitrogen; or nitrogen in the atmosphere Is converted into one or more nitrogenous p) compounds, including but not limited to, ammonia, ammonium salts, urea, add nitrates.

As used herein, the term “nitrogen fixing organism" is intended to refer to any organism: (e,g., diazOtrpphs) capable of converting molecular nitrogen or nitrogen in the atmosphere into one or more hitrogenOyl (N) cornpoundS:, including but not limited to, ammonia, ammonium;: salts, urea, and nitrates.

As used herein terms ’’phosphate solubilization", or "phosphate solubilizing*,: etc. are intended to mean the conversion of insoluble phosphate rock pbosphate, etc.) iiitb a soluble phosphate form.

As used herein, the term "phosphate solubilizing organism” is intended to refer to any organism capable of converting Insoluble phosphate into a soluble phosphate form.

As used herein, the terms "pianifsf and "plant parifs)" are intended to refer to ail plants ami plant populations such as desired and undesired wild plants or crop plants (inciudlng 'naturally occurring crop plants). Crop plants can be plants, which can be obtained by conventional plant breeding and. ^optimization· .methods, or by biotechnological and genetic engineering methods or by combinations of these methods, including the transgenic plants and including the plant cuftivars protectable or not protectable by plant breeders' rights. Plant parts are to be understood as meaning ali parts and organs of plants above and below the ground, such as shoot, leaf, flower and root, examples which may be mehtioned being leaves, needles, siaifcs, stems, flowers:, fruit bodies* fruits, seeds, roots, nodules, tubers,. add: rhizomes. The plant parts also include harvested materiel and .vegetative and .generative propagation material; (e.g., cuttings, tubers, rhizomes, off-shoots and seeds, etc,).

As used herein, the term module1'' is intended to include, but not be limited to, determinate nodules, Indeterminate noddles, or a combination thereof. Examples of deiefmihat© nodules and indeterminate nodules are well known In the art and described in Denison, R, R:e 2000, The Amen Naturalist 156:(6): 597-576, Determinate nodules are found on Glycine, Lotus, or Phaseeius species and are round and/spherical in shapes (Denison, 2000), Determinate nodules grow only for a limited period of time — '-typically a few weeks. (Denison, 2060) In contrast to determinate nodules, indeterminate nodules are found on Medicago, TrtfoMum, and Pmum species, have an elongated shape and grow continuously, (Denison, 2000) The term “nodule occupancy” is a term known in the art McDermott T.R. & Graham

Appi. and Environ. Microbiol $5(10): 2403*2408. It is well known in the art that, notwithstanding the rare exception, a single nodule: will contain only one: bacterial strain. Johnston, A.W.B,, et a!., 1974, J, Gem Microbiol 87: 343*350: Dunham, D , ti &amp; Baldwin, I.L, 1931, Soil Science 32; 235-249;; Johnson, RW., et ah, 1963, Agmm 3. 55: 269-271; Dudman, W,F. &amp; Brockweil, J„ 1968, J. Agricui. Rm. 19: 739-747; IStiooi, H.%Thorton, Η.Θ., 1941, Pmc, Roy. .;Sco, 8 136, 32-09 Hughes, D.G., &amp; Vincent, J,P., 1942, Pmc. of the IJmwn&amp;n See, of New South Wales 67. 1-2-152; and Vincent, J.M. &amp; Waters, LM,< 1953, J. Gan. Microbiol. 9: 367-376.

As used herein, term "enhanced plant growth" Is Intended to refer to increased plant yield increased btamass. increased fruit number* or a combination thereof as measured by bushels: per acre), increased root number, increased root mass, increased foot volume, increased leaf area, increased plant stand, increased plant vigor, increased weight of a plant (e.g. total dry weight of a plant or plant part, total fresh weight or a plant or plant part, etc.), or combinations thereof.

As used herein, "Enhanced competitiveness" and/or "enhanced Population."' Is defined to mean bacterial strain(s) possessing a percent nodule occupancy, e.g. at least 56%, at: least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, up to 100% nodule occupancy.

As used herein, the term “temperature tolerance” is intended to mean the range of temperatures at which a bacterial strain(s) are able to grow, e.g., the maximum and minimum temperatures at which a bacterial strain can grow.

As used herein, the term “commercially available strain(s)” is intended to mean commercially available bacterial strains, e.g., USDA 532C, USDA 110, USDA 123, USDA 127, US DA 129, etc. Cregan, P.B., et al., 1989, Appl. and Enviro. Microbiol. 55 (10): 2532-2536.

As used herein, the term “micronutrient(s)” is intended to refer to nutrients which are needed for plant growth, plant health, and/or plant development.

As used herein, the term “biostimulant(s)” is intended to refer to any substance capable of enhancing metabolic or physiological processes within plants and soils.

As used herein, the term “wetting agent(s)” is intended to refer to any substance capable of lowering and/or reducing the surface tension of water.

STRAINS

In one embodiment, the isolated strain(s) described herein is a nitrogen fixing bacterial strain(s). In another embodiment, the strain(s) is a Bradyrhizobum sp. strain(s). In a further aspect, the strain is derived from a strain of Bradyrhizobium, including but not limited to a strain selected from the group consisting of Bradyrhizobium bete, Bradyrhizobium canariense, Bradyrhizobium elkanii, Bradyrhizobium iriomotense, Bradyrhizobium japonicum, Bradyrhizobium jicamae, Bradyrhizobium liaoningense, Bradyrhizobium pachyrhizi, and Bradyrhizobium yuanmingense. In yet another embodiment, the strain(s) is a Bradyrhizobum japonicum strain(s).

In yet another embodiment, the isolated strain(s) is a strain(s) of Bradyrhizobium sp. having enhanced/increased bacterial survival rate in a substantially moisture free environment when the survival rate of the isolated Bradyrhizobium strain(s) is compared to the survival rate of a parental strain(s), e.g., parental strain Bradyrhizobium japonicum USDA 532C, over a period of time, e.g., at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year or more.

In still another embodiment, the isolated strain(s) is a strain(s) of Bradyrhizobium sp. having an enhanced/increased survival rate in a substantially moisture free environment, wherein an increased survival rate in a substantially moisture free environment includes an increased bacterial survival rate in an environment that is at least 70% moisture free, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, up to a 100% moisture free environment, when the survival rate of the isolated Bradyrhizobium strain(s) is compared to the survival rate of a parental strain(s), e.g., parental strain Bradyrhizobium japonicum USDA 532C.

In another embodiment the isolated strain(s) is a strain(s) of Bradyrhizobium sp. having the following enhanced/superior characteristics when compared to commercially available strains, e.g., commercial strain Bradyrhizobium japonicum USDA 532C, wherein enhanced/superior characteristics include, but are not limited to: a. enhanced competitiveness for colonizing a soybean plant; and b. enhanced effectiveness at promoting soybean plant growth.

In still another embodiment, the isolated strain(s) is a strain(s) of Bradyrhizobium sp. having enhanced/superior competitiveness for colonizing a plant. In yet another embodiment, the isolated strain(s) is a strain(s) of Bradyrhizobium sp. having enhanced/superior effectiveness at promoting plant growth. In still another embodiment, the isolated strain(s) is a strain(s) of Bradyrhizobium sp. having enhanced/superior competitiveness for colonizing a plant and enhanced/superior effectiveness at promoting plant growth.

In yet another aspect of the present invention, the isolated strain(s) is a strain(s) of Bradyrhizobium sp. having enhanced/superior temperature tolerance.

In still another aspect of the present invention, the isolated strain(s) is a strain(s) of Bradyrhizobium sp. having natural resistance to glyphosate.

In still another embodiment, the strains are Bradyrhizobum japonicum strains selected from the group consisting of: the strain having the deposit accession number NRRL B-50608; the strain having the deposit accession number NRRL B-50609; the strain having the deposit accession number NRRL B-50610; the strain having the deposit accession number NRRL B-50611; and the strain having the deposit accession number NRRL B-50612. in a particular embodiment, the strain(s| may be one or more of the above mentioned deposited strains {e g., including at least two of the above strains, at least three of the above strains, at least four of the above strains, up to and including all of the above strains), ih an embodiment, the strain is the strain having thedeposit accession NRRL 8-50668,

In an embodiment, the siram is the strain having tie deposit accession number NRRL 8-50609,

In an embodiment, the stram is the strain having tie depose accession number NRRL 8-50616,: in: ah emhodimeht, the strain Is the strain having the deposit accession number N RRL 8-50611,

In an embodiment the strain ls:ibe strain having the deposit accession number NRRL B-50812.

In another embodiment, the bacterial cuiturets) has properties/characteristics identicai to at least pne of the deposited strains or a combination of at least two of the above deposited strains, InoiUdlng more than two, such as, at least three of the above strains, at least four Of the above strains, up to: and including all of the above strains,: Properties/characteristics of hie bacterial culture include, but are not limited to, feaeienai strains having enhanced and/or superior resistance to deslecatiom in stilt another embodiment, the stralnis) is a strain|s) of Bmdyrhimbium having enhanced and/or superior desiccation resistance- when the desiccation resistance is compared to the desiccation resistance and/or tolerance of a parental strain/s) of bacteria, e.g,, parental stmn Bradyftiizobium japonicum USDA 532C. in another aspect, the isolated bacterial slrain(s) of the present invention includes sirain(s) that are closely related ίο any of the above strains on the basis of ibS rONA sequence identity. See Stac-kehrandt E, et al,, “Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology," hit J Syat Evo! Micmbtoi, 52(3):1643-2 {2082) regarding use of IBS rDNA sequence identity for determining reiatedness in bacteria, In an embodiment, the at least one strain is at least 95%: identical to any of the above strains oh the basis of IBS rDNA sequence identity, at least 98% identical to any of the above strains oh the basis of 16S rDNA sequence identity, at least 97% identicai to any of the above strains on the basis of 16S rDNA Sequence identity, at ieast 98% to any of the above strains on the basis of 16$ rDNA sequence identity, at least §8.5% identical to any of the above strains on the basis of IBS rDNA sequence identify, at ieast §9% identical to any of the above strains on the basis of 168 rDNA sequence identity or at least 90.5% to any of the above strains on the basis of 188 rDNA sequence identity.

The Bradyrhizobium bacterium described herein, and in particular, the strains having deposit accession numbers NRRL 8-50608, NRRL 8-50609, NRRL B-50610, NRRL 8-50611, and NRRL 6-50812, can be grown according to methods known in the art.

The resulting material may be used directly in a composition, as a seed treatment, or the spores may be harvested, concentrated by centrifugation, formulated, and then dried using air drying, freeze drying, or fluid bed drying techniques (Friesen T., Hill G., Pugsley T., Holloway G., and Zimmerman D. 2005, Experimental determination of viability loss of Penicillium bilaiae conidia during convective air-drying Appl Microbiol Biotechnol 68: 397-404) to produce a wettable powder.

Above mentioned deposited strains were deposited on November 30, 2011, as indicated in more detail below in the “Materials &amp; Methods” section, under terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at Agricultural Research Culture Collection (NRRL), International Depositary Authority, 1815 N. University Street, Peoria, Illinois 61604, U.S.A. COMPOSITIONS:

In another aspect, the invention relates to a composition comprising a carrier and an inoculum of one or more of the deposited strains (either spore form or strains in a vegetative state) described herein. In certain embodiments, the composition may be in the form of a liquid, a slurry, a solid, or a powder (wettable powder or dry powder). In another embodiment, the composition may be in the form of a seed coating. Compositions in liquid, slurry, or powder (e.g., wettable powder) form may be suitable for coating seeds. When used to coat seeds, the composition may be applied to the seeds and allowed to dry. In embodiments wherein the composition is a powder (e.g., a wettable powder), a liquid, such as water, may need to be added to the powder before application to a seed. Example of yet other carriers include moistened bran, dried, sieved and applied to seeds prior coated with an adhesive, e.g., gum arabic.

Carriers:

The carriers described herein will allow the deposited bacterial strain(s) to remain efficacious (e.g., capable of fixing nitrogen) and viable once formulated. Non-limiting examples of carriers described herein include liquids, slurries, or solids (including wettable powders or dry powders). In an embodiment, the carrier is a soil compatible carrier as described herein.

In one embodiment, the carrier is a liquid carrier. Non-limiting examples of liquids useful as carriers for the compositions disclosed herein include water, an aqueous solution, or a non-aqueous solution. In one embodiment, the carrier is water. In another embodiment the carrier is an aqueous solution, such as sugar water. In another embodiment, the carrier is a non-aqueous solution. If a liquid carrier is used, the liquid (e.g., water) carrier may further include growth media to culture the deposited bacterial strains. Non-limiting examples of suitable growth media for the deposited bacterial strains include arabinose-gluconate (AG), yeast extract mannitol (YEM), G16 media, or any media known to those skilled in the art to be compatible with, and/or provide growth nutrients to the deposited bacterial strains.

In another embodiment, the carrier is a slurry. In an embodiment, the slurry may comprise a sticking agent, a liquid, or a combination thereof. It is envisioned that the sticking agent can be any agent capable of sticking the inoculum (e.g., one or more of the deposited strains) to a substrate of interest (e.g., a seed). Non-limiting examples of sticking agents include alginate, mineral oil, syrup, gum arabic, honey, methyl cellulose, milk, wallpaper paste, and combinations thereof. Non-limiting examples of liquids appropriate for a slurry include water or sugar water.

In another embodiment, the carrier is a solid. In a particular embodiment the solid is a powder. In one embodiment the powder is a wettable powder. In another embodiment, the powder is a dry powder. In another embodiment, the solid is a granule. Non-limiting examples of solids useful as carriers for the compositions disclosed herein include peat, wheat, wheat chaff, ground wheat straw, bran (e.g., moistened bran, non-moistened bran), vermiculite, cellulose, starch, soil (pasteurized or unpasteurized), gypsum, talc, clays (e.g., kaolin, bentonite, montmorillonite), and silica gels.

Optional Agriculturally Beneficial Ingredients:

The compositions disclosed herein may comprise one or more optional ingredients. Nonlimiting examples of optional ingredients include one or more biologically active ingredients, micronutrients, biostimulants, preservatives, polymers, wetting agents, surfactants, or combinations thereof.

Biologically Active Inqredient(s):

The compositions described herein may optionally include one or more biologically active ingredients as described herein. Non-limiting examples of biologically active ingredients include signal molecules (e.g., lipo-chitooligosaccharides (LCO), chitooligosaccharides (CO), chitinous compounds, flavonoids, jasmonic acid or derivatives thereof, linoleic acid or derivatives thereof, linolenic acid or derivatives thereof, karrikins, etc.) and beneficial microorganisms (e.g., Rhizobium spp., Bradyrhizobium spp., Sinorhizobium spp., Azorhizobium spp., etc.).

Signal Molecule(s):

In an embodiment, the compositions described herein include one or more signal molecules. In one embodiment, the one or more signal molecules are one or more LCOs. In another embodiment, the one or more signal molecules are one or more chitinous compounds. In still another embodiment, the one or more signal molecules are one or more COs. In yet_ another embodiment, the one or more signal molecules are one or more flavonoids or derivatives thereof, in still yel another embodiment, the one or more signal molecules are one or more non-fiavonoid nod; gene inducers (e.g., jasmonic acid, linoielc acid, iinolenie acid, and derivatives thereof). In still yet another embodiment, the one or more signal molecules are one or more karnkins or derivatives:thereof, in stilt another embodiment, the one or more signal molecules are one or more LCDs, one or more ehittnouscompouncfs, one or more COs, one or more flavonoids and derivatives thereof, one or more non-fiavondid nod gene inducers and derivatives thereof, one or more karnkins and derivatives thereof, or any signal molecule combination thereof, LCDs:

Upo-chitooligoaacbhande compounds (1.(3©$), also known Sri the art as symbiotic Nod signals or Nod factors, consist of an oligosaccharide backbone of p-i,4-ilnked N-acetybD-giucosamine CGicNAc”) residues with an N-iinked fatty acyl chain condensed at the non-reducing end. LCO's differ in the number of GIcNAc residues in the backbone, in the length and degree of saturation of the fatty acyl chain, and in the substitutions of reducing and non-reducing sugar residues. An example of an ICO is presented below as formula I:

in which: G Is a hexosamine which can be substituted, for example, by an acetyl: group on the nitrogen, a sulfate group, an acetyl group and/or an ether group on an oxygen, R-i, R?.. R&amp; R->, R« and Rr, which may be identical or different, represent H, CHs CO--. C>: HyCO- where x is an integer between 0 and 1?, and y is an integer between 1 and 35, or any other acyl group such as for example a carbamyl,

Ha represents a mono-, dl- or triunsaturated aliphatic chain containing at least 12 carbon atoms, and n Is an integer between 1 and 4, LCDs may lie obtained (isolated and/or purified) from bacteria such as RMmhia, e,g„ RNzabiam spp,, Bm^yrhizobkim sop,, Sirnrhizobitm spp. and Azorhizobkim spp, LCD struoture tS: chpraetarlstlb for each such bacterial species,: and each strain may: produce multipip LCD’s; with: different 'Structures, For example, specific LCDs from S. mettfoti have also been: described In LLS· Patent 0,549,718: as having the formula jib

in which R represents H or CHgCO- and n is equal to 2 Or 3,

Even more specific LGOs include NodRM, NodRM-l, NodRM-3. When acetyiaied (the R~CH;5 00--), they1 become AeNddR:M-1s and: AcNodRM-3, respectively (U.S. Patent: 5,645,218). LCDs from BmdyrhiEbMiit® feputiibum ate described In U.S. Patents 5,125,149 and 5,321,011, Broadly, they are pentasaccharide phytohormones comprising methylfdcose. A number of LCDs are described: BjNod-V (C^r); BjNod-V/(&amp;s Cie;t>*

BjRod'-V (C }S;j)> sod BjNod-V (Ac. C<$£) with: “V" indicating the presence of five hi-acetylgiucosamines;: ("Ac* an acetylation; the number following the ’O* indicating the number of carbons in the fatly acid side chain; and the number following the the number of double bonds. LCDs used In compositions of the invention may be obtained {Le.. isolated and/or purified) from bacterial strains that produce LCD's, such:: as strains of Azorhizobmni Bmdyrhkobktm (including &amp;· iaponicim), Marnrhizobimn, Rhizolmm (including R. iegummosarum), Smorhizobium (including $. maiiiot:i% and bacterial strains genetically engineered to produce LSD's.

Aiso encompassed by the present invention are comodsitidns using LCDs obtained (be., Isolated and/or purified} from a mycorrblzai fungus, such as fungi of the group Giomsrbq^cota, eygb Ghmus iniraradicus. The structures of representative:LCDs obtained from these: fungi are described in WO 2010/049751 and WO 2010/049751 (the LCDs described therein also referred to as bViyc factors").

Further encompassed by compositions of the present invention Is use of synthetic LCD compounds, such as those described in WO 2005/063784, and recombinant LCD’s produced through genetic engineering. The basic, naturally occurring LCD structure may contain modifications or substitutions found in natural!'/ occurring LCD’s, such as those described in Spaink, Grit, Rev. Plant ScL S4;2S7~288 (2GQQ) and D’Haeze, et.ai, Glycofoioiegy i2:79R-105R (2002), Precursor oligosaccharide molecules (CDs, which as described below, are also useful as plant signal molecules Id the present inventieh) for the construction of LCDs may also he synthesized by genetically engineered: organisms, e.g.: as in Samaln, ef a/.. Garb, Res. 302:35-42 (1997);: Samain, et ai,s 1 BiofechnoL 72:33-47 (1999). LCD’s may be utilized: in: various forms of purify and may be used alone or In the form of a culture of LCO-producing bacteria or fungi. ^Methods to provide: substantially pure LDO’s include simply removing the microbial cells from: a mixture of LCDs and the microbe, or continuing to isolate and purify the LCD molecules through:: LCD solvent phase separation followed by HP LG chromatography as described, for example, in U.S. Patent 5,549,713. Purification can be: enhanced by repeated HPIC, and the purified LCD molecules can be freeze-dried for long-term storage,

GhltooSigosaccharides (CDs) are: known In the art as β-1-4 linked N actyl glucosamine structures Identified as chitin oligomers, also as N-acetySchitobligosaccharides, CD's have unique and different side chain decorations which make them different from chitin molecules KCgHisNOs/h, CAS No. 1398-81-4), and ehltosan molecules ((CsH«N04)n, GAS No. 9012-78-4). .Representative literature describing the structure and production of CDs is as follows: Van der

Hoist, et ato Current Opinion-' in Structural Biology, 1.1:808-818 {2001}; Robina, et a!,.. Tetrahedron 53.521-630 (2002); Hanoi, elaf, Plants 232:787-806 (2010); Rouge, el a/. Chapter 27, "The Molecular Immunology of Complex Carbohydrates* In Advances in Experimental:: Medicine and Biology, Springer Science; Wan, at at. Plant Ceil 21:1053-69 (2009); PC17F100/00803 (9/21/2000); and pemonf-Caufet etaL Piant Physiol /20(1):83-92 (1999), The CDs may fee synthetic or recombinant Methods for preparation of recombinant CDs are known in the art. See, a a. Saroalh, et at (supra-); Cbftato ef at, Meth, Eng. 7(^:341-7 (2006} and Safnaln, et al, d. Siofechnoi, 72:53-47 (1999). OMinous Compounds:

Chltlns and chifosaos, which are major components of the cell walls of fungi and the exoskelefons of insects and crustaceans,: are also composed of QIcNAc resldfees, Chitinous com pounds include: ch in, (| UPAC; N · (5-[f3-acety la min c~ 4,5-d !hydmxy-6--(nydroxyrnethy1}oxan-· 2yi}methoxymethy!|-'2diS~acefyiamlno-4s6~dihydroxw2~{hydroxynfi8fhyl)oxan-3'' yl}methoxymefhylj~4~hydroxy~6~(hydroxymethyl)oxan~3-ysjetfianamide), and chitosan, (IUPAC: 5~amino-6-[5-'8mino-6~[S'3miao-'4J~dlhydroxy--2(hydroxymethytWxan-3“y0oxy-4-hydroxy-'2·* (hydrGxymethyi)oxan-3--yl]oxy-2(hydroxymefhyi)oxane-3s4-dioi),

These compounds may fee obtained commercially, e.g,, from Sigma-Aldrich, or prepared from insects, crustacean shells, or fungal ceil wails. Methods for the preparation of chide and chitosan are known In the art, and have been described, for example, in li.S. Patent 4,636,297 (preparation from crustacean shells), Pochanavanjoh, ef a/., Lett Appi, Microbiol 36:17-21 (2002) (preparation from fungal cell walls), and U.S. Patent 5,965,545 (preparation from crab .shells and hydrolysis of commercial chitosan),: Deaceiyiated chitlns and ohitosans may be obtained that range; from less than 35% to greater than 99% deacelytafion, and coyer a broad spepl^hlip^fhdl^ulaf weights, e.g,, low: molecular weight chitosan oligomers ef iess than 15kD and chitln oligomers of 0.S to 2kD; "practical grade" chitosan with a mpleoyiar weight of about iSkD; and high molecular weight chitosan of up to 70kD. Chitln and chitosan compositions; formulated for seed treatment are also commercially available. Commercial products include, for exampie, ELE§A® (Plant Defense Boosters, Inc,) and BEYOND - (Aghhouse. Inc,).

Fiavonoids:

Fiavonoids are phenolic compounds having the general structure of two aromatic rings connected by a three-carbon bridge. Fiavonoids are produced by plants and have many functions, e#, as beneficial signaling molecules, and as protection against insect, 'animate, fungi and bacteria. Classes of fiavonoids include chalcones, anlhocyanidins, coumarins, flavones, flavanols, flavonols, flavanones, and isoflavones. See. Jain, et al., J. Plant Biochem. &amp; Biotechnol. //:1-10 (2002); Shaw, etal., Environmental Microbiol. //: 1867-80 (2006).

Representative flavonoids that may be useful in compositions of the present invention include luteolin, apigenin, tangeritin, quercetin, kaempferol, myricetin, fisetin, isorhamnetin, pachypodol, rhamnazin, hesperetin, naringenin, formononetin, eriodictyol, homoeriodictyol, taxifolin, dihydroquercetin, dihydrokaempferol, genistein, daidzein, glycitein, catechin, gallocatechin, catechin 3-gallate, gallocatechin 3-gallate, epicatechin, epigallocatechin, epicatechin 3-gallate, epigallocatechin 3-gallate, cyanidin, delphinidin, malvidin, pelargonidin, peonidin, petunidin, or derivatives thereof. Flavonoid compounds are commercially available, e.g., from Natland International Corp., Research Triangle Park, NC; MP Biomedicals, Irvine, CA; LC Laboratories, Woburn MA. Flavonoid compounds may be isolated from plants or seeds, e.g., as described in U.S. Patents 5,702,752; 5,990,291; and 6,146,668. Flavonoid compounds may also be produced by genetically engineered organisms, such as yeast, as described in Ralston, etal., Plant Physiology 137:1375-88 (2005).

Non-Flavonoid Nod-Gene Inducer(s):

Jasmonic acid (JA, [1 R-[1a,2p(Z)]]-3-oxo-2-(pentenyl)cyclopentaneacetic acid) and its derivatives, linoleic acid ((Z,Z)-9,12-Octadecadienoic acid) and its derivatives, and linolenic acid ((Z,Z,Z)-9,12,15-octadecatrienoic acid) and its derivatives, may also be used in compositions of the present invention. Jasmonic acid and its methyl ester, methyl jasmonate (MeJA), collectively known as jasmonates, are octadecanoid-based compounds that occur naturally in plants. Jasmonic acid is produced by the roots of wheat seedlings, and by fungal microorganisms such as Botryodiplodia theobromae and Gibbrella fujikuroi, yeast (Saccharomyces cerevisiae), and pathogenic and non-pathogenic strains of Escherichia coli. Linoleic acid and linolenic acid are produced in the course of the biosynthesis of jasmonic acid. Jasmonates, linoleic acid and linolenic acid (and their derivatives) are reported to be inducers of nod gene expression or LCO production by rhizobacteria. See, e.g., Mabood, Fazli, Jasmonates induce the expression of nod genes in Bradyrhizobium japonicum, May 17, 2001; and Mabood, Fazli, "Linoleic and linolenic acid induce the expression of nod genes in Bradyrhizobium japonicum," US DA 3, May 17, 2001.

Useful derivatives of linoleic acid, linolenic acid, and jasmonic acid that may be useful in compositions of the present invention include esters, amides, glycosides and salts. Representative esters are compounds in which the carboxyl group of linoleic acid, linolenic acid, or jasmonic acid has been replaced with a -COR group, where R is an -OR1 group, in which R1 is: an alkyl group, such as a CrC8 unbranched or branched alkyl group, e.g., a methyl, ethyl or_ propyl group;; an alkenyl group, such as a SrCs unbfahohed or branched alkenyl group; an aikynyl group, such as a 0^1¾ unbranehed or branched aikynyl group; an aryl group Paving, for example, 6 to 10 carbon atoms* or a hoteroaryl group having, for example, 4 to 9 carbon atoms, wherein the heteroatoms so the heteroaryl group can fee, for example, N„ Q, P, or S, Representative amides are compounds in which the carboxyl group of linoieic acid, linolenic acid, or jasmonsc acid has been replaced with a —C0R group, where R is an NR*RJ group, in which R2 and R3 are independently: hydrogen: an; alkyl group, such as a C^Cs ubhrahehed or branched alkyl group, e.g,, a methyl, ethyl or propyl group; ah alkenyl group, such as a; CrCs unbranched or branched alkenyl group; an aikynyl group, such as a CrQ. unbrahohed or branched aikynyl group; an aryl group having, for example, 6 to 10 carbon atoms; dr a heieroaryl group having, for example, 4 to 9 carbon atoms, wherein the heteroatoms in the heteroaryl group can be, for example, N, Ο, P, dr S. Esters may be prepared by known methods, such as acid-catalyzed nucleophilic addition, wherein the carboxylic acid is reacted with an alcohol m the presence of a catalytic amount of a mineral acid. Amides may also be prepared by known methods, such as by reacting the carboxylic acid with the appropriate amide in the presence of a coupling agent such as dlcyciohexyl carbodilmide (DCC), under neutral conditions, Suitable salts of linoieic acid, linolenic acid, and jasmonsc acid include e.g„ base addition salts. The bases that may be used as reagents to prepare metaboilcaily acceptable base salts of these compounds include those derived from cations such as alkali metal cations (e,0.s potassium and sodium) and alkaline earth metal cations fag., calcium and magnesium). These salts may be; readily prepared by mixing together a solution of linoieic acid, linolenic acid, or jasmonsc acid with a solution of the base. The salt may be; precipitated from solution and be collected by filtration or may be recovered by other means such as by evaporation of the solvent

Karrikini s):

Kanikins are vinylogous 4H-pyrones o.g., 2H-iuroj2,3-cjpyran-2-ones Inciudlng denyatiyes and analogues thereof. Examples of these compounds are represented by the following structure:

wherein; Z Is 0, S or NRg.JR,, f% %· and R* are each rniledeoaentfy H, alkyl, alkersyi, ajkynyi, phenyl* benzyl, hydroxy, hydraxyalkyl, aikcxy. phenyioxy. benzyloxy, .'CM, CDRS, COORm halogen, NRsR?, or hiOj»;: and R5:, l% and R? are each:independently B, alkyl or alkenyl, or a biologically acceptable salt thereof,: Examples of 'biologically acceptable salts of these compounds may include acid addition salts formed with biologically acceptable acids, examples of which include hydrochloride, hydidbromide, suiphaie or feisyiphate, phosphate or hydrogen phosphate, acetate, benzoate, succinate, fumarste, mafeate, lactate, citrate, tartrate, gluconate; methahesulpbonate, behzenesuiphonate and p-ldeenesuiphonic acid, Additional biologically acceptable metal salts may include alkali metal salts, with bases, examples of which include the sodium and potassium salts. Examples of compounds embraced by the structure and which may be suitable for use in the present Invention include the following: 3~mmhyi-2H4um[2,3~ cfpymn~2~one (where RpCHv Rg, R* 2.H-furo|2,:3~cj^ran^ene (where R,, R?, % R4*H), ?~methyi''2Hdur0(2s3~c3pyran“2-one (where Rit R2, Rs-GMg}, S-methyi^H- furo[2,3*c|pyran-2-one (where Rr, % ΒφΗ,. R^CM*}, 3,:?~dlmethyh2N4iiro[2,Mpyran^on:e (where R1s Rg~CR3, R2s R*-H), 3sS-dimeihyi~2H~furo[2s3-c3pyran»2-one (where Rif R^CH* Rs, R3"H), 3,5J4rimethy!~2H~furo|'2,3~cJpyran~2--one (where: R*, R3, R^CHs, RjpH),·· S-metho%memyl“3“niethyi"2li-hiro[2,S~o|pyran-2*one (where RpCH3, R*, R3^H, R^CHjOCB^ 4~brbmo-3,?~dimethyi~2Hdurb(2,3“c3pyran-2*ohe (where R*,, R^Br, 3- methyifurop,3m]pyrldin-2(3H>bne {Where 2~MH, ^~ΟΗ3, R* R3:, R^B), B^-dimethylfuroiaj» o]pyridin-2{eH)-one (where Z«N~CB3, R^CPR R2. R3, R^H). See, U.S. Patent 7,576,213. These molecules are also known as karrikins, See, Haiford, “Smoke Signals,” in Chem. Eng. News (April 12, 2010), at pages 37-38 (reporting that karrikins or hutenoiides which are contained in smoke act as growth stimulants and spur seed gemmation after a forest fire, and can Invigorate1 seeds such as cbm, tomatoes, lettuce and onions that had been stored), these molecules are the subject of U J. Patent 7,573,213»

In an embodiment, the compositions described herein may comprise one or more benefidiai microorganisms. The one or more beneficial microorganisms may have one or more beneficial properties :{e.g,·, produce one or more of the signal molecules described herein, enhance nutrient: and water uptake, enhance growth, enhance seed germination, enhance seedling emergence, break fhe dormancy or quiescence of a plant etc.),

Ih one embodiment, the beneficial microorgahismts) comprise one or more bacteria: that produce one or mere :bf the slgnas molecules described herein.: In still: another embodiment the bacteria are bacteria from the genera RpizobiumSpp. (a.g,, R calluiPsilytictim, R: daejsQpanm, R ef§: R gafegae, R gallimrn, R glardim, R bamanapsp, R huaptiensm R im%gfp0py:R.., legumM&amp;sarum, R, loas$ans&amp;f R. iupink R lusitamm, R melileik R ''mongolmm, R mtuomns&amp;< R suSbe,: R M>pl&amp;, R, undicoia, andiot R. yanglingense), Azorhizobium spp. ie.g... A cauiinodms and/or A timbem&amp;ipraei,: $MtkH%&amp;biu0· spp, (e,g„ R abrl, 8, adh&amp;pf&amp;ns, &amp; ammeanum, 8, aborts, 8, fredil, 8, iadimase, 8. kostmnsa, R kummamwmSi Sr m&amp;dicae, R meliloti. S. maxicanus, S. moreiensa, S. saheii, S. iarangaa, and/or &amp; xinpangansa), Mesarbimbium spp., (M Plbizlm, M. pm&amp;fphmf M cbacoenee^ M, cicert, M hmkuii, Μ ίσΙί,Μ : madliei'raaeum, M, p/u#artrtm, Μ. 8$ρ$&amp;ρίΡοη&amp;Ι&amp;%··Μ temperatum, and/or M: bansbanense), and combinations thereof. In a particular embodiment, the beneficial microorganism Is selected from the group consisting of" Mi Isgummsarum, R mpliloU, S. metoR and combinations ihereoffn another embodiment, the beneficial microorganism is R l&amp;gumiaosarum. in another embodiment, the beneficial microorganism is R m&amp;iiiotL in another embodiment, the beneficial microorganism is 6'. meliloti.

In another embodiment, the one or more beneficial microorganisms comprise one or more phosphate solubilizing microorganisms. Phosphate solubilizing microorganisms include fungal and bacterial strains, in an embodiment, the phosphate solubilizing microorganism includes species from a genus selected from the group consisting of Acineiobacier spp. {a.g., Adnetabacier calcoaceilcus, etc,), Arthrobacier spp, Artlmbofrys spp. ia.g.. Arihrohotrys oHgmpora, etc,), Aspetgikm spp. (age, Asimgiitm aigag etc.), AMspkiiiim #R (@#s Azo-spirillum haippmaf&amp;mns, etc.). Bacillus spp. is.g., EpcJiius arayioliquefadans; Bacillus atropbaaus, Eaeilius cirmlpm., Badlkm iiPbeaikirmfd Badlkm sub&amp;is, etc,), BPtitfwId&amp;ria 'spp.· (ag., BwkPoldam cepacR Burkhoidaria wemam/ensReieR Candida spp. " (e.g.: Candida krissii, etc/), CPrysaomonas Spp. Chryseomoims iutaola, etc.), Ealarobader spp,ip,g., Entefobacter aerogaPas, Bni&amp;wbacterashuriaacEntambadarspp,, Eni&amp;mbact&amp;ftaylome, etc.), Eupeaidiiium spp. (e.g.r Eupanidilium parvum,· etc.), Exigmbactaripm spp., Klebsiella spp,,

Mayvera spp. fag., 't&amp;iiyvem .ΰφθΰϋΘ$ύ&amp;η'$, etc.}, Microbactemmi sp/?;:s Mucorspp, Mugor mmosissimus, etc.), Paediomyces spp, (p.g„ Paediomyceshepiaiki, Paediomyces marquandli, etc.;)* :&amp;mnjb&amp;z$Im· .spp. (©#, Paenlbaciiiu$ macemns, Pamib&amp;ctjfas mudifagfaowSs· etc.),· P&amp;mclUlum spp< (eg., PemeMium hiiaiaa -{formerly known as PenicMum Mali), PenicMum albidUm, PemcMum autaritirygnsaum, PenicMum· cbrysogenum, PenicMum dUeonigmm, PenicMum citnnum, PemcMum dlgitaium, PenicMum frequenias, PenicMum lit scum. Penieiiikm gaesirivorus, PenicMum giaPrum, Pemdiiium griseofuivum, PenicMum impiicatum,, Panidilhmpfiihmeiium, PenicMum Hiacmum, PenicMum Minioiut&amp;um, PenicMum montatmPse, P&amp;nicMum nigricans, PenicMum oxaiicum, PenicMum pirietorum, P&amp;nidliium pinopblium, PenicMum purpurogenum, PenicMum radicans, PenicMum redicum, Penidifium: raJsinckii, PenicMum ruguiosutm Penldlitum simpiisissimumj, PenicMum solifum^ Penisiiiium vadeMef PemcMum v&amp;iutmumt P&amp;nloMam viridicaium, PeumMum gisucum, Pemdiiium fussiporm, and Pemdiiium expansum, etcd, Pseudemanas spp, Pseudomonas corrugate, Ps&amp;udemenas tiuorescens, Pseudomonas iuiea, Pseudomonas poae, Pseudomonas puiida, Pseudomonas stuizerp Pseudomonas iriviaiis, etc.}, S&amp;rratia spp.ie.g.. Serraiia maroescebSi etc,), Sienoimphomonas spp. fe,g.(: StenQimpbomonas maiiopiMia, etc.), Strapiomyces spp., Stmpiosporangium^ spp,, Sw&amp;minaibania spp,fe,g,s Bwammathama saiiioierans, etc,), Tbiob&amp;cilius spp, Thiobadiius fermoxidans, etc.), toruiospora spp. (e.g,, 1‘omiospom igiobosm etc.), Vibrio spp, (e.g., Vibrio proieoiyiicus, eb*)* Xsathobacter am {&amp;,§., Xanthobacter agiiis, etc.), Xanthomon&amp;s spp. (e.g., Xanibomonas campesfriSt etc,), and combinations thereof. in a particular embodiment the one or more phosphate solubilizingmicroorganisms is a strain of the fungus PenicMum, In another embodiment, the one or more PemcMum species Is P, bilaiae, P. gaestrivorus, or combinations thereof. in andther embodiment the beneficial microorganism is one or mere myeorrhisa. in particular, the one or more mycorrhiza is an ehdomycorrbiza (also called vesicular arhuseuiar mycorrblzas, 'VMM, arbuscuiar myeorrhizas, or AMs), an eciomycorrhiza, of a combination thereof, in one embodiment, the one or more mycorrhiza is an endomycorrhiza of the phyium e/omeromyeofa and genera Gbmus and Gigasfxtra. in still a further embodiment, the endomyeorrhiza Is a strain of Glomus aggregatum, Glomus brasiiianum, Glomus datum, Glomus dasarlicoia, Glomus etuaicaium, Glomus imcieulatwm Glomus intramdices, Glomus monosporum, or Glomus mosseae, Gigaspora margarita, or a combination thereof. in another embodiment, the one or more mycorrhiza is an eciomycorrhiza of the phyium 8asidiomycota, Ascomycota, and Zygomycoia. in stiii yet another embodiment, the ectomycorrhiza is a strain of laceana bfcoior, Larnana iaccaia, PimiHbmtinctonm, Rhizopogon amybpogon, Mimpogm 'fiiMghba, Rbimpogon fateoim, RbimpogmWopufL Sclerod&amp;ma or a combination thereof. in,: still another embodiment, the one or more mycorrhiza is an ceroid mycorriliza, an arbutoid mycorrhiza, or a mooPtropoid mycorrhiza, ArbuSeuiar and eciomycorrhizas form ericoid myeorrhlza with many plants: belonging to the order Hfeles,: while some Ericales form arbutoid and monotropokl mycorrhizas. All orchids are mycoheterobophic at some stage during their lifecycle and form orchid mycorrhizas with a range of basfdidmyoetp fungi in one embodiment, the mycorrhiza may be an cricoid myoorrhiza, proferadiy of the phylum Ascornycola, such as Hymenoscyphovs encaa or Qldbd&amp;nProa sp,- in another embodiment, the mycorrhiza also may be an arbytoid mycorrhiza, preferably of the phylum Bssbbmyaota. In yet another embodiment the mycorrhiza may be a monotripoid mycorrhiza, preferably of: the phylum: Basbbmycoia. in -Still yet another embodiment, the mycorrhiza may be- an orchid mycorrhiza, preferably of the genus Rhizoctoaia. in still another embodiment, the compositions described herein may comprise one or more beneficial micronutrients, ffendimiifng:: examples of micronotnents for use in the compositions described herein include vitamins, vitamin A, vlamfn B complex {La., vitamin 8ti vitamin By, vitamin By vitamin 83, 8¾ vitamin 83, vitamin 8¾. choilne) vitamin Q, vitamin D, vitamin Ev vitamin ti carotenoids (e-carotene, β-carotene, cryptoxanthin, lutein., lycopene, zeaxanfhln, etc.), macrominerais (e,g., phosphorous, caicium, magnesium, potassium, sodium, iron, etc,), trace minerals (eg,, boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, zinc, etc.), organic acids Ce.g., acetic add, citric acid, lactic add, malic aciid. taurine, etc.), and combinations thereof, in a particular embodiment, tie compositions may comprise phosphorous, boron, chlorine, copper, iron, manganese, molybdenum, zinc or combinations thereof. in certain embodiments, where trie compositions described herein may comprise phosphorous, it is envisioned that any suitable source of phosphorous may be provided. In one embodiment, the phosphorus may be derived from a source, in another embodiment, suitable sources of phosphorous include phosphorous sources capable of solubilization by one or more microorganisms (e.g>, PeniciPium Piiaiae. etc.).

In one embodiment, the phosphorus may be derived from a rock phosphate source, in another embodiment the phosphorous may be derived from fertilizers comprising one or more phosphorous sources. Commercially available manufactured phosphate fertilizers are of many types. Some common ones are these containing rack phosphate, monoammonium phosphate^ diammonium phosphate, moGocafeturo phosphate, super phosphate, triple super phosphate, and/or ammonium polyphosphate. Alt of these, fertilizers are producedby chemical processing of insoluble natural rock phosphates in largo scale fertilizer-manufacturing facillftes and the product is expensive. By means of the present invention il ls possible to,redueelthe amount of these fertilizers applied to the soil while still maihiaihing the sameamqunt of phosphorus uptake: from the soil. in still another embodiment, the phosphorous may be derived from an organic; phosphorous source. In a: further particular embodiment, the source of phosphorus mdy Inciuda an organic fertilizer. An Organic fertilizer refers to a soil amendmeht denyed from natural sources that guarantees, at least, the minimum percentages of nitrogen, phosphate, and potash. Nan-limltlng examples of organic fertilizers include plant and animat by-products, rock powders, seaweed, inocuiants, and conditioners, These are often available at garden centers and through horticultural supply companies. In particular the organic source of phosphorus is from bone meal, meat meal, animal manure, oompost, sewage sludge, or guan o, or combinations thereof, in sti another embodiment, the phosphorous may be derived from a combination of phosphorous Sources including, but not limited to, rock: phosphate,; fertilizers comprising one or more phosphorous sources feg., monoammonium phosphate, diammooium phosphate, monocalcium phosphate* super phosphate, triple super phosphate, ammonium polyphosphate, etc.) one or more organic phosphorous sources, and combinations thereof.

Biostimuianifs); in one embodiment, the compositions described herein may comprise one or more beneficial hiostimularifs. Biostirouiants may enhance metabolic or physiological processes such as respiration, photosynthesis, nucleic acid uptake, ion uptake, nutrient delivery, or a combination thereof. Non-limiting exampies of biostimuiants include seaweed extracts (e.g., ascophyflum nodosum}., humic acids {o.g„ potassium humate), fulvic acids, myo-inositoi, glycine, and combinations thereof. In another embodiment, the compositions comprise seaweed extracts, humic acids, fuiyie acids, myo-inositoi, glycine^ and combinations thereof.

PolvmerfsX in one embodiment, the compositions described herein may further comprise one or more polymers, Non-limiting uses of polymers In the agricultural Industry include agrochemical delivery, heavy metai removal, water retention and/or water delivery, and combinations thereof. Pouch ef J. Agri. &amp; Biol. Sci., 3(?p99~314 {2008}. in one embodiment, the one or more polymers is a natural polymer {e,g„ agar, starch, alginate, pectin, cellulose, etc,}, a synthetic polymer, a Biodegradable polymer (e.g„ polycaprolactone, polylactide, poly (vinyl alcohol), etc,), or a combination thereof.

For a non-limiting list of polymers useful for the compositions described herein, see Roues, ei a£, Am, J. Agri. &amp; Biol, Sci„ 3(7):299-314 (2008), in one embodiment, the compositions described herein comprise cellulose, cellulose derivatives* methyiceiluiose, methyiceiluiose derivatives, starch, agar, alginate, pectin, polyvinylpyrrolidone, and combinations thereof.

In one embodiment the compositions described herein may further comprise one or more wafting agents. Watting agents are commonly used on soils, partidulariy hydrophobic soils, to improve the infiltration' and/or penetration of water into a soil. The watting agent may be an adjuvant, oil, surfactant* buffer, acldifier, or combination thereof. In an embodiment, the wetting agent is a surfactant. In ah embodiment, the wetting agent is one or more nonionic surfactants, one or more anionic surfactants, or a combination thereof, in yef another embodiment, the wetting agent is one or more nonionic surfactants.

Surfactants suitable for the compositions described herein are provided in the “Surfactants’' section.

Surfactants suitable for the compositions described herein may be non-ionic surfactants (e,g., semi~po|ar and/or anionic and/or cationic and/or swstterionic). It is envisioned that the surfactant(s) will cause as little harm to the activity of the one or mom deposited strains andior the one or more beneficial microorganisms as possible. The surfactants can wet and emulsify seii(s) and/or dirt(s). It is envisioned that the surfactants used in described composition have low toxjeiily contained within the formulation, it is further envisioned, that the surfactants ."used in the described obmposition have a low phytotoxicity (/.©., the degree of; toxicity a substahoe or combination of substances has on a plant), A single surfactant or a blend of seyerai surfactants can be used.

Anionic surfactants

Anionic Surfactants or mixtures of anionic and nonionic surfactants may aiso be used in the compositions. Anionic surfactants are surfactants having a hydrophiiio moiety in an anionic or negatively charged state in aqueous solution. The compositions described herein may comprise one or more anionic surfactants. The anionic snrfaGtant(s) may be either water soluble anionic surfactants, water insoluble anionic surfactants, or a combination of water soluble anionic surfactants and water insoluble anionic surfactants. Non-limiting examples of anionic surfactants Include sulfonic adds:, sulfuric add esters, carboxylic acids, and salts thereof; Non-llmking examples; of water soluble anionic surfactants include alkyl sulfates, alkyl ether sulfates, alkyl amide ether sulfates, alkyl aryl polyether sulfates, alkyl aryl sulfates, alkyl aryl sulfonates, monoglycersde sulfates, alkyl sulfonates, alkyl amide sulfonates, alkyl aryl sulfonates, benzene sulfonates,:toluene sulfonates, xylene sulfonates, cumene: sulfonates, alkyl banxene sulfonates, alkyl dlphenyioxide sulfonate, alpha-olefin sulfonates, alkyl naphthalene sulfonates, paraffin sulfonates, lignin sulfonates, alkyl suifosucdhates, ethoxylated suifosuecinaies, alkyl ether sulfosuodnates, alkylamide. sulfOSucdnates, alkyl sulfosyccinamaie, alkyl sulfoaeelates,, alkyl phosphates! phosphate ester, alkyl ether phosphates, acyl sareohsinateS, acyl Isethionates, N-acyl taurates, N-e:cyi~N~alkyltaurates, alkyl carboxylates, or a combination thereof,

Nenionic surfactants

Nonionb surfactants am surfactants haying no electrical charge when dissolved or dispersed in an agueoas medium. In at least one embodiment of the composition: described therein, one or more nonionic surfactants are used: as they provide: the desired wetting and emulsification actions end do not significantly inhibit spore stability and: activity. The nonionic surfaciant(s) may be either water soluble nonionic: surfactants, water insoluble nonionic surfactants, or a cembinefion of water soluble nonionic surfactants and water insoluble nonionic surfactants.

Water insoluble nonionic surfactants

Non-limiting examples of water insoluble nonionic surfactants include alkyl and aryl; glycerol ethers, glycol ethers, efhanoiamides, sulfoanylamides, alcohols, amides, alcohol efhoxylates, glycerol esters, glycol esters, etlioxyiates of glycerol ester and glycol esters, sugar-based alkyl polyglycosides, poiyoxyethyienated fatty acids, aikanoiarnine condensates, aikanolamides, tertiary acetylenic glycols, poiyoxyethyienated mercaptans, carboxylic add esters, poiyoxyethyienated poiyoxyproylene glycols, serhitan fatty esters, or combinations thereof. Also included are EO/PO block copolymers (EO is ethylene oxide, PO is propylene oxide), E© polymers and copolymers, poiyamines, and polyvinylpyholidones.

Water soluble nonionic surfactants

Non-limiting examples of water soluble nonionic surfactants include sofbifao fatty acid alcohol ethoxylates and sorbitan fatty acid ester efhoxylates,

Combination of nonionic surfactants b one embodiment the compositions described herein: comprise at ieasf one or more nonionic surfactants, in one embodiment, the compositions comprise at least one water insolubie nonionic surfactant and at least one water soluble nonionic surfactant- inf-still another embodiment,: the compositions comprise a combination of· nonionic surfactants having hydrocarbon oftains of substantially the same length. in another embodiment, the compositions described heneb may also comprise organosiilcone surfactants, siiicone-based aniifoams used as surtactants in silicone-based and mineral-oil based antifoams. in yet another embodiment, the compositions described herein may also comprise alkali metai salts of fatty acids {&amp;.$,, water soluble alkali metal safe of fatty adds and/or water insoluble alkali metal salts of fatty acids).

In one embodiment, die conipositions described herein may further comprise one or more herbiddes, in a particular embodiment, the herbicide may be a pre-emergent herbicide,: a post-emergent herbicide, or a combination thereof.

Suitable herbicides Include chemical herbicides, natural heriaiddes (e,§,, blOherbiddes:, organic herbicides, etc.}, or combinations thereof. Non-limiting examples of suitable herbicides include bentazon, acifiuorfen, chlorimuron, lactofen, doraazone, fiuaxlfop, glufosinate, glypbosate, sethoxydtm, Imazetbapyr, imazamox, fomesafe, femlciorac, imazaquin, cletbodim, pendlmethaiin; 3,4-Dimethyb2;6-dlnitro-A/-pentan~3~yi-anisine; U-{ 1 -ethylpropyl|~2,6*dinitro-3s4-xylidine; pronamide; propyzamide; 3,5~Dictfero-N~(l ,l~d!metbylpropynyi}benzamide; 3,5-Dichloro-N-{ 1,1 -dimethyl-2-pfopynyl)benzamide; M~( 1,1 * Dime! hyf propyny 1)-3,5- di eh lo r ob e n za mid e: S^ethyl N-ethy 1th iocy d oh e xa n e ca r ba m ate: trifiuraiin; 2,8~Dinitro-A/,iV-dipropyi4~{trifiuQromdhyi)an|l|ne; glyphosate; :N“(pbosphonomethyl}g!ydne; and derivatives thereof, in one embodiment, the one or more herbicides for: use in accordance with this disdosure include pronamide (commerciaiiy referred to as Kerb€>}; propyzamide; 3,5~Dlch:loro~hK1s1-dimethyipropynyObenzamide; 3:5-Dicdoro-N~(1,1-dirnethy!~2-propynyi}benzarnfde; 14-(1,1-Dimetbylpropynyij-SJ-dichiorobenzamldej oycioate, S-ethyi N-ethyltbiocyciohexafiecarbamate (commerciaiiy referred to as Ro^NeetiS); trifiuraiin; ^B-Dinitro-Af^dipfOpyi-d-(trifiuoromefhyi}aniiine; glypbosate; N~{phosphonornethyi)giycine; and derivatives thereof. Commercial products containing each of these compounds are readily available. Herbicide concentration in the composition will generally correspond to the labeled use: rate for a particular herbicide.;

Funcjicide(s): in one embodiment, the compositions described herein may further comprise one or more fungicides. Fungiddes useful to the compositions described herein will suitably exhibit activity against a broad range of pathogens, including but no! limited to PiiytoptithorB, Rhkoctonia, fusBriumy Pyibtum, Phofnofxsis or Se/emfiwa: and PhBkopsom Bnd combinations thereof.

Non-limiting examples of commercial fungicides which may be suitable for the compositions disclosed herein include PRQTEGfe, RIVAL or ALLEGIANCE FL or LS (Gustafson, Plano, TX), WARDEN R'TA (Agriiance, St. Paul, MN). APRON XL. APRON MAXX RTA or RFC. MAXIM APS Or XL (Syngenta, Wilmington, DE), CAPIAN (Arveste, Guelph, Ontario) and PROTREAT (Nttragln Argentina, Buenos Ares, Argentina). Active ingredients in these and other commercial fungicides include, but are not iimited to, fludloxonii, mefenoxam, azoxystrobin and metalaxyl. Commercial fungicides are most suitably used in accordance with the manutaeiunirts instructions a! the recommended concentrations. in one embodiment, the compositions described herein may further comprise one or more insecticides. insecticides useful to the compositions described herein vviii suitably exhibit activity against a broad range of insects including, but not iimited to, wlrevvorms. cutworms, grubs, com rooiworm, seed corn maggots, fiea beetles, chinch bugs, aphids, leaf beetles, stink bugs, and combinations thereof.

Non-limiting examples of commercial Insecticides which may be suitable for the cxj^l^sl^ohs-idlsdlidsed·., herein. feeiiude CRUISER (Syngenta, Wilmington;, DE), GAUCHO and PONCHO (Gustafson, Plano, TX), Active ingredients in these and other commercial Insecticides include tbiamelhoxam, clothianidin, and tmidacioprld. Commercial insecticides are: most suitably used in accordance with: the manufacturer's instructions at the recommended concentrations.

METHODS in another aspect,: methods of using the deposited strains and compositions described herein are disclosed. in one embodiment a method for enhancing plant growth is described, The method comprises contacting a pi ant or plant part with an inoculum of one or more bacterial strains selected from the group consisting of:

In a particular embodiment, the inoculum may comprise one or more of the above mentioned deposited strains (©.g,, including at least two of the above strains, at least three of the above strains, at feast four of the above strains, up to and including all of the above strains), in an embodiment, tbe inoculum comprises the strain having the deposit accession number NRRL 8-50608, in an embodiment, the inoculum comprises the strain having the deposit accession number NRRL 8-50609. in an embodiment, the inoculum comprises the strain baying the deposit accession number NRRL 8-80610, in an embodiment, the inoculum comprises the strain having the deposit accession number NRRL 8-50811. in an embodiment, the inoculum comprises the strain having the deposit accession number NRRL B -50812. in still another embodiment the step of contacting a plant or plant: part with an inoculum of one or more of the deposited bacterial strains comprises contacting a plant or plant part with one or more of the compositions described herein. The Inocuium(s) or compositions may be made to contact the plant or plant part according to methods known to those skilled In the art, Non-iimfting examples include in-furrow introduction, coating seeds, etc. in a particular embodiment, the contacting step comprises in-furrow introduction of tbe inoculum or compositions described herein, in a particular embodiment, the contacting step comprises on-seed (seedi coating) introduction of the inoculum or compositions described herein.

In certain embodiments, the step of contacting a plant or plant part with an Inoculum of one or more of the deposited bacterial strains comprises introducing the inoculum info the soil in an amount of 1 x 10*- 1 x 10^ more preferably; 1 x 10s ™ 1 x: 10*3 colony forming: units per hectare, in other certain embodiments, the step of contacting: a, plant or plant: part with an; inoculum of one or more of the deposited bacterial strains comprises introducing the deposited bacterial strains as a seed coated: with 1 x 10* --1 x 10s, more preferably 1 x 10s - 1 :x 1 colony "forming units per seed. id another aspect, the method comprises growing plants in a soil comprising one or more of the bacterial strain. The method comprises: a) treating the soil an inoculum of one or more: bacterial strains selected from the group consisting of: the strain having the deposit accession hurnber NRRL 8-50808: the: strain having the deposit accession number NRRL B-50609; the strain having the deposit accession number NRRL 8-50610; the strath having the deposit accession number NRRL 8-50611; the strain having the deposit accession number NRRL 8-50812; or a mixture of two or more of the strains; and: b} growing a plant in the treated soil in a particular embodiment, the inoculum may comprise one or more of the above mentioned deposited strains (e,g., including, at least two of the above strains, at least three of the above strains, at least four of the above strains, up to and including all of the above strains). in an embodiment, the inoculum comprises the strain having the deposit accession number NRRL B-50608, in an embodiment the inoouium comprises the strain having the deposit accession number NRRL B-6Q609. In an embodiment the inoouium comprise the strain having the deposit accession number NRRL 8-50610. in an embodiment, the inoouium comprises the strain having the deposit accession number NRRL 8-50811. In an embodiment, the inoculum comprises the strain having fee deposit accession number NRRL 8-50612.

The step of treating the soil with an inoculum of one or more of the deposited bacteria! strains comprises treating the soil with one or more of the compositions described herein. The inoculum(s) or compositions may be introduced into the soii according to methods known to those skilled in the art. Noh-limiting examples include in-furrow treatment, coating seeds, etc. In a particular embodiment, the treating step comprises in-furrow treatment of the inoculum or compositions described herein. In a particular embodiment, the ir^ating step comprises on-seed (seed coatihg)ireatmdnt of the Inoculu m: or compositions described hereim in a particular embodimenL the step of treating the soil wife an inormlum of one or riiofe of the deposited bacteria! strains comprises treating fee soli with one or more of the compositions described herein, in certain embodiments, the step of treating the soil with an Inoculum of one or more of the deposited bacterial strains comprises treating the soil with an inoculum in an amount of 1 x 1G1-- 1 X ίΟδΙ more preferably 1 x 10s- 1 x 10—colony forming units per hectare, in other certain embodiments, fee step of treating the soli with an inoculum of one or mom of the deposited bacteria! strains comprises introducing the deposited bacteria! strains as a seed coated with 1 x 1 χ 10¾ more preferably 1 x 10—-1 x l 0'5 colony forming units per seed. in anolfter embodiment, the method further comprises the step of piantfog a planter plant part, the planting step can occur before, after or during the treating step, in one embodiment, the planting step occurs before the treating step, in another embodiment, the planting step occurs during the treating step (e.g., the planting step occurs simuiianeousiy with the treating step, the planting step occurs substantially' simultaneous with the treating step, etc.}, in still another embodiment, the planting step occurs after the treating step. in another embodiment, the method further comprises the step of subjecting the soil to one or more agriculturally beneficial ingredients described herein. In one embodiment, the step of subjecting the soil to one or more agriculturally beneficial ingredients occurs before, during, after, or simultaneously with the treating step. In one embodiment, ibe step of subjecting the soil to one or more agriculturally beneficial ingredients as described herein occurs before the treating step, in another embodiment, tine step of subjecting the soil to one or more agrtcuituraily beneficial Ingredients as described herein occurs during the treating step, in still another embodiment, the step of subjecting the soil to one or more agriculturaiiy beneficial ingredients as described herein occurs after the treating step, in yet another embodiment, the step of subjecting the soi! to one or more agriculturaiiy beneficial ingredients as described herein occurs simultaneously with the treating step fag., treating the soil with one or more of the compositions described herein, etc,}. in yet another embodiment, the invention includes a method for treating seeds comprising applying to the seeds an inoculum of one or more bacteria! strains selected from the group consisting of: the strain having the deposit accession number NRRL 8-50808' the strain having the deposit accession number NRRL 6-60889; the strain having the deposit accession number NRRL 8-50610; the strain having the deposit accession: number NRRL 8-6Q611; the strain having the deposit accession number NRRL 8-5081¾ or a mixture of two or more of the strains, in a particular embodiment, the method for treating seeds: may comprise one or more of the above mentioned deposited strains (e,g.s including at least two of the above strains, at least three of the above strains, at least four of the above strains, up to and Including all of tbe above strains). in an embodiment, the method of treating seeds comprises applying to the seed the strain having the deposit accession number NRRL 8-50608. in an embodiment, the method of treating seeds comprises applying to the seed: the strain having the deposit:accession number NRRL 6-56809, In an embodiment, the method of treating seeds comprises applying; to the seed the strain having the deposit accession number NRRL 8-50610· in an embodiment, the method of treating seeds comprises applying to the seed the strain having Use deposit accession number NRRL 8-50811. in an embodiment, the method of treating seeds comprises applying to the seed the strain having the deposit accession number NRRL B-50812. in yet anether embodiment, the method further comprises the step of apply ing to the seeds one or" more agriculturally benefieiai Ingredients to the seed, in another embodiment, the method comprises applying to the seeds any of the compositions described herein to the seeds. in stilt another embodiment, the method comprises storing seeds with an inoculum of at least one or more of the Isolated bacterial straihs in a substantiaily moisture free environment for a period of lime, e.g,, at ieast 1 day, at least 2 days, at least 3 days, at least 4 days, at least 6 days, at ieast 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at ieast 4 months, at least 5 menths, at least 6 months;, at least 1 year or more. In one aspect of the method, the seeds are leguminous plant seeds. In another aspect, the leguminous plant seeds are soybean seeds.

The methods described herein am potentially useful for Improving growth conditions resulting in increased phosphorous uptake and/or yield for any type of plant. in one particular embodiment the plant is selected from the^nuup consisting of non-legumes. legumes, Brassiaa spp., cereals, fruits, vegetables, nuts, flowers, and tuff. Particularly the cereals are wheat, corn, rice, oat, rye, bariey. Particularly legumes are lentil, chickpeas, beans, soybeans, peas, and alfalfa. in another particular embodiment the plants are selected from the group consisting of alfalfa, rice, wheat, barley, rye, oat, cotton, sunflower, peanut, corn, potato, sweet potato,, bean, pea, chickpeas, lentil, chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip, turnip, cauliflower, broccoli, turnip, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple, soybean, tobacco, tomato,sorghum, and sugarcane, mstssmirn

In another aspect, seeds are coated with one or more-bacterial strains selected from the group consisting of: the strain having the deposit accession number NRRL 8-50808; the strain having the deposit accession number NRRL 8-50809; the strain having the deposit accession number NRRL B-50610; the strain having the deposit accession number NRRL B-50611; the strain having the deposit accession number NRRL B-50612; or a mixture of two or more of the strains. in a particular embodiment, the seed(s) is coated with one or more of the above mentioned deposited strains (e.g.. including at ieast two of the above strains, at ieast three of the above strains, at least four of the above strains, up to and including ail of the above strains).

In an embodiment, the seed(s) is coated with the strain having the deposit accession number NRRL 8-50608, In: an embodiment, the seed(s} is coated with:, the strain having the: deposit::accession number NRRL 8-50809. In an embodiment the seed(s) is: coated with thO: strain having the deposit accession number NRRL B-50810, in an embodiment, the seedfs) Is coated with the strain having; the deposit accession: number NRRL B-SCBIi. in an: embodiment, the seed^s) is coated with the strain having the deposit aceeSsiori number NRRL 8-50812,

Id one embodiment, seeds may he treated: with any of the composttiohts) described heroin in several ways but preferably via spraying or dripping. Spray and drip treatment: may be conducted by formulating compositions described herein end spraying dr dripping the com position (s) onto a seed{s} via a continuous: treating System (which is calibrated: to apply treatment at a predefined rate ip: proportion to the continuous flow of seed), Such as a drum-type of treater. Batch systems, in which a predetermined hatch size of seed and eompCsitionts) as described herein are delivered1 into a mixer, may also he employed. Systems; and dpparati for performing these processes are commercially available from numerous suppliers, e,g.s Bayer CropScience (Gustafson).

In another embodiment, the treatment entails coating seeds. One such process involves coating the Inside wall of a round container with the eoffiposlttonfs) described hereto, adding seeds, then rotating the container to cause the seeds to contact the wail and the compesitionts), a process known in the art as "container coating”. Seeds can he coated by combinations of coating methods. Soaking typically entails using liquid forms of the compositions described. For example, seeds can be soaked for about 1 minute to about 24 hours (e.g,5 for at least 1 min, S min, 10 min, 20 min, 40 min, 80 min, 3 hr, 8 hr, 12 hr, 24 hr), in certain embodiments, a seed(s) coated with one or more of the compositions described herein wiii comprise 1 x 10* - 1 x 10s, more preferably 1 x IQ2--l x 10sCoiony forming units of one or more of the deposited bacterial strains per seed.

EXAMPLES

The following examples are provided for illustrative purposes and are not intended to limit the scope of the invention as claimed herein. Any variations in the exemplified examples which occur to the skilled artisan are intended to fali within the scope of the present invention.

Materials &amp; Methods Deposit of Bloiooicai Material

The following biological materiai has been deposited under the terms of the Budapest Treaty at the iViierobiai Genomics and Bioprocessing Research Unit (NRRL) National Center for .Agricultural Utilization Research· ISIS N. University Street, Peoria, It 81804, USA and given tile following accession number:

Table 1: Deposit of Biological Material

The strains have boon-deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 87 CJTR. §1,14 and pi LI.S-C. §122, The deposit represents a 'pure- culture of the deposited strain. The deposit is available as required by foreign patent Jaws in countries wherein counterparts Of the subject application or Its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Media

Table 2: Components of G16 medium.

Table 3: Trace element Stock Store at 4°C for up to 6 months.

"Trace elements are added with all other components before sterilization.

Table 4: Vitamin Stock - filter sterilized followed by storage at 4°C for up to 6 months.

**Vitamins are added after the media has been sterilized and has cooled, typically at time of inoculation.

Table 5: Components of Yeast Extract Mannitol (YEM) Medium.

Example I: Determine 99.99% Kill Rate for USDA 532C

The following experiment(s), consisting of three (3) studies, was performed to determine the 99.99% kill rate for parental strain Bradyrhizobia japonicum USDA 532C.

Parental strain USDA 532C was grown in two 10ml G16 (Tables 2-4) and YEM (Table 5) disposable culture tubes (VWR, 18x150mm, #47729-583) for two days and harvested to obtain the highest cell concentration. This was achieved by combining both culture tubes into one tube and concentrating the cells down to 2ml. Approximately fifty soybean seeds (variety Stine RR 1108-4) were surfaced sterilized in 50ml sterile, disposable centrifuge tube (Fisher brand, #06-443-18) containing 5% household bleach solution for 30 seconds and rinsed with sterile deionized (Dl) water. The sterilization step was repeated for five times. The seeds were immediately placed in a sterilized Petri dish and dried under the laminar hood. Once the seeds were completely dried and transferred to a 250ml beaker, 1.5ml of the concentrated parental strain USDA 532C culture was added to the seeds. The seeds were swirled in the beaker to evenly coat the seeds and allowed to dry under the hood. The beaker, containing the seeds, was wrapped with blue, sterilization paper and left in the hood until the experiment was completed. Time points were taken at zero time, every two days for one week, and every week until complete cell death occurred. Results are provided in Table 6.

Table 6: CFU per seed and percent kill rate for study 1.

As shown in Table 6 the initial CFU per seed for parental strain USDA 532C was 3.06 x 108 and at days 37 the CFU was at 2.64 x 104. The percent kill rate from times 0 to 37 days was calculated to be 99.99%.

The procedure was repeated except G16 was used as the initial growing medium. The results are provided in Table 7.

Table 7: CFU per seed and percent kill rate for study 2.

As shown in Table 7S when G18 was used as the initial growing medium it took from 29 to 37 days for the kill rate to reach 99.99%. A third desiccation study was completed to determine if G16 and YEM media affected the rate of desiccation of parental strain USDA 5320. The results are provided in Tables 8 and 9 respectively.

Table 8: CFU per seed and percent klli rate for parental strain USDA 5320 grewn 016 medium.

Table 9: OF-ϋ per seed and percent kill rate for parental strain USDA 5320 grown YEM medium.

As shown in Tables 8 and 9;, there was no difference in the desiccation rate for parental strain USDA 532C when grown in G18 or YEM, The 99.99% kill rate was observed for the third study at approximately 28 days which Is similar to what was observed in studies one (i) and two (2) supra,

Example if: Determine the Kill Rate of USDA S32C Using Ethyl Methanesuifonate fEMS)

The following expenment(s) were performed to determine the application rate of the mutagen, ethyl methanosulfonate (EMS) that would give a 99.9-99.99 percent kill rate for parental strain USDA. 5320. This rate -determination will become part of the mutagenesis protocol used: to generate desiccation-resistant putative mutants although the method of mutagenesis: may evolve for efficiency. inocula preparation:

Parental strain USDA 5320 was grown in six 10ml YEM disposable culture tubes for two days and 5mi of the culture was inoculated info four 250ml flasks containing 50ml YEM medium. The flasks were incubated:·'for two days at 3CTG shaker. The culture from the flasks were; subsequently centrifuged in 50ml disposable sterile tubes at 8,000rpm for ten minutes Sn a Servall RQ 6 Pius® centrifuge and combinedIntoione tube. The pellet was re-suspended in;#mi of fresh YEM medium and separated into four 1.5ml mlpfocenirifuge tubes, The tubes would each :represent different application rates used for the mutagenesis process.

MuMOEiesisMocess:

Once the culture have been allquoted into separate tubes and the mutagen: EMS: (Sigma, C3H803S, FW 124.16:. #M0B8CMG) added to each tube, the tubes were vortexed vigorously and placed in an empty 260ml bask. The bask: containingfhe reaction tubes were incubated: for 30 minutes at 38“C in a shaker, immediately following: the Incubation period,: the tubes were washed five times with 0,161V! sbdium thiosulfate (SIS* Fisher Chemical,, Na2S203*5H20s. FW 248.18, #8446-3)) solution to inactivate the mutagen. After washinQ,::the cells in the: reaction lubes were Sheared with 21 gauge syringe needle (BO 1m! 2101 i-atex Free Syringe PrecisioriGiide® iNeedle, 0>8mmx25mm, #309624) and dilutions were completed and plated on YEMA plates. The eel! counts were available after five days incubation at SOX and the percent kill of the EMS application rate was calculated. To calculate the percent kill rate for each application rate, the following equation was used: {[cell count Dpi EMS (control) - (ceil count of μ I EMS (treatment)) * ceil count ΟμΙ EMS (control)] x 106%). At! other experiments following this experiment used this equation to calculate the percent kit rate. Results are provided in Table 10,

Table 18: initial rates of EMS to determine upper limit of mutagenesis for parental strain U5DA S32C

As shown in Table 10t the Initial rates of EMS used were 0 pi, Ϊ pi. 10pl and ΙΟΟμΙ. There was no differenoe among Opl) ΙμΚ and i0p| EMS, but 100μΐ EMS resulted in 180% kill.

Experiments were repeated add refined 'to determine the acceptable kill rate. See Tables 11-17.

Table 11: Refining the rates of EMS to determine 89.9% kill rate for parental strain USQA 63:20

From the initial·filing, the amount of EMS used was narrowed to Ομί, 15μΙ, 2Spi and SQpS EMS., As shown in fable 11, the percfontkili rate was 10.17% to 30.92% for 1δμΙ and 25pi EMS applications and 100% for 50pl.

Table 12; Additional refinement of the application rates of EMS for parental strain USDA 532C

it was determined horn Table 11 that 25u! EMS was still too lew of a kill rate. The application amounts for Table 12 were Opl, SSpI, 35pi, and δΟμ! EMS. The application af 25pi EMS had a higher kill rafa than the results in Table 11 due to the decreased washing; therefore, the hill rate vvas higher than expected. However, the kill rate was still too low at 86.58% kill even when 35μΙ EMS was used. See Table 12.

Table 13: Additional refinement of the applioation rates of EMS for parental Strain USDA 632G

The amount of EfoS used was increased to 4ΰμΙ, 45μΙ: and 0tlpl EMS. The EMS dose rates resulted in percent kill ranges of 82.28%~99J9%:, See Table 13.

Table: 14; Repeat applfo^od rate used In Table 13;

The results provided in: Table 13 were repeated again in Table 14 using the same EMS rates and this time the percent kill was 99.81% for 40μΙ EMS and 89,93% for 4|μΙ EMS and 100% for 50μ! EMS. The desired kit rate of 98.9% was observed when 45μί EMS was used so the application will be repeated See Table 15-

Table 15: Repeat application rate used in Table 14,

The application rate of 4Spi EMS was duplicated to determine if the results in Table 14 were repeatabie. The kT rate for 45μ i EMS was 99.98%, See Table 15.

Example til: Mutagenesis

The following experiments} were performed td generate putative desiccaitomresistant mutants of strains parental Strain USDA 5320 using classical, e,g.; chemical mutagenesis. A cell suspension of parental strain USDA 532C was made by taking a loop of cellsTrom a fresh plate of USDA532C by using a 10ui sterile plastic loop (Fisher brand, #22-883-809) and mixing the cells in imi sterilized deionized (Dl) water in 1,5ml disposable microcentrifuge· tube. The ceil suspension was· inoculated: into two 250mi flasks containing 50ml YEfvf medium to achieve a final optical density (0D) OPteA -of 0,01,,, The flasks were incubated at 30*C for three days and the cultures of the two flasks were combined. The culture was centrifuged for twenty minutes at 8.000 rpmin a Sorvaii RC 6 Pius centrifuge- The supernatant was discarded and the pbifei from r@~suspended In 30ml Di water, CD was taken of the concentrated culture and was inoculated into ten 250ml flasks containing 50ml YEM medium at OD~0<05, These flasks were incubated at 3p:aC shaker for two days prior to the ouiture being used tor mutagenesis,:

The cultures from the ten flasks were combined into a 11 centrifuge bottle. Optical density of the combined cultures was recorded and the cultures were centrifuged for 20 minutes at 8,000 rpm In the Sorvaii RC 8 Plus €> centrifuge. The majority of the supernatant was discarded leaving approximately 30ml of the supernatant In the centrifuge boitie. The supernatant was mixed with the pellet and transferred info a 50ml steriie disposable centrifuge tube. The PD of the concentrated culture was taken and recorded, 1m) of the concentrated, '.culture was placed Into six 1,5ml disposabie microcentrifuge tubes. The microcentrifuge tubes were centrifuged and the supernatant was discarded- This was repeated three more times or until the size of the pellet had reach the 0.1ml mark on the microcentrifuge tube. The cells were mixed well with 1ml fresh YEM medium using a sterile Imi 21 gauge syringe needle prior to the addition of the mutagen, ethyl methanesulfonate (EMS;). The rates of mutagen added to each tube eontaineda High and a low dose with medium: dosages between the high and low doses:;®® indicated in Experiment Ii, immediately after the addition of EMS, the reaction tubes were placed in an empty 2S0ml flask and incubated: at 3CTC for 30 minutes, After incubation, the reaction tubes were centrifuged for one minute at 13,200 rpm using the Eppendorf Centrifuge 64150, Tbs supernatant of the reaction tubes was discarded. The mutagen In the reaction tubes was Inactivated by washing five times with tmi of 0,1 BM sodium thiosulfate (STS) and mixed vigorously by vortexing the tubes. For each wash cycle, the reaction tubes were centrifuged after vortexing and the supernatant was discarded. After the fifth time, the reaction tubes were ali combined into one 15ml disposable tube for use in the enrichment process.

Example t¥: Enrichment and Desiccation

The fcilowing experimentfs) were performed to enrich and desiccate the mutated cells of parental strain USDA 632C to eitmlnate wild type escapes and increase tip putative mutant population and make it easier to isolate the mutant(s) that have desiocation-resistant characteristics,:

Parental strain USDA 532C was subjected to the mutagenesis process mentioned in Example 111, The mutated population of parental strain U8DA:632C was enriched by inoculating; 0.6m! of the reaction Into two 60mi YEM flasks and incubating the cells for two days: at 30*C Shaker, After two days, the cultures were desiccated by coating: the ceils onto soybean seeds and membrane filters and subjected to dryingi cpndltsons, The culture from the: enrichment step: was adjusted ta of 0.5 before it was used: toreoat both soybean seeds; and membrane fillers.:

Forty sterilized soybean seeds were coated with 0,5m! of the culture. The:seeds were placed in a 100m i sterile beaker to dry under the laminar hood and covered with autoclave paper. Triplicate samples of the seeds were taken to get an Initial GFU of the seeds. For each sample, three seeds were placed in a 15ml disposable tube containing Smi sterile Di water and allowed to expand in the tube for approximately two hours before the suspension was serially diluted and spread onto YEMA plates. The remaining seeds left in the covered, beaker was placed underthe hood for four days before the seeds were enriched,

To enrich the seeds, twenty seeds were placed into a 250mi flask containing fresh 60m! YEM. A final CFU Was also takeh when the seeds were enriched to determine the percent kill rate for the ceil population. The same sampling was completed for the second time point as the initial time point. After the culture containing the seeds was incubated for two days, the culture was harvested by removing any seed debris by allowing the debris to settle before removing the supernatant The supernatant of the culture was centrtfu§ed and the pallet washed with sterile D! water prior to the culture being used to coal new sets of soybean seeds. This process was repeated until the calculated percent kiil rate of the cell culture was less than 80%, Once 80% was achieved, the ceil population v^s ready for; isolation of the putative mutants for confirmation experiment.

Coating of membrane filters:

To control for soybean seed inconsistency and contamination issues, membrane filters were used as am alternative mediu m for cell coating . For membrane filters, 1ml of the culture was used to coat both durapore fMiliipom. 0.22 pm, PVDF, #GVWP02500) and isopore (fvliilipore. 0.4 pm, polycarbonate, # HTTP02500) membrane filters. For each type of filter, fifteen filters were coated by using a 25mm Easy Pressure Syringe Filter Holder (VWR, #28144-109) and the filters were placed in sterile Petri dish containing two pieces of stedie. guaiitati\® 125mm Whatman paper (Whatman, #1001125). Once the filters were dried under the laminar hood, triplicates Initial CPU were taken for each type of filter by placing one filter Into a 15mi disposable tube containing 6mI sterile Df water and mixed by vortexing. After two hours soaking in the 15ml tube, the filter suspension was diluted and plated onto YEMA plates. After the filters have dried under the hood for three days, eight filters were added into 25dml flask with SQfnl" fresh YEMand incubated1 at 3G*€ for three days. A final CPU was taken at the same time the filters were enriched la get the percent kill rate:; calculation. The same method was used for the final CPU as the initial CPU. The process of coating and drying was repeated until the percent kill rate was less than 80%. After 80% was achieved, single colony isolates were selected for further confirmation.

Example ¥: Confirmation of Putative Mutants

The following experiment(s) were to confirm the putative desiccation-resistant mutants by comparing their on-seed survivability after seed application with the original parent strain Sradyrhizobiuni japonimw strain US DA 532C,

When 80% kill rate was observed for the seeds or filters, single colonies were randomly picked: from the final time point and the putative mutants of a set of mutagenesis were analyzed for desiccation tolerance characteristic. The twenty single colonies picked from each set of mutagenesis results were individually grown in 250mi flask containing 50ml YEM medium. Each putative mutant strain was incubated at 30riC shaker for three days and OD for each strain was adjusted to 0,5. Each strain was used to coat thirty unsteriie soybean seeds with 0.5ml culture in a IQOmi beaker covered by autoclave paper. Time points were taken at T~(3J: T-3, and T~7 days for the first round. Triplicate seed samplings were taken for each time point where each sample consisted of three seeds placed In 5ml sterile Di water in 15ml disposable tube. The seeds were allowed to expand for two hours before each sample was diluted and plated onto YEMA plates. After comparing the amount of cells recovered from each time point in relation to parental strain USDA 5320, any strain that performed better than the parent strain was subjected to a second round of confirmation. See Fid. 1. For the second round, the strains that had the best desiccation tolerance compared to the wild type were tested for desiccation aga'ih. See Fie. 2. Time points were taken at T~0, T~?s and T~14 days. The set of putative mutants ftx>m the second round was turther confirmed for desiccation tolerance two more times. See Flos. 3-6.

Twenty putative mutants of parental strain USDA 5320 were Isolated and screened for desiccation resistance. See Figs. 1-3. Of the twenty putative strains tested, five putative mutant strains were confirmed for their desiccation tolerance characteristics when compared to the desiccation tolerance of parental strain, Bradyrhizobium japmtcum strain USDA S32C. See Figs 4-6.

Example Vi: Greenhouse testing of Confirmed Mutant Strains

The following experiment^) were performed to test the putative mutant strains in the greenhouse to test the performance of the mutant strains against the performance of the parent strain. USDA 532C.

The mutant strains having the best desiccation tolerance compared to the parent strain USDA 532C were tested in the greenhouse for performance against parental strain USDA 532C. The mutant and parent strains were grown in 50ml YE1VI for two days before seed coating. Each strain was planted three different times; T~Q, f~7, and: T~14 days after seed coating. All time points were set up at the same time but the seeds were planted at the specified times. To set up for the time points, thirty soybean seeds were coated with 0.5ml erf culture at 00^,,,,,--0.5 in 100ml beaker and the T~0 day time point was allowed to sit under the hood for 30 minutes before planting. The other two time points were allowed to dry completely and covered with autoclave paper. The seeds from the last two time points were planted at a later date. At each time point, two seeds were planted per pot for ten pots per strain. The leftover seeds were used to take a CFU for comparison with T~Q. After nine weeks of growing in the greenhouse, the soybean pods were harvested horn each plant from each time point and the dry weights were analyzed fer statistical significance.

When the soybean pod weights of the mutant strains were compared to the parent strain at any of time points, there was no statistical significance at 95% confidence. This indicates that there was no performance difference between the mutant strains and parent strain that would affect the production of soybean pods when the mutant strains were used to coat soybean seeds.

SUMMARY PARAGRAPHS

The present invention is defined in the claims and accompanying description. Additional aspects of the present invention are presented herein by way of numbered paragraphs. 1. A biologically pure culture of a Bradyrhizobium japonicum strain selected from the group consisting of: the strain having the deposit accession number NRRL B-50612; the strain having the deposit accession number NRRL B-50611; the strain having the deposit accession number NRRL B-50610; the strain having the deposit accession number NRRL B-50609; the strain having the deposit accession number NRRL B-50608, or a combination of at least two or more of the strains. 2. The strains of paragraph 1, wherein said strains have a superior desiccation resistance. 3. The strains of paragraph 2, wherein said desiccation resistance is compared to said desiccation resistance of a parental strain of said isolated strains, e.g., parental strain Bradyrhizobium japonicum US DA 532C. 4. The strains of paragraph 2, wherein said superior desiccation resistance includes an increased bacterial survival rate in a substantially moisture free environment when said survival rate of said isolated Bradyrhizobium strain(s) is compared to said survival rate of a parental strain of said isolates, e.g., parental strain Bradyrhizobium japonicum USDA 532C, over a period of time, e.g., at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year or more. 5. The strains of any of paragraphs 2-4, wherein said increased survival rate in said substantially moisture free environment includes an increased bacterial survival rate in an environment that is at least 70% moisture free, e.g., at least 75%, at least 80%, at least 85%, at least 00%, at least 91%, at least 92%, at least 93%, at least 94%, at least 98%, at least 98%, at least §7%, at least 98%, at least 99%, up to a 100% moisture free environment, when said survival rate of said isolated BradyrhmQbium strainfs) is oompared to said syfvival rate of a parental strain of said isolates, e.g,< parental· strain' Bmd^izobium'japwiwm USDA S32C. 8. A composition comprising one or more of said isolated bacterial strain(s) according, to paragraphs 1-5 and an agrieulturaify suitable Garner, 7. The composition of paragraph 6, wherein the composition includes M least one agneuituraily beneficial ingredient. 8. The composition of paragraph 7, wherein said at least one agriculturally beneficiai ingredient includes one or more plant signal molecules. 9. The composition of paragraph 8, wherein the plant signal molecule is a iipo~ chitooligosacchailde (LCD). 10 . the composition of paragraph 9 , wh erein the ICO. is synthetic. 11. The composition of paragraph 9,: wherein the LCD is recombina nt. 1&amp; The composition: of paragraph 9, wherein the LCD is naturally Dceurring, 13, The composition of paragraph 9, wherein the LCD Is obtained from a species of Rhfeobis selected from: Rhizobiism spp., SirmhizeMum spp, and: AzodMQhkm spp, 14; The composition of paragraph 9, wherein the, LCD is obtained from Bmdyrhimbtum japonicurn, 1:5. The .composition of paragraph 9, wherein the LCD is obtained from an arbuscuiar" mycorrhfeal fungus. 16. The composition of paragraph 8;: wherein the plant signal molecule is a chiiinous compound. 17. The composition of paragraph 16, wherein the chitinous compound is a chito-oligomer (CO). 18. The composition of paragraph 17, wherein the CO is synthetic. 19. The composition of paragraph 18, wherein the CO is recombinant. 20. The composition of paragraph 18, wherein the CO is naturally occurring. 21. The composition of paragraph 8, wherein the plant signal molecule is a flavonoid. 22. The composition of paragraph 21, wherein the flavonoid is selected from the group consisting of luteolin, apigenin, tangeritin, quercetin, kaempferol, myricetin, fisetin, isorhamnetin, pachypodol, rhamnazin, hesperetin, naringenin, formononetin, eriodictyol, homoeriodictyol, taxifolin, dihydroquercetin, dihydrokaempferol, genistein, daidzein, glycitein, catechin, gallocatechin, catechin 3-gallate, gallocatechin 3-gallate, epicatechin, epigallocatechin, epicatechin 3-gallate, epigallocatechin 3-gallate, cyanidin, delphinidin, malvidin, pelargonidin, peonidin, petunidin, or derivatives thereof. 23. The composition of paragraph 8, wherein the plant signal molecule is jasmonic acid or a derivative thereof. 24. The composition of paragraph 8, wherein the plant signal molecule is linoleic acid or a derivative thereof. 25. The composition of paragraph 8, wherein the plant signal molecule is linolenic acid or a derivative thereof. 26. The composition of paragraph 8, wherein the plant signal molecule is a karrikin. 27. The composition of any of paragraphs 8-26, wherein the composition includes at least two different plant signal molecules. 28, The composition of paragraph 27, wherein the agriculturally beneficial ingredient is a herbicide, insecticide or a fungicide. 29, The composition of paragraph 27, wherein the agricuturally beneficial ingredient Is at. least one phosphate sciuhliislng micraorganism, 30, The composition of paragraph 29, wherein lire at least one phosphate solubifiefng microorganism comprises a strain of the 31, The composition of paragraph 30, wherein the at least one phosphate solubilizing microorganism comprises a strain ofR Ma/ae. 32, The composition of paragraph 31, wherein the strain of P. Mfaia&amp; is selected from the group consisting of NRRL 50162, NRRL 50169, ATCC 20851, Mm 22348, and ATCC 18309. 33, The composition of paragraph 30, wherein the at least one phosphate solubilising microorganism comprises a strain of Al gaesfdyares, 34, The composition of paragraph 33, wherein the strain of P. gaestmmrm Is NRRL 50170. 35, A method for treating seeds comprising applying to said seeds an inoculum of one or more of the isolated bacterial straln(s) according to paragraph 1. 38. The method according to: paragraph 35, wherein a composition according to any of paragraphs 6-34 are applied to a seed. 37. The method according to any of paragraphs 35-36, wherein the method further comprises the step of storing said treated seeds In a substantially moisture free environment for a period of time, e,g,, at least T day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 8 weeks, at'least 4 weeks, at least 1 month, at feast 2 months, at least 3 months, at ieasf 4 months, at least 5 months, at least 6 months:, at least I: year or more. :38, The method according; to paragraph 37, Wherein said seeds are leguminous piant seeds. 39, The method according to paragraph 38, wherein said leguminous plant seeds are soybean seeds. 40, A method of enhancing plant growth, comprising applying to plants, plant seeds, or soil surrounding plants or plant seeds a composifion according to any of paragraphs 8-34, 41, The method according to paragraph 40, wherein said seeds are leguminous plant seeds. 42, The method according to paragraph 41, wherein said leguminous plant seeds are soybean seeds, 43, A method for enhancing the growth of a plant or plant part comprising contacting a plant or plant part with an inoculum of one or more of the strains of paragrapn 1. 44, The method of paragraph 43, wherein the method further comprises the step of subjecting the soil to one or more agriculturally benefldal ingredients, 45, The method of paragraph 43, wherein the treating step comprises iritrodudng the inoculum of one or more strains of paragraph 1 as a composition. 46, The method of paragraph 43, wherein the composition is the composition of any of paragraphs 6-34. 47, The method of paragraph-43,, wherein· the plant pari is: a,plant seed, 48, The method according to paragraph 47,: wherein said seeds are leguminous plant seeds, 49, The method according to paragraph 48, wherein said leguminous plant; seeds are soybean seeds. 50, A method for enhancing the growth of a plant or plant part comprising a. treating a soil with an inoculum of one or more Of the strains of paragraph If and b. growing a plant or plant part in the treated soli. 51, The method ¢4 paragraph 50, wherein the method further comprises the step of planting a plant or plant! part before, during, or after the treating step. 52, The method of paragraph SO, wherein the method.further comprises the step of su^ecting the soil to one or mom agriculturally feenelidal ingredients. 53, Tiie method of paragraph 50, wherein the treating step comprises introducing the one or more strains of paragraph las a composition, 54, Tiie method of paragraph 50. wherein the composition is the composition of any of paragraphs 6-34. 55, A seed coated with a composition of any of paragraphs 6-34,

The invention described amt claimed herein is not to be iimtted in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention, Indeed, various modifications of the Invention in: addition to those shown and described herein: will become apparent to those skilled in the art from the foregoing: description. Such modifications are aiso intended to fail within: the' scope of the appended claims. Sh the Case of conflict the present disclosure including: definitions: will control.

Various references are cited· herein, the disclosures of which are incorporated by reference in their entireties.

Claims (40)

1. A method of treating a seed, comprising applying to said seed an inoculum that comprises at least one mutagenized Bradyrhizobium japonicum strain having the deposit accession number NRRL B-50612.

2. The method of claim 1, wherein said inoculum further comprises one or more plant signal molecules.

3. The method of claim 1 or claim 2, wherein said inoculum further comprises a lipo-chitooligosaccharide (LCO).

4. The method of claim 3, wherein said LCO is obtained from a strain of Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, or Sinorhizobium.

5. The method of claim 3, wherein said LCO is obtained from a strain of Bradyrhizobium japonicum.

6. The method of claim 3, wherein said LCO is obtained from a strain of Rhizobium leguminosarum.

7. The method of claim 3, wherein said LCO is obtained from a strain of Sinorhizobium meliloti.

8. The method of claim 3, wherein said LCO is obtained from a mycorrhizal fungus.

9. The method of any one of claims 1 to 9, wherein said inoculum further comprises a chitinous compound.

10. The method of claim 9, wherein said chitinous compound is a chito-oligomer (CO).

11. The method of claim 10, wherein said CO is a chitooligosaccharide.

12. The method of any one of claims 1 to 11, wherein said inoculum further comprises a flavonoid.

13. The method of claim 12, wherein said flavonoid is selected from the group consisting of luteolin, apigenin, tangeritin, quercetin, kaempferol, myricetin, fisetin, isorhamnetin, pachypodol, rhamnazin, hesperetin, naringenin, formononetin, eriodictyol, homoeriodictyol, taxifolin, dihydroquercetin, dihydrokaempferol, genistein, daidzein, glycitein, catechin, gallocatechin, catechin 3-gallate, gallocatechin 3-gallate, epicatechin, epigallocatechin, epicatechin 3-gallate, epigallocatechin 3-gallate, cyanidin, delphinidin, malvidin, pelargonidin, peonidin, petunidin, and derivatives thereof.

14. The method of any one of claims 1 to 13, wherein said inoculum further comprises jasmonic acid or a derivative thereof.

15. The method of any one of claims 1 to 14, wherein said inoculum further comprises linoleic acid or a derivative thereof.

16. The method of any one of claims 1 to 15, wherein said inoculum further comprises linolenic acid or a derivative thereof.

17. The method of any one of claims 1 to 16, wherein said inoculum further comprises a karri kin.

18. The method of any one of claims 1 to 17, wherein said inoculum further comprises a herbicide, an insecticide and/or a fungicide.

19. The method of any one of claims 1 to 18, wherein said inoculum further comprises one or more additional beneficial microorganisms.

20. The method of claim 19, wherein said one or more additional beneficial microorganisms comprises one or more strains of Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and/or Sinorhizobium.

21. The method of claim 19 or claim 20, wherein said one or more additional beneficial microorganisms comprises one or more strains of Bradyrhizobium japonicum.

22. The method of any one of claims 19 to 21, wherein said one or more additional beneficial microorganisms comprises one or more strains of Mesorhizobium cicero.

23. The method of any one of claims 19 to 22, wherein said one or more additional beneficial microorganisms comprises one or more strains of Rhizobium leguminosarum.

24. The method of any one of claims 19 to 23, wherein said one or more additional beneficial microorganisms comprises one or more strains of Sinorhizobium meliloti.

25. The method of any one of claims 19 to 24, wherein said one or more additional beneficial microorganisms comprises one or more phosphate-solubilising microorganisms.

26. The method of claim 25, wherein said one or more phosphate-solubilising microorganisms comprises one or more strains of Penicillium.

27. The method of claim 25, wherein said one or more phosphate-solubilising microorganisms comprises one or more strains of P. bilaiae.

28. The method of claim 25, wherein said one or more phosphate-solubilising microorganisms comprises the P. bilaiae strain having the deposit accession number NRRL 50162, the P. bilaiae strain having the deposit accession number NRRL 50169, the P. bilaiae strain having the deposit accession number ATCC 20851, the P. bilaiae strain having the deposit accession number ATCC 22348, and/or the P. bilaiae strain having the deposit accession number ATCC 18309.

29. The method of any one of claims 25 to 28, wherein said one or more phosphate-solubilising microorganisms comprises one or more strains of P. gaestrivorus.

30. The method of any one of claims 25 to 28, wherein said one or more phosphate-solubilising microorganisms comprises the P. gaestrivorus strain having the deposit accession number NRRL 50170.

31. The method of any one of claims 1 to 30, further comprising the step of storing treated seed in a substantially moisture free environment for a period of at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months, or at least 1 year or more.

32. The method of any one of claims 1 to 31, wherein said seed is non-leguminous plant seed.

33. The method of any one of claims 1 to 31, wherein said seed is leguminous plant seed.

34. The method of any one of claims 1 to 31, wherein said seed is wheat seed.

35. The method of any one of claims 1 to 31, wherein said seed is barley seed.

36. The method of any one of claims 1 to 31, wherein said seed is sorghum seed.

37. The method of any one of claims 1 to 31, wherein said seed is oat seed.

38. The method of any one of claims 1 to 31, wherein said seed is pea seed.

39. The method of any one of claims 1 to 31, wherein said seed is corn seed.

40. The method of any one of claims 1 to 31, wherein said seed is chickpea seed.