RU2588483C2 - Bradyrhizobium strain for improving plant growth (versions), composition containing said strain and seed coated with composition - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: following is presented to improve plants growth: Bradyrhizobium japonicum NRRL b-50608, Bradyrhizobium japonicum NRRL b-50609, Bradyrhizobium japonicum NRRL b-50610, Bradyrhizobium japonicum NRRL b-50611, Bradyrhizobium japonicum NRRL b-50612. These strains are stimulating growth risobacteria (PGPR) which have excellent tolerance to dehydration/resistance to dehydration and improve total efficiency of plant growth. There is also disclosed a composition containing an acceptable for agriculture carrier and inoculum, containing the above strains either separately or in combination. Also disclosed is a seed, coated with the specified composition.

EFFECT: group of inventions refers to strains of Bradyrhizobium japonicum used for improving growth of plants, a composition based on the said strains and a seed coated with the said composition.

71 cl, 6 dwg, 15 tbl, 6 ex

Description

LINK TO DEPOSITED BIOLOGICAL MATERIAL

This application contains a reference to deposited biological material, which is incorporated herein by reference. See table 1 for complete details.

FIELD OF THE INVENTION

The present invention relates to an isolated Bradyrhizobium bacterium having improved characteristics, including but not limited to improved dehydration resistance.

BACKGROUND OF THE INVENTION

To maintain healthy growth, plants must extract many elements from the soil on which they grow. These elements include nitrogen and the so-called micronutrients (for example, copper, iron and zinc), but many types of soil are deficient in such elements or contain them only in forms that cannot be easily absorbed by plants (as a rule, they believe that necessary elements cannot be easily absorbed by plants if they are not present in dissolved form in the soil). Nitrogen is an essential element for most plants because it plays a role in the synthesis of amino acids, proteins, nucleotides, nucleic acids, chlorophyll, coenzymes and in the overall growth and health of a plant. To counter this deficiency, sources of scarce elements are usually introduced into the soil to improve the growth rate and yield obtained from agricultural plants. For example, nitrate and / or ammonium is often added to the soil to counter the lack of available nitrogen.

In the field of crop production, it is well known that for many crops it is necessary that the soil provides the plant with relatively large amounts of nitrogen. Notable exceptions from plants requiring nitrogen from the soil are legume plants.

Specifically, legumes are unique compared to non-leguminous plants in their ability to fix atmospheric nitrogen into ammonia. The plant's ability to fix atmospheric nitrogen into an applicable nitrogen source eliminates the need for the plant to receive nitrogen from the soil. To fix nitrogen, however, a symbiotic relationship between a legume plant and a natural bacterium inside the soil is needed. One of the most intensively studied partners in this symbiotic relationship are 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 usually achieved through a complex two-way signal exchange between a plant and a microorganism and between a microorganism and a plant. Typically, plant factors, such as flavonoids and flavonoid-like substances, induce colonization of bacteria in the root nodule of the legume plant. (Gresshoff, 1999). After the colonization of bacteria in the root nodule, the bacteria cause morphological changes in the plant, namely, twisting of the root hair and the development of a new root organ - the nodule. (Gresshoff, 1999). The nodule allows the formation of a new physiological environment for the nodule, which induces the differentiation of bacteria into nitrogen-fixing endosymbiont, or bacteriod, for a colonized plant. (Gresshoff, 1999).

To facilitate a symbiotic two-way signal exchange between the plant and the microorganism, bacteria such as Bradyrhizobia sp. Often cover the seeds. To prolong the viability of the microorganism on the seed, it is desirable that the microorganism is resistant to dehydration and, in general, to dry environmental conditions.

There remains a need for microorganisms with improved resistance to dehydration.

SUMMARY OF THE INVENTION

New bacterial strains having improved dehydration resistance are described herein, especially when the new strains are compared to their parent strain, for example, the parent strain of Bradyrhizobium sp., USDA 532C. The inventors have isolated and tested a significant number of bacterial strains by their dehydration resistance properties.

As discussed throughout this document, the isolated strains are strains from the genus Bradyrhizobium spp. In particular, the isolated strains are Bradyrhizobium japonicum strains. Even more specifically, the isolated strains are isolated Bradyrhizobium japonicum strains selected from the group consisting of:

strain with depository access number NRRL B-50608;

strain with depository access number NRRL B-50609;

strain with accession number NRRL B-50610;

strain with depository access number NRRL B-50611;

strain with accession number NRRL B-50612, or a combination of at least two or more of the above deposited strains.

Also provided herein are compositions comprising a carrier and one or more of the bacterial strains described herein. In one embodiment, the composition comprises one or more plant signaling 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 another embodiment, the composition comprises at least one flavonoid. In another embodiment, the composition comprises jasmic acid or a derivative thereof. In another embodiment, the composition comprises linoleic acid or a derivative thereof. In another embodiment, the composition comprises linolenic acid or a derivative thereof. In another embodiment, the composition comprises curricin.

In addition, this document describes a method for improving the growth of a plant or part of a plant, comprising contacting a plant or part of a plant with one or more of the bacterial strains described herein. The method includes introducing into the soil of the inoculum one or more of the bacterial strains described herein. In another embodiment, the method includes introducing into the soil of the inoculum one or more of the bacterial strains in the form of a seed coating.

This document also describes a method for improving nitrogen fixation in a plant (s), comprising growing a plant (s) in soil containing one or more of the bacterial strains described herein. In one embodiment, the plant (s) is a bean plant (s), not a bean plant (s), or a combination thereof. In another embodiment, the plant is a plant selected from the group consisting of soy, beans, alfalfa, clover, corn, lettuce, tomatoes, potatoes, cucumbers, and combinations thereof.

In addition, seeds coated with the bacterial strains described herein are described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of a preliminary screening of compared dehydration resistant mutants when compared with the dehydration resistance of the parent strain USDA 532C.

FIG. 2 is a graphical representation of a second screening of the compared dehydration resistant mutants when compared with the dehydration resistance of the parent strain USDA 532C.

FIG. 3 is a graphical representation of a third screening of the compared dehydration resistant mutants when compared with the dehydration resistance of the parent strain USDA 532C.

FIG. 4 is a graphical representation of a fourth screening of the compared dehydration resistant mutants when compared with the dehydration resistance of the parent strain USDA 532C.

FIG. 5 is a bar graph representing the dehydration resistance of the selected compared dehydration resistant mutants when compared with the dehydration resistance of the parent strain USDA 532C on days 0 (0) and fourteenth (14).

FIG. 6 is a bar graph representing the dehydration resistance of the selected compared dehydration resistant mutants when compared with the dehydration resistance of the parent strain USDA 532C on the seventh (7) and fourteenth (14) days.

DETAILED DESCRIPTION OF THE INVENTION

The bacterial strains described are isolated and tested for their ability to solubilize phosphorus. This is described in detail in the Examples section below. In addition, the described embodiments relate to compositions, seed coatings, methods of increasing the availability of phosphorus to be absorbed by a plant from the soil, and methods of increasing the absorption of phosphorus by plants, including growing plants in soil containing a source of phosphorus.

Definitions:

As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly requires otherwise.

As used herein, the term “biologically pure culture” is intended to mean a culture that is generally free of biological contamination and has genetic uniformity, so that for the various subcultures taken from it, substantially identical genotypes and phenotypes are shown (for example, cultures having purity 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 at least 92%, at least 93%, by exchange necks least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% up to 100% purity).

As used herein, the term “isolate, isolates, isolation and / or isolation, etc.” is intended to mean that the material referred to is removed from the environment in which it is found to be normal.

As used herein, the term “inoculum” is intended to mean any form of bacterial cells or spores that are capable of propagating on or in the soil when conditions of temperature, humidity, etc., are favorable for bacterial growth.

As used herein, the term “dispute” has its usual meaning, which is well known and understood by those skilled 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 under the soil conditions under consideration, does not make this element completely accessible for absorption by the plant.

As used herein, the terms "effective amount", "effective concentration", or "effective dose" are intended to mean the amount, concentration or dose of one or more bacterial isolates sufficient to cause an increase in the growth of a plant or part of a plant or an increase in nitrogen fixation . The absolute value of the actual effective dose depends on factors, including, but not limited to, the size (for example, area, total area in acres, etc.) of the field for introducing bacterial isolates, synergistic or antagonistic interactions with other active or inert ingredients that may increase or decrease the activity of bacterial isolates, and the stability of bacterial isolates in compositions and / or in seed treatment forms. An "effective amount", an "effective concentration" or an "effective dose" of a bacterial composition can be determined, for example, by means of a conventional dose-dependent experiment.

As used herein, the terms “carrier” or “agronomically acceptable carrier” are intended to mean any material that can be used to deliver active agents (eg, a bacterial strain) to a seed, soil, plant or part of a plant.

As used herein, the term “soil compatible carrier” is intended to mean any material that can be added to the soil without causing / having an adverse effect on plant growth, soil structure, soil drainage, or the like.

As used herein, the term “seed-compatible carrier” is intended to mean any material that can be added to a seed without causing / adversely affecting a seed, seed-growing plant, seed germination, or the like.

As used herein, the term “agricultural benefit ingredient (s)” is intended to mean any agent or combination of agents capable of inducing or providing a beneficial and / or beneficial effect in agriculture.

As used herein, “at least one biologically active ingredient” is intended to mean biologically active ingredients, for example, signaling molecules, other microorganisms, etc.) other than one or more of the bacterial isolates described herein.

As used herein, the term "dehydration" is intended to indicate a condition of unusual dryness, for example, conditions without moisture and / or water. The terms “dehydration resistance” and / or “dehydration tolerance” are intended to include the body’s ability to withstand conditions and / or survive in conditions and / or withstand conditions of extreme dryness.

As used herein, the terms “nitrogen fixation”, “atmospheric nitrogen fixation”, or “nitrogen fixation”, etc. are intended to include biological processes in which molecular nitrogen or nitrogen in the atmosphere is converted to one or more nitrogen (N) compounds, including, but not limited to, ammonia, ammonium salts, urea, and nitrates.

As used herein, the term “nitrogen-fixing organism” is intended to mean any organism (eg, diazotrophs) capable of converting molecular nitrogen or nitrogen in the atmosphere into one or more nitrogen (N) compounds, including, but not limited to, ammonia, salts ammonium, urea and nitrates.

As used herein, the terms “solubilization of phosphate”, or “increasing the solubility of phosphate”, etc. are intended to indicate the transfer of insoluble phosphate (e.g., mineral phosphate, etc.) to a soluble form of phosphate.

As used herein, the term “solubilizing phosphate organism” is intended to mean any organism capable of converting insoluble phosphate into a soluble form of phosphate.

As used herein, the terms “plant (s)” and “plant part (s)” are intended to mean all plants and plant populations, such as desirable and undesired wild plants or agricultural plants (including naturally occurring agricultural plants). Agricultural plants can be plants that can be obtained by conventional methods of plant breeding and optimization, or by methods of biotechnology and genetic engineering, or by a combination of these methods, including transgenic plants and including cultivars of plants protected or not protected by the rights of plant breeders. Parts of a plant should be understood to mean all parts and organs of plants above and below the ground, such as shoot, leaf, flower and root, examples of which are leaves, needles, cuttings, trunks, flowers, fruiting bodies, fruits, seeds, roots , nodules, tubers, and rhizomes. Parts of the plant also include harvested material and material for vegetative and generative propagation (e.g., cuttings, tubers, rhizomes, shoots and seeds, etc.).

As used herein, the term “nodule” is intended to mean, but not limited to, determined nodules, non-deterministic nodules, or a combination thereof. Examples of deterministic nodules and non-deterministic nodules are well known in the art and are described in Denison, R. F., 2000, The Amer. Naturalist. 156 (6): 567-576. Deterministic nodules were found in Glycine, Lotus, or Phaseolus species and are round and spherical in shape. (Denison, 2000) Deterministic nodules grow only for a limited period of time — typically a few weeks. (Denison, 2000) Unlike deterministic nodules, non-deterministic nodules are found in the species Medicago, Trifolium and Pisium, have an elongated shape and grow continuously. (Denison, 2000) The term “nodule colonization” is a term known in the art. McDermott T.R. & Graham, P. H., Appl. and Environ. Microbiol. 55 (10): 2493-2498. It is well known in the art that, despite rare exceptions, a single nodule contains only one bacterial strain. Johnston, A.W. B., et al., 1974, J. Gen. Microbiol 87: 343-350; Dunham, D. H. & Baldwin, I. L., 1931, Soil Science 32: 235-249; Johnson, H. W., et al., 1963, Agrono. J. 55: 269-271; Dudman, W.F. & Brockwell, J., 1968, J. Agricul. Res. 19: 739-747; Nicol, H. & Thorton, H. G., 1941, Proc. Roy. Soc. B 130, 32-59; Hughes, D.Q. & Vincent, J. M., 1942, Proc. of the Linnenan Soc. of New South Wales 67: 142-152; and Vincent, J.M. & Waters, L. M., 1953, J. Gen. Microbiol. 9: 357-370.

As used herein, the term “enhanced plant growth” is intended to mean an increased plant yield (eg, increased biomass, increased number of fruits, or a combination thereof, as measured in bushels per acre), increased number of roots, increased root mass, increased volume roots, increased leaf area, increased plant density, increased plant power, increased plant mass (e.g., total dry weight of a plant or part of a plant, total light weight its plants and parts of plants, etc.), or combinations thereof.

As used herein, “enhanced competitiveness” and / or “enhanced nodule formation” is a definition for a bacterial strain (s) having a population percentage of nodules, for example at least 50%, at least 55%, at least at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 9 6%, at least 97%, at least 98%, at least 99%, up to 100% of the population of nodules.

As used herein, the term "temperature stability" is intended to mean the temperature range at which the bacterial strain (s) are able to grow, for example, the maximum and minimum temperatures at which the bacterial strain can grow.

As used herein, the term “commercially available strain (s)” is intended to mean commercially available bacterial strains, for example, USDA 532C, USDA 110, USDA 123, USDA 127, USDA 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 mean the nutrients necessary for plant growth, plant health and / or plant development.

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

As used herein, the term “moisturizing agent (s)” is intended to mean any substance capable of reducing 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, strain (s) is strain (s) Bradyrhizobum sp. In a further aspect, the strain is derived from a Bradyrhizobium strain, including, but not limited to, a strain selected from the group consisting of Bradyrhizobium bete, Bradyrhizobium canariense, Bradyrhizobium elkanii, Bradyrhizobium iriomotense, bradyrhizobium yrhymidium, Bradyrhizobium japonicum, Bradyrhizobium japonicum, Bradyrhizobium japonicum, Bradyrhizobium japonicum, Bradyrhizobium japonicum, Bradyrhizobium japonicum, Bradyrhizobium japonicum, Bradyrhizobium japonicum, Bradyrhizobium japonicum In another embodiment, strain (s) is Bradyrhizobum japonicum strain (s).

In another embodiment, the isolated strain (s) is Bradyrhizobium sp. Strain (s) having an improved / increased percentage of bacterial survival in a substantially anhydrous environment, when the percent survival of the isolated Bradyrhizobium strain (s) is compared with the percent survival of the parent strain (s) ), for example, the parent strain of Bradyrhizobium japonicum USDA 532C, for a period of time, for example at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 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 another embodiment, the isolated strain (s) is Bradyrhizobium sp. Strain (s) having an improved / increased survival rate in a substantially anhydrous environment, where an increased survival rate in a substantially anhydrous environment includes an increased percentage of bacterial survival in an external environment that is at least 70% anhydrous, for example at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 91%, at least and 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% anhydrous external environment, when the percentage of survival of the isolated strain (s) of Bradyrhizobium is compared with the percentage of survival of the parent strain (s), for example, the parent strain of Bradyrhizobium japonicum USDA 532C.

In another embodiment, the isolated strain (s) is Bradyrhizobium sp. Strain (s) having the following improved / superior characteristics when compared to commercially available strains, for example, the commercial Bradyrhizobium japonicum USDA 532C strain, where the improved / excellent characteristics include, but without limitation:

a. improved competitiveness for the colonization of soy plants and

b. improved effectiveness in stimulating soybean plant growth.

In another embodiment, the isolated strain (s) is Bradyrhizobium sp. Strain (s) having improved / superior competitiveness for plant colonization. In another embodiment, the isolated strain (s) is Bradyrhizobium sp. Strain (s) having improved / excellent potency for stimulating plant growth. In another embodiment, the isolated strain (s) is Bradyrhizobium sp. Strain (s) having improved / excellent competitiveness for colonizing a plant and improved / excellent effectiveness for promoting plant growth.

In another aspect of the present invention, the isolated strain (s) is Bradyrhizobium sp. Strain (s) having improved / excellent temperature resistance.

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

In another embodiment, the strains are Bradyrhizobum japonicum strains selected from the group consisting of:

strain with depository access number NRRL B-50608;

strain with depository access number NRRL B-50609;

strain with accession number NRRL B-50610;

strain with depository access number NRRL B-50611; and

strain with accession number NRRL B-50612.

In a specific embodiment, the strain (s) may be one or more of the aforementioned deposited strains (for example, including at least two of the aforementioned strains, at least three of the aforementioned strains, at least four of the aforementioned strains, up to all and including all of the above strains).

In one embodiment, the strain is a strain with accession number NRRL B-50608. In one embodiment, the strain is a strain with accession number NRRL B-50609. In one embodiment, the implementation of the strain is a strain with a depository access number NRRL B-50610. In one embodiment, the implementation of the strain is a strain with a custody access number NRRL B-50611. In one embodiment, the strain is a strain with accession number NRRL B-50612.

In another embodiment, the bacterial culture (s) possesses properties identical to at least one of the deposited strains or a combination of at least two of the aforementioned deposited strains, including more than two, for example at least three of the aforementioned strains, at least four of the above strains, up to and including all of the above strains. The properties / characteristics of the bacterial culture include, but are not limited to, bacterial strains having improved and / or excellent resistance to dehydration. In another embodiment, the strain (s) is Bradyrhizobium strain (s) having improved and / or excellent dehydration resistance, when dehydration resistance is compared to dehydration resistance and / or dehydration tolerance of the parent bacterial strain (s), for example, parent strain of Bradyrhizobium japonicum USDA 532C.

In another aspect, the isolated bacterial strain (s) of the present invention includes strain (s) closely related to any of the above strains based on the identity of the 16S rDNA sequence. See Stackebrandt E, et al., “Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology”, Int J Syst Evol Microbiol. 52 (3): 1043-7 (2002) regarding the use of 16S rDNA sequence identity to determine bacterial affinity. In one embodiment, the at least one strain is at least 95% identical to any of the above strains based on the 16S rDNA sequence identity, at least 96% identical to any of the above strains based on the 16S rDNA sequence identity, at least 97% identical to any of the above strains based on the identity of the 16S rDNA sequence, at least 98% identical to any of the above strains based on the identity of the rdN sequence 16S, at least 98.5% identical to any of the above strains based on the 16S rDNA sequence identity, at least 99% identical to any of the above strains based on the 16S rDNA sequence identity, or at least 99.5% identical to any of the above strains based on the identity of the 16S rDNA sequence.

The Bradyrhizobium bacterium described herein, and in particular strains with depository access numbers NRRL B-50608, NRRL B-50609, NRRL B-50610, NRRL B-50611 and NRRL B-50612, can be grown by methods known in the art.

The resulting material can be used directly in the composition, as a seed treatment, or spores can be collected, concentrated by centrifugation, formulated and then dried using methods of air drying, lyophilization or drying in a fluidized bed (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 obtain a wettable powder.

The aforementioned deposited strains were deposited on October 1, 2012, as described in more detail below in the Materials and Methods section, under the terms of the Budapest Agreement on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure in the American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108, USA.

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 in the form of spores or strains in the vegetative state) described herein. In specific embodiments, the composition may be in the form of a liquid, suspension, solid, or powder (wettable powder or dry powder). In another embodiment, the composition may be in the form of a seed coating. Compositions in the form of a liquid, suspension or powder (eg, wettable powder) may be suitable for coating seeds. When used to coat seeds, the composition can be applied to seeds and allowed to dry. In embodiments, when the composition is a powder (eg, a wettable powder), a liquid, such as water, may be necessary to add to the powder before application to the seeds. Examples of other carriers include moistened bran, dried, sieved and applied to the seeds prior to coating with an adhesive, such as gum arabic.

Carriers:

The carriers described herein allow the deposited bacterial strain (s) to remain effective (e.g., capable of fixing nitrogen) and viable after formulation. The carriers described herein include, but are not limited to, liquids, suspensions, or solids (including wettable powders or dry powders). In one embodiment, the carrier is a soil compatible carrier as described herein.

In one embodiment, the carrier is a liquid carrier. Liquids suitable as carriers for the compositions described herein include, but are not limited to, 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 an aqueous solution of sugar. In another embodiment, the carrier is a non-aqueous solution. If a liquid carrier is used, the liquid (e.g., aqueous) carrier may further include growth medium for culturing the deposited bacterial strains. Non-limiting examples of suitable growth media for deposited bacterial strains include arabinose gluconate (AG) medium, yeast extract and mannitol (YEM) medium, G16 medium, or any medium known to those skilled in the art as being compatible with nutrients and / or providing nutrients for deposited bacterial strains.

In another embodiment, the carrier is a suspension. In one embodiment, the suspension may comprise an adhesive, liquid, or a combination thereof. It is contemplated that the adhesive may be any agent capable of adhering the inoculum (e.g., one or more of the deposited strains) to a substrate of interest (e.g., seed). Adhesives include, but are not limited to, alginate, mineral oil, syrup, gum arabic, honey, methyl cellulose, milk, wallpaper glue, and combinations thereof. Suitable suspension liquids include, but are not limited to, water or an aqueous sugar solution.

In another embodiment, the carrier is a solid. In a specific 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 granules. Solids suitable as carriers for the compositions discussed herein include, but are not limited to, peat, wheat, wheat chop, ground wheat straw, bran (e.g., moist bran, non-moist bran), vermiculite, cellulose, starch , soil (pasteurized or not pasteurized), gypsum, talc, clay (e.g. kaolin, bentonite, montmorillonite) and silica gels.

Optional agriculturally preferred ingredients:

The compositions described herein may contain one or more optional ingredients. Optional ingredients include, but are not limited to, one or more biologically active ingredients, trace elements, biostimulants, preservatives, polymers, moisturizers, surfactants, or combinations thereof.

Biologically active ingredient (s):

The compositions described herein may optionally include one or more biologically active ingredients, as described herein. Biologically active ingredients include, but are not limited to, signaling molecules (e.g., lipo-chitooligosaccharides (LCO), chitooligosaccharides (CO), chitin compounds, flavonoids, jasmic acid or its derivatives, linoleic acid or its derivatives, linolenic acid or its derivatives, carricins and etc.) and advantageous microorganisms (e.g. Rhizobium spp., Bradyrhizobium spp., Sinorhizobium spp., Azorhizobium spp., etc.).

Signal molecule (s):

In one embodiment, the compositions described herein comprise one or more signaling molecules. In one embodiment, one or more signal molecules is one or more LCOs. In another embodiment, one or more signaling molecules is one or more chitin compounds. In another embodiment, one or more signal molecules are one or more CO. In another embodiment, one or more signaling molecules are one or more flavonoids or their derivatives. In another embodiment, one or more signaling molecules are one or more non-flavonoid inducers of the nod gene (e.g., jasmic acid, linoleic acid, linolenic acid, and derivatives thereof). In another embodiment, one or more signaling molecules are one or more carricins or their derivatives. In another embodiment, one or more signal molecules are one or more LCO, one or more chitin compounds, one or more CO, one or more flavonoids and their derivatives, one or more non-flavonoid inducers of the nod gene and their derivatives, one or several carrickins and their derivatives, or any combination of signaling molecules from them.

LCO:

Compounds of lipo-chitooligosaccharides (LCOs), also known in the art as Nod symbiotic signals or Nod factors, consist of an oligosaccharide backbone of из-1,4-linked N-acetyl-D-glucosamine residues (“GlcNAc”) with an N-linked fatty acyl chain condensed with a non-reducing end. LCOs differ in the number of GlcNAc 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 LCO is presented below in the form of formula I:

,

,

wherein:

G represents hexosamine, which may be substituted, for example, by an acetyl group on a nitrogen atom, a sulfate group, an acetyl group and / or an ether group on an oxygen atom,

R ₁ , R ₂ , R ₃ , R ₅ , R ₆ and R ₇ , which may be identical or different, are H, CH ₃ CO—, C _x H _y CO—, where x is an integer between 0 and 17, and y is an integer between 1 and 35, or any other acyl group, for example, such as carbamyl,

R ₄ is a mono-, di- or trisaturated aliphatic chain containing at least 12 carbon atoms, and n is an integer between 1 and 4.

LCO can be obtained (isolated and / or purified) from bacteria such as Rhizobia, for example, Rhizobium spp., Bradyrhizobium spp., Sinorhizobium spp. and Azorhizobium spp. The LCO structure is characteristic of each such type of bacteria, and each strain can produce many LCOs with different structures. For example, specific LCOs from S. meliloti are also described in US Pat. No. 5,549,718 as having the formula II:

,

,

in which R represents H or CH ₃ CO-, and n is equal to 2 or 3.

Even more specific LCOs include NodRM, NodRM-1, NodRM-3. Upon acetylation (R = CH ₃ CO-), they become AcNodRM-1 and AcNodRM-3, respectively (US Patent 5,545,718).

LCOs from Bradyrhizobium japonicum are described in US Pat. Nos. 5,175,149 and 5,321,011. Broadly, they are pentasaccharide phytohormones containing methyl fucose. A number of these originating from B. japonicum LCO: BjNod-V (C _{18: 1} ); BjNod-V (A _{C, C 18: 1),} BjNod-V (C _{16: 1);} and BjNod-V (A _C , C _{16: 0} ), where “V” denotes the presence of five groups of N-acetylglucosamine; "Ac" - acetylation; the number after “C” indicates the number of carbon atoms in the side chain of the fatty acid; and the number after “:” is the number of double bonds.

LCOs used in the compositions of the invention can be obtained (i.e., isolated and / or purified) from bacterial strains producing LCO, such as strains of Azorhizobium, Bradyrhizobium (including B. japonicum), Mesorhizobium, Rhizobium (including R. leguminosarum ), Sinorhizobium (including S. meliloti), and bacterial strains genetically modified for LCO production.

The present invention also relates to compositions using LCO obtained (i.e., isolated and / or purified) from mycorrhizal fungi, such as fungi from the Glomerocycota group, for example, Glomus intraradicus. The structures of representative LCOs obtained from these fungi are described in WO 2010/049751 and WO 2010/049751 (the LCOs described in this document are also referred to as “Myc factors”).

In addition, the use of synthetic LCO compounds, such as those described in WO 2005/063784, and recombinant LCOs obtained by genetic engineering is included in the compositions of the present invention. The primary, natural LCO structure may contain modifications or substitutions found in natural LCOs, such as those described in Spaink, Crit. Rev. Plant Sci. 54: 257-288 (2000) and D'Haeze, et al., Glycobiology 72: 79R-105R (2002). Oligosaccharide precursor molecules (CO, which, as described below, are also suitable as plant signaling molecules of the present invention) for constructing LCO, can also be synthesized by genetically modified organisms, for example, as in Samain, et al., Carb. Res. 302: 35-42 (1997); Samain, et al., J. Biotechnol. 72: 33-47 (1999).

LCO can be used in forms of varying purity, and they can be used alone or in the form of a culture of LCO producing bacteria or fungi. Methods for producing substantially pure LCOs include simply removing microbial cells from a mixture of LCOs and microorganisms, or continuing to isolate and purify LCOs by phase separation of the LCOs — solvent followed by HPLC chromatography as described, for example, in US Pat. No. 5,549,718. Purification can be improved by repeated HPLC, and purified LCO molecules can be lyophilized for long-term storage.

CO:

Chitooligosaccharides (CO) are known in the art as -1-4 linked N-actylglucosamine structures identified as chitin oligomers, as well as N-acetylchitooligosaccharides. CO possess unique and various additional side chain groups that make them different from chitin molecules [(C ₈ H ₁₃ NO ₅ ) n, CAS No. 1398-61-4], and chitosan molecules [(C ₅ H ₁₁ NO ₄ ) n, CAS No. 9012-76-4]. Representative literature describing the structure and production of CO are: Van der Hoist, et al., Current Opinion in Structural Biology, 77: 608-616 (2001); Robina, et al., Tetrahedron 58: 521-530 (2002); Hanel, etal., Planta 232: 787-806 (2010); Rouge, et al. Chapter 27, “The Molecular Immunology of Complex Carbohydrates” in Advances in Experimental Medicine and Biology, Springer Science; Wan, et al., Plant Cell 27: 1053-69 (2009); PCT / F100 / 00803 (9/21/2000); and Demont-Caulet, et al., Plant Physiol. 120 (1): 83-92 (1999). CO can be synthetic or recombinant. Methods for producing recombinant CO are known in the art. See, for example, Samain, et al. (above.); Cottaz, et al., Meth. Eng. 7 (4): 311-7 (2005) and Samain, et al., J. Biotechnol. 72: 33-47 (1999).

Chitin compounds:

Chitins and chitosans, which are the main components of the cell walls of fungi and exoskeletons of insects and crustaceans, also consist of GlcNAc residues. Chitin compounds include chitin, (IUPAC: N- [5 - [[3-acetylamino-4,5-dihydroxy-6- (hydroxymethyl) oxan-2-yl] methoxymethyl] -2 - [[5-acetylamino-4 , 6-dihydroxy-2- (hydroxymethyl) oxan-3-yl] methoxymethyl] -4-hydroxy-6- (hydroxymethyl) oxan-3-is] ethanamide), and chitosan, (IUPAC: 5-amino-6- [ 5-amino-6- [5-amino-4,6-dihydroxy-2-hydroxymethyl) oxan-3-yl] oxy-4-hydroxy-2- (hydroxymethyl) oxan-3-yl] oxy-2 (hydroxymethyl) oxane-3,4-diol).

These compounds can be obtained commercially, for example, from Sigma-Aldrich, or obtained from insects, shells of crustaceans or cell walls of fungi. Methods for producing chitin and chitosan are known in the art and are described, for example, in US Pat. No. 4,536,207 (production of shellfish), Pochanavanich, et al., Lett. Appl. Microbiol. 35: 17-21 (2002) (production from fungal cell walls), and US Pat. No. 5,965,545 (production of crab shells and hydrolysis of commercial chitosan). Deacetylated chitins and chitosans can be obtained in the range from less than 35% to more than 90% deacetylation, and they cover a wide range of molecular weights, for example, low molecular weight chitosan oligomers less than 15 kDa and chitin oligomers 0.5-2 kDa; "practical class" chitosan with a molecular weight of approximately 15 kDa; and high molecular weight chitosan up to 70 kDa. Chitin and chitosan formulations formulated for seed treatment are also commercially available. Commercial products include, for example, ELEXA (Plant Defense Boosters, Inc.) and BEYOND (Agrihouse, Inc.).

Flavonoids:

Flavonoids are phenolic compounds having a common structure of two aromatic rings connected by a bridge of three carbon atoms. Flavonoids are produced by plants and have many functions, for example, as primary signaling molecules, and as protection against insects, animals, fungi and bacteria. Classes of flavonoids include chalcones, anthocyanidins, coumarins, flavones, flavanols, flavonols, flavanones and isoflavones. See, Jain, et al., J. Plant Biochem.  Biotechnol. 77: 1-10 (2002); Shaw, et al. Environmental Microbiol. 77: 1867-80 (2006).

Representative flavonoids that may be suitable in the compositions of the present invention include luteolin, apigenin, tangeritin, quercetin, kempferol, myricetin, fisetin, isoramnetin, pachipodol, ramnazine, hesperetin, naringenin, formononetinodiodeinodiodeiodioderiodioderiodioderiodioderiodioderiodioderiodiodeiodioderiodioderiodioderiodiodeiodioderiodioderiodiodeiodioderiodioderiodioderiodioderiodiodeiodioderiodiodeliodioderiodioinodioderoinodiodeinodiodeinodiodeiodioderiodiodelihydrodinolide dihydrocampferol, genistein, daidzein, glycitein, catechin, gallocatechin, 3-gallate catechin, 3-gallate gallocatechin, epicatechin, epigallocatechin, 3-gallate epicatechin, 3-gallate epigallocatechin, cyanidine, dolphinidine, lvidine, pelargonidine, peonidine, petunidine or their derivatives. Flavonoid compounds are commercially available, for example, from Natland International Corp., Research Triangle Park, NC; MP Biomedicals, Irvine, CA; LC Laboratories, Woburn MA. Flavonoid compounds can be isolated from plants or seeds, for example, as described in US Patents 5,702,752; 5,990,291; and 6146668. Flavonoid compounds can also produce genetically modified organisms, such as yeast, as described in Ralston, et al., Plant Physiology 737: 1375-88 (2005).

Flavonoid-Free Nod Gene Inducer (s):

Jasmic acid (JA, [1R- [1, 2 (Z)]] - 3-oxo-2- (pentenyl) cyclopentane acetic acid) and its derivatives, linoleic acid ((Z, Z) -9,12-octadecadiene acid) and its derivatives, and linolenic acid ((Z, Z, Z) -9,12,15-octadecatrienoic acid) and its derivatives can also be used in the compositions of the present invention. Jasmine acid and its methyl ester, methyl jasmonate (MeJA), collectively known as jasmonates, are octadecane-based compounds found naturally in plants. Jasmic acid is produced by the roots of wheat seedlings, and fungal microorganisms such as Botryodiplodia theobromae and Gibbrella fujikuroi, yeast (Saccharomyces cerevisiae), and pathogenic and non-pathogenic Escherichia coli strains. Linoleic acid and linolenic acid are formed during the biosynthesis of jasmic acid. It has been reported that jasmonates, linoleic acid and linoleic acid (and their derivatives) are inducers of nod gene expression or LCO production by rhizobacteria. See, for example, 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”, USDA 3, May 17, 2001.

Suitable derivatives of linoleic acid, linolenic acid and jasminic acid, which may be suitable in the 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 jasminic acid is replaced by a —COR group, where R is a —OR ¹ group in which R ¹ is an alkyl group such as C ₁ -C _{8 a} straight or branched alkyl group, for example a methyl, ethyl or propyl group; an alkenyl group such as a C ₂ -C ₈ straight or branched alkenyl group; an alkynyl group such as a C ₂ -C ₈ straight or branched alkynyl group; an aryl group having, for example, 6-10 carbon atoms; or a heteroaryl group having, for example, 4-9 carbon atoms, where the heteroatoms in the heteroaryl group can be, for example, N, O, P or S. Representative amides are compounds in which the carboxyl group of linoleic acid, linolenic acid or jasmine the acid is replaced with a —COR group, where R is an NR ² R ³ group in which R ² and R ³ independently are: hydrogen; an alkyl group such as a C ₁ -C ₈ unbranched or branched alkyl group, for example a methyl, ethyl or propyl group; an alkenyl group such as a C ₂ -C ₈ straight or branched alkenyl group; an alkynyl group such as a C ₂ -C ₈ straight or branched alkynyl group; an aryl group having, for example, 6-10 carbon atoms; or a heteroaryl group having, for example, 4-9 carbon atoms, where the heteroatoms in the heteroaryl group can be, for example, N, O, P or S. Esters can be obtained by known methods, such as acid-catalyzed nucleophile addition, where the reaction is carried out carboxylic acid with alcohol in the presence of a catalytic amount of inorganic acid. Amides can also be prepared by known methods, such as reacting a carboxylic acid with a suitable amine in the presence of a facilitating coupling reaction of an agent such as dicyclohexylcarbodiimide (DCC) under neutral conditions. Suitable salts of linoleic acid, linolenic acid and jasminic acid include, for example, base addition salts. Bases that can be used as reagents to produce metabolically acceptable base salts of these compounds include bases derived from cations, such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium). These salts can be easily obtained by mixing together a solution of linoleic acid, linolenic acid or jasmic acid with a solution of the base. Salt can be precipitated from the solution and collected by filtration or can be isolated by other methods, such as evaporation of the solvent.

Carrickin (s):

Carrickins are vinylogical 4H-pyrons, for example, 2H-furo [2,3-c] pyran-2-ones, including their derivatives and analogues. Examples of these compounds are represented by the following structure:

,

,

where Z represents O, S or NR ₅ ; R ₁ , R ₂ , R ₃ and R ₄ each independently represents H, alkyl, alkenyl, alkynyl, phenyl, benzyl, hydroxy, hydroxyalkyl, alkoxy, phenyloxy, benzyloxy, CN, COR ₆ , COOR =, halogen, NR ₆ R ₇ , or NO ₂ ; and R ₅ , R ₆ and R ₇ each independently represents H, 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, hydrobromide, sulfate or bisulfate, phosphate or hydrogen phosphate, acetate, benzoate, succinate, fumarate, maleate, lactate, citrate, tartrate, gluconate; methanesulfonate, benzenesulfonate and p-toluenesulfonic acid. Additional biologically acceptable metal salts include alkali metal salts, with bases, examples of which include sodium and potassium salts. Examples of compounds which are encompassed by the structure and which may be suitable for use in the present invention include the following: 3-methyl-2H-furo [2,3-c] pyran-2-one (where R ₁ = CH ₃ , R ₂ , R ₃ , R ₄ = H), 2H-furo [2,3-c] pyran-2-one (where R ₁ , R ₂ , R3, R4 = H), 7-methyl-2H-furo [ 2,3-c] pyran-2-one (where R ₁ , R ₂ , R ₄ = H, R ₃ = CH ₃ ), 5-methyl-2H-furo [2,3-c] pyran-2-one (where R ₁ , R ₂ , R ₃ = H, R ₄ = CH ₃ ), 3,7-dimethyl-2H-furo [2,3-c] pyran-2-one (where R ₁ , R ₃ = CH ₃ , R ₂ , R ₄ = H), 3,5-dimethyl-2H-furo [2,3-c] pyran-2-one (where R ₁ , R ₄ = CH ₃ , R ₂ , R ₃ = H ), 3,5,7-trimethyl-2H-furo [2,3-c] pyran-2-one (where R ₁ , R ₃ , R ₄ = CH ₃ , R ₂ = H), 5-methoxymethyl-3 -methyl-2H-furo [2,3-c] pyran-2-one (g de R ₁ = CH ₃ , R ₂ , R ₃ = H, R ₄ = CH ₂ OCH ₃ ), 4-bromo-3,7-dimethyl-2H-furo [2,3-c] pyran-2-one ( where R ₁ , R ₃ = CH ₃ , R ₂ = Br, R ₄ = H), 3-methylfuro [2,3-c] pyridin-2 (3H) -one (where Z = NH, R ₁ = CH ₃ , R ₂ , R ₃ , R ₄ = H), 3,6-dimethylfuro [2,3-c] pyridin-2 (6H) -one (where Z = N-CH ₃ , R ₁ = CH ₃ , R ₂ , R ₃ , R ₄ = H). See US Patent 7,576,213. These molecules are also known as carricins. See Halford, “Smoke Signals,” in Chem. Eng. News (April 12, 2010), on pages 37-38 (which states that carricken or butenolides contained in smoke act as stimulants for the growth and germination of seed shoots after a forest fire, and can stimulate seeds, for example, corn, tomatoes, lettuce and onion after storage). These molecules are the subject of US Pat. No. 7,576,213.

Beneficial microorganism (s):

In one embodiment, the compositions described herein may contain one or more beneficial microorganisms. One or more beneficial microorganisms may have one or more beneficial properties (for example, producing one or more of the signaling molecules described herein, enhancing the absorption of nutrients and water, enhancing growth, enhancing seed germination, enhancing seedling emergence, and interrupting a dormant state or dormancy of a plant, etc.).

In one embodiment, the beneficial microorganism (s) comprise one or more bacteria producing one or more of the signaling molecules described herein. In another embodiment, the bacteria are bacteria of the genus Rhizobium spp. (e.g. R. cellulosilyticum, R. daejeonense, R. etli, R. galegae, R. gallicum, R. giardinii, R. hainanense, R. huautlense, R. indigoferae, R. leguminosarum, R. loessense, R. lupini , R. lusitanum, R. meliloti, R. mongolense, R. miluonense, R. sullae, R. tropici, R. undicola and / or R. yanglingense), Azorhizobium spp. (e.g. A. caulinodans and / or A. doebereinerae), Sinorhizobium spp. (e.g. S. abri, S. adhaerens, S. americanum, S. aboris, S. fredii, S. indiaense, S. kostiense, S. kummerowiae, S. medicae, S. meliloti, S. mexicanus, S. morelense , S. saheli, S. terangae and / or S. xinjiangense), Mesorhizobium spp., (M. albiziae, M. amorphae, M. chacoense, M. ciceri, M. huakuii, M. loti, M. mediterraneum, M . pluifarium, M. septentrionale, M. temperatureum and / or M. tianshanense) and combinations thereof. In a specific embodiment, the beneficial microorganism is selected from the group consisting of R leguminosarum, R meliloti, S. meliloti, and combinations thereof. In another embodiment, the beneficial microorganism is R leguminosarum. In another embodiment, the beneficial microorganism is R meliloti. In another embodiment, the beneficial microorganism is S. meliloti.

In another embodiment, one or more beneficial microorganisms comprise one or more phosphate solubilizing microorganisms. Solubilizing phosphate microorganisms include strains of fungi and bacteria. In one embodiment, the solubilizing phosphate microorganism includes species from a genus selected from the group consisting of Acinetobacter spp. (e.g. Acinetobacter calcoaceticus, etc.), Arthrobacter spp, Arthrobotrys spp. (e.g. Arthrobotrys oligospora, etc.), Aspergillus spp. (e.g., Aspergillus niger, etc.), Azospirillum spp. (e.g. Azospirillum halos praeferans, etc.), Bacillus spp. (e.g., Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans, Bacillus licheniformis, Bacillus subtilis, etc.), Burkholderia spp. (e.g. Burkholderia cepacia, Burkholderia vietnamiensis, etc.), Candida spp. (e.g. Candida krissii, etc.), Chryseomonas spp. (e.g. Chryseomonas luteola, etc.), Enterobacter spp. (e.g. Enterobacter aerogenes, Enterobacter asburiae, Enterobacter spp., Enterobacter taylorae, etc.), Eupenicillium spp. (e.g. Eupenicillium parvum, etc.), Exiguobacterium spp., Klebsiella spp., Kluyvera spp. (e.g. Kluyvera cryocrescens, etc.), Microbacterium spp., Mucor spp. (e.g., Mucor ramosissimus, etc.), Paecilomyces spp. (e.g. Paecilomyces hepialid, Paecilomyces marquandii, etc.), Paenibacillus spp. (e.g. Paenibacillus macerans, Paenibacillus mucilaginosus, etc.), Penicillium spp. (E.g., Penicillium bilaiae (formerly known as Penicillium bilaii), Penicillium albidum, Penicillium aurantiogriseum, Penicillium chrysogenum, Penicillium citreonigrum, Penicillium citrinum, Penicillium digitatum, Penicillium frequentas, Penicillium fuscum, Penicillium gaestrivorus, Penicillium glabrum, Penicillium griseofulvum, Penicillium implicatum, Penicillium janthinellum, Penicillium lilacinum, Penicillium minioluteum, Penicillium montanense, Penicillium nigricans, Penicillium oxalicum, Penicillium pinetorum, Penicillium pinophilum, Penicillium purpurogenum, Penicillium radicans, Penicillium radicum, Penicillium raistrickii, Penicillium rugulosum, Penicillium simplicissimum, Penicillium solitum, Penicillium variabile, Penicillium velutinum, Penicillium viridicatum, Penicillium glaucum, Penicillium fussiporus and Penicillium expansum, etc.), Pseudomonas spp. (e.g. Pseudomonas corrugate, Pseudomonas fluorescens, Pseudomonas lutea, Pseudomonas poae, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas trivialis, etc.), Serratia spp. (e.g. Serratia marcescens, etc.), (e.g. Stenotrophomonas maltophilia, etc.), Streptomyces spp., Streptosporangium spp., Swaminathania spp. (e.g. Swaminathania salitolerans, etc.), Thiobacillus spp. (e.g. Thiobacillus ferrooxidans, etc.), Torulospora spp. (e.g. Torulospora globosa, etc.), Vibrio spp. (e.g. Vibrio proteolyticus, etc.), Xanthobacter spp. (e.g., Xanthobacter agilis, etc.), Xanthomonas spp. (e.g. Xanthomonas campestris, etc.) and combinations thereof.

In a specific embodiment, one or more phosphate solubilizing microorganisms is a Penicillium fungus strain. In another embodiment, one or more Penicillium species is P. bilaiae, P. gaestrivorus, or a combination thereof.

In another embodiment, the beneficial microorganism is one or more mycorrhiza. In particular, one or more mycorrhiza is endomycorrhiza (also called vesicular treelike mycorrhiza, VAM, treelike mycorrhiza, or AM), ectomycorrhiza, or a combination thereof.

In one embodiment, one or more mycorrhiza is an endomycorrhiza of the phylum Glomeromycota and the genera Glomus and Gigaspora. In another embodiment, the endomycorrhiza is a strain of Glomus aggregatum, Glomus brasilianum, Glomus clarum, Glomus deserticola, Glomus etunicatum, Glomus fasciculatum, Glomus intraradices, Glomus monosporum or Glomus mosseae, Gigaspora margarita, or a combination thereof.

In another embodiment, one or more mycorrhiza is an ectomycorrhiza of the phylum Basidiomycota, Ascomycota, and Zygomycota. In another embodiment, the ectomycorrhiza is a strain of Basidiomycota, Ascomycota, and Zygomycota. In another embodiment, the ectomycorrhiza is a strain of Laccaria bicolor, Laccaria laccata, Pisolithus tinctorius, Rhizopogon amylopogon, Rhizopogon fulvigleba, Rhizopogon luteolus, Rhizopogon villosuli, Scleroderma cepa, Scleroderma cit.

In another embodiment, one or more mycorrhiza is ericoid mycorrhiza, arbutoid mycorrhiza, or monotropic mycorrhiza. Arbuscular mycorrhiza and ectomycorrhiza form ericoid mycorrhiza with many plants belonging to the Ericales order, while some Ericales form arbutoid and monotropoid mycorrhiza. All orchids are mycoheterotrophic at some stages during their life cycle and form orchid mycorrhiza with a number of basidiomycete fungi. In one embodiment, the mycorrhiza may be an ericoid mycorrhiza, preferably Ascomycota phylum, for example, Hymenoscyphous ericae or Oidiodendron sp. In another embodiment, the mycorrhiza may also be an arbutoid mycorrhiza, preferably a phylum of Basidiomycota. In another embodiment, the mycorrhiza may be a monotropoid mycorrhiza, preferably a phylum of Basidiomycota. In another embodiment, the mycorrhiza may be orchid mycorrhiza, preferably of the genus Rhizoctonia.

Micronutrient (s):

In another embodiment, the compositions described herein may contain one or more micronutrient benefits. Trace elements for use in the compositions described herein include, but are not limited to, vitamins (e.g., vitamin A, vitamin B complex (i.e., vitamin B ₁ , vitamin B ₂ , vitamin B ₃ , vitamin B ₅ , vitamin B ₆ , vitamin B ₇ , vitamin B ₈ , vitamin B ₉ , vitamin B ₁₂ , choline) vitamin C, vitamin D, vitamin E, vitamin K, carotenoids (α-carotene, β-carotene, cryptoxanthin, lutein, lycopene , zeaxanthin, etc.), macrominerals (e.g. phosphorus, calcium, magnesium, potassium, sodium, iron, etc.), trace elements (e.g. boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, zinc, etc.), organic acids (e.g., acetic acid, citric acid, lactic acid, malic acid, taurine, etc.) and their combinations In a particular embodiment, the compositions may contain phosphorus, boron, chlorine, copper, iron, manganese, molybdenum, zinc, or combinations thereof.

In specific embodiments, where the compositions described herein may contain phosphorus, it is contemplated that any suitable source of phosphorus may be provided. In one embodiment, phosphorus may be obtained from a source. In another embodiment, suitable phosphorus sources include phosphorus sources capable of solubilizing with one or more microorganisms (e.g., Penicillium bilaiae, etc.).

In one embodiment, phosphorus can be obtained from a source of mineral phosphate. In another embodiment, phosphorus can be obtained from fertilizers containing one or more sources of phosphorus. There are many types of commercially available industrial phosphate fertilizers. Some common ones are fertilizers containing phosphorite, monoammonium phosphate, diammonium phosphate, calcium monophosphate, superphosphate, triple superphosphate and / or ammonium polyphosphate. All these fertilizers are obtained by chemical processing of insoluble natural phosphorites at large-scale fertilizer manufacturing enterprises, and the product is expensive. By the methods of the present invention, it is possible to reduce the amount of these fertilizers applied to the soil, while still retaining the same amount of phosphorus from the soil.

In another embodiment, phosphorus can be obtained from an organic source of phosphorus. In a further specific embodiment, the phosphorus source may include organic fertilizer. Organic fertilizer refers to a soil supplement derived from natural sources that provides at least minimal percentages of nitrogen, phosphate and potassium carbonate. Non-limiting examples of organic fertilizers include plant and animal by-products, mineral powders, algae, modifiers, and structurants. They are often available at horticultural centers and through horticulture supply companies. In particular, the organic source of phosphorus is a source of bone meal, meat meal, manure, compost, sewage sludge or guano, or combinations thereof.

In another embodiment, phosphorus can be obtained from a combination of phosphorus sources, including, but not limited to, mineral phosphate, fertilizers containing one or more phosphorus sources (e.g., monoammonium phosphate, diammonium phosphate, calcium monophosphate, superphosphate, triple superphosphate, ammonium polyphosphate and etc.), one or more organic sources of phosphorus and their combinations.

Biostimulant (s):

In one embodiment, the compositions described herein may contain one or more beneficial biostimulants. Biostimulants can enhance metabolic or physiological processes, such as respiration, photosynthesis, absorption of nucleic acids, absorption of ions, delivery of nutrients, or a combination thereof. Biostimulants include, but are not limited to, algae extracts (e.g., ascophyllum nodosum), humic acids (e.g., potassium humate), fulvic acids, myoinositol, glycine, and combinations thereof. In another embodiment, the compositions comprise algae extracts, humic acids, fulvic acids, myoinositol, glycine, and combinations thereof.

Polymer (s):

In one embodiment, the compositions described herein may further comprise one or more polymers. Non-limiting uses of polymers in agricultural production include delivery of agrochemicals, removal of heavy metals, water retention and / or water delivery, and combinations thereof. Pouci, et al., Am. J. Agri. &  Biol. Sci., 3 (7): 299-314 (2008). In one embodiment, 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), and t .d.) or a combination thereof.

A non-limiting list of polymers suitable for the compositions described herein, see Pouci, et al., Am. J. Agri. &  Biol. Sci., 3 (7): 299-314 (2008). In one embodiment, the compositions described herein comprise cellulose, cellulose derivatives, methyl cellulose, methyl cellulose derivatives, starch, agar, alginate, pectin, polyvinylpyrrolidone, and combinations thereof.

Moisturizer (s):

In one embodiment, the compositions described herein may further comprise one or more moisturizing agents. Moisturizers are generally accepted for use in soils, in particular in hydrophobic soils, to improve the infiltration and / or penetration of water into the soil. The moisturizing agent may be an adjuvant, oil, surfactant, buffer, acidifier, or a combination thereof. In one embodiment, the moisturizing agent is a surfactant. In one embodiment, the moisturizing agent is one or more nonionic surfactants, one or more anionic surfactants, or a combination thereof. In another embodiment, the moisturizing agent is one or more non-ionic surfactants.

Surfactants suitable for the compositions described herein are presented in the section "Surfactants".

Surfactant (s):

Surfactants suitable for the compositions described herein may be nonionic surfactants (e.g., semipolar and / or anionic and / or cationic and / or zwitterionic). It is contemplated that the surfactant (s) can do as little harm to the activity of one or more deposited strains and / or one or more advantageous microorganisms as possible. Surfactants can moisturize and emulsify the soil (s) and / or soil (s). It is contemplated that the surfactants used in the composition described have low toxicity to the microorganisms contained in the composition. In addition, it is contemplated that the surfactants used in the described composition have low phytotoxicity (i.e., the degree of toxicity of the substance or combination of substances available to the plant). You can use a single surfactant or a mixture of several surfactants.

Anionic Surfactants

Anionic surfactants or mixtures of anionic and nonionic surfactants can also be used in the compositions. Anionic surfactants are surfactants having a hydrophilic group in an anionic or negatively charged state in an aqueous solution. The compositions described herein may contain one or more anionic surfactants. The anionic surfactant (s) may be water-soluble anionic surfactants, water-insoluble anionic surfactants, or a combination of water-soluble anionic surfactants and insoluble anionic surfactants. Non-limiting examples of anionic surfactants include sulfonic acids, sulfuric acid esters, carboxylic acids and their salts. Water soluble anionic surfactants include, but are not limited to alkyl sulfates, alkyl ether sulfates, sulfates alkilamidoefirov, sulfates, alkylaryl, alkylaryl, alkylaryl sulfonates, sulfates, monoglyceride sulfates, alkylsulfonates, alkilamidsulfonaty, alkylaryl sulfonates, benzene sulfonates, toluene sulfonates, xylene sulfonates, cumene sulfonates, alkyl benzene sulfonates, alkildifeniloksidsulfonaty, alpha olefin sulfonates, alkylnaphthalene sulfonates, paraffinsulfonates, lignes sulphonates, alkyl sulphosuccinates, ethoxylated sulphosuccinates, sulphosuccinates of alkyl ethers, alkyl sulphosuccinates, alkyl sulphosuccinamates, alkyl sulphoacetates, alkyl phosphates, esters phosphates, alkyl esters of alkyl esters, N-alkyl acylates, acyl n-acyls,

Nonionic Surfactants

Nonionic surfactants are surfactants that do not have an electric charge when dissolved or dispersed in an aqueous medium. In at least one embodiment of the composition described herein, one or more nonionic surfactants are used as they provide the desired effects of moisturizing and emulsification and do not significantly inhibit spore stability and activity. The non-ionic surfactant (s) can be water-soluble non-ionic surfactants, water-insoluble non-ionic surfactants, or a combination of water-soluble non-ionic surfactants and water-insoluble non-ionic surfactants.

Water-insoluble non-ionic surfactants

Non-limiting examples of water-insoluble non-ionic surfactants include alkyl and aryl: glycerol esters, glycol ethers, ethanolamides, sulfonamides, alcohols, amides, alcohol ethoxylates, glycerol esters, glycol esters, glycerol esters and glycol esters ethoxylates sugar-based alkyl polyglycosides, polyoxyethylene fatty acids, condensed compounds alkanolamine, alkanolamides, tertiary acetylene glycols, polyoxyethylene mercaptans, complex carboxylic acid esters, polyoxyethylene polyoxypropylene glycols, sorbitan fatty esters, or combinations thereof. Also included are EO / PO block copolymers (EO is ethylene oxide, PO is propylene oxide), EO polymers and copolymers, polyamines and polyvinylpyrrolidones.

Water Soluble Nonionic Surfactants

Non-limiting examples of water-soluble non-ionic surfactants include fatty acid ethoxylates, alcohols and sorbitan, and fatty acid ester ethoxylates and sorbitan.

Combination of nonionic surfactants

In one embodiment, the compositions described herein comprise at least one or more non-ionic surfactants. In one embodiment, the compositions comprise at least one water-insoluble non-ionic surfactant and at least one water-soluble non-ionic surfactant. In another embodiment, the compositions comprise a combination of nonionic surfactants having hydrocarbon chains of substantially the same length.

Other surfactants

In another embodiment, the compositions described herein may also contain organosilicon surfactants, silicone-based antifoams used as surfactants in silicone-based antifoams and in mineral oil-based antifoams. In another embodiment, the compositions described herein may also contain alkali metal salts of fatty acids (for example, water-soluble salts of alkali metals of fatty acids and / or water-insoluble salts of alkali metals of fatty acids).

Herbicide (s):

In one embodiment, the compositions described herein may further comprise one or more herbicides. In a specific embodiment, the herbicide may be a pre-emergence herbicide, a post-emergence herbicide, or a combination thereof.

Suitable herbicides include chemical herbicides, natural herbicides (e.g. bioherbicides, organic herbicides, etc.), or a combination thereof. Non-limiting examples of suitable herbicides include bentazone, acifluorophene, chlorimuron, lactophen, clomazone, fluazifop, glufosinate, glyphosate, setoxime, imazetapyr, imazamox, fomezafen, fumiclorac, imazquin, celodim, pendimetal; 3,4-dimethyl-2,6-dinitro-N-pentan-3-yl-aniline; N- (1-ethylpropyl) -2,6-dinitro-3,4-xylidine; pronamide; propisamide; 3,5-dichloro-N- (1,1-dimethylpropinyl) benzamide; 3,5-dichloro-N- (1,1-dimethyl-2-propinyl) benzamide; N- (1,1-dimethylpropinyl) -3,5-dichlorobenzamide; S-ethyl-N-ethylthiocyclohexanecarbamate; trifluralin; 2,6-dinitro-N, N-dipropyl-4- (trifluoromethyl) aniline; glyphosate; N- (phosphonomethyl) glycine; and their derivatives. In one embodiment, one or more herbicides for use in accordance with this description include pronamide (commercial designation Kerb); propisamide; 3,5-dichloro-N- (1,1-dimethylpropinyl) benzamide; 3,5-dichloro-N- (1,1-dimethyl-2-propinyl) benzamide; N- (1,1-dimethylpropinyl) -3,5-dichlorobenzamide; cycloate, S-ethyl-N-ethylthiocyclohexanecarbamate (commercial designation Ro-Neet); trifluralin; 2,6-dinitro-N, N-dipropyl-4- (trifluoromethyl) aniline; glyphosate; N- (phosphonomethyl) glycine; and their derivatives. Commercial products containing each of these compounds are readily available. The concentration of herbicides in the composition typically corresponds to the frequency of use of a particular herbicide indicated on the label.

Fungicide (s):

In one embodiment, the compositions described herein may further comprise one or more fungicides. Fungicides suitable for the compositions described herein suitably have activity against a wide range of pathogens, including but not limited to Phytophthora, Rhizoctonia, Fusarium, Pythium, Phomopsis or Selerotinia and Phakopsora, and combinations thereof.

Non-limiting examples of commercial fungicides that may be suitable for the compositions described herein include PROTÉGÉ, RIVAL or ALLEGIANCE FL or LS (Gustafson, Piano, TX), WARDEN RTA (Agrilance, St. Paul, MN), APRON XL , APRON MAXX RTA or RFC, MAXIM 4FS or XL (Syngenta, Wilmington, DE), CAPTAN (Arvesta, Guelph, Ontario) and PROTREAT (Nitragin Argentina, Buenos Ares, Argentina). Active ingredients in these and other commercial fungicides include, but are not limited to, fludioxonil, mefenoxam, azoxystrobin, and metalaxyl. Commercial fungicides are most suitably used in accordance with the manufacturer's instructions at recommended concentrations.

Insecticide (s):

In one embodiment, the compositions described herein may further comprise one or more insecticides. Insecticides suitable for the compositions described herein suitably have activity against a wide range of insects, including, but not limited to wireworms, scoops, vermiform larvae damaging the roots of corn larvae, sprout fly larvae, earthen fleas, ground bugs, aphids, leaf-eaters, shield insects and their combination.

Non-limiting examples of commercial insecticides that may be suitable for the compositions described herein include CRUISER (Syngenta, Wilmington, DE), GAUCHO and PONCHO (Gustafson, Piano, TX). Active ingredients in these and other commercial insecticides include thiamethoxam, clothianidin, and imidacloprid. Commercial insecticides are most suitably used in accordance with the manufacturer's instructions at recommended concentrations.

WAYS

In another aspect, methods of using deposited strains and compositions described herein are described.

In one embodiment, a method for enhancing plant growth is described. The method includes contacting a plant or part of a plant with an inoculum of one or more bacterial strains selected from the group consisting of:

strain with depository access number NRRL B-50608;

strain with depository access number NRRL B-50609;

strain with accession number NRRL B-50610;

strain with depository access number NRRL B-50611;

strain with accession number NRRL B-50612, or

mixtures of two or more strains.

In a specific embodiment, the inoculum may contain one or more of the aforementioned deposited strains (for example, 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 one embodiment, the inoculum contains strains with an accession number of access NRRL B-50608. In one embodiment, the inoculum comprises a strain with accession number NRRL B-50609. In one embodiment, the inoculum comprises a strain with an accession deposit number of NRRL B-50610. In one embodiment, the inoculum comprises a strain with accession number NRRL B-50611. In one embodiment, the inoculum comprises a strain with accession number NRRL B-50612.

In another embodiment, the step of contacting the plant or part of the plant with the inoculum of one or more of the deposited bacterial strains includes contacting the plant or part of the plant with one or more of the compositions described herein. Inoculum (s) or compositions may be prepared for contact with a plant or part of a plant in accordance with methods known to those skilled in the art. Non-limiting examples include introduction to furrows, seed coating, etc. In a specific embodiment, the contacting step comprises introducing into the furrows the inoculum or compositions described herein. In a specific embodiment, the contacting step comprises applying to the seed (seed coating) the inoculum or compositions described herein.

In specific embodiments, the step of contacting the plant or part of the plant with the inoculum of one or more of the deposited bacterial strains includes introducing the inoculum into the soil in an amount of 1 × 10 ¹ -1 × 10 ⁸ , more preferably 1 × 10 ⁶ -1 × 10 ¹² colony forming units per hectare. In other specific embodiments, the step of contacting the plant or part of the plant with the inoculum of one or more of the deposited bacterial strains includes administering the deposited bacterial strains in the form of a seed coated with 1 × 10 ¹ -1 × 10 ⁸ , more preferably 1 × 10 ² - 1 × 10 ⁶ colony forming units per seed.

In another aspect, the method includes growing plants in soil containing one or more bacterial strains. The method includes:

a) soil treatment with an inoculum of one or more bacterial strains selected from the group consisting of:

strain with depository access number NRRL B-50608;

strain with depository access number NRRL B-50609;

strain with accession number NRRL B-50610;

strain with depository access number NRRL B-50611;

strain with depository access number NRRL B-50612; or mixtures of two or more strains; and

b) growing a plant in cultivated soil.

In a specific embodiment, the inoculum may contain one or more of the above deposited strains (for example, 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 one embodiment, the inoculum comprises a strain with accession number NRRL B-50608. In one embodiment, the inoculum comprises a strain with accession number NRRL B-50609. In one embodiment, the inoculum comprises a strain with an accession deposit number of NRRL B-50610. In one embodiment, the inoculum comprises a strain with accession number NRRL B-50611. In one embodiment, the inoculum comprises a strain with an accession deposit number of NRRL B-50612.

The step of treating the soil with an inoculum of one or more of the deposited bacterial strains includes treating the soil with one or more of the compositions described herein. The inoculum (s) or compositions may be introduced into the soil in accordance with methods known to those skilled in the art. Non-limiting examples include introduction to furrows, seed coating, etc. In a specific embodiment, the treatment step includes introducing into the grooves the inoculum or compositions described herein. In a specific embodiment, the processing step includes applying to the seed (seed coating) the inoculum or compositions described herein.

In a specific embodiment, the step of treating the soil with an inoculum of one or more of the deposited bacterial strains includes treating the soil with one or more of the compositions described herein. In specific embodiments, the step of treating the soil with an inoculum of one or more deposited bacterial strains includes treating the soil with an inoculum in an amount of 1 × 10 ¹ -1 × 10 ⁸ , more preferably 1 × 10 ⁶ -1 × 10 ¹² colony forming units per hectare. In other specific embodiments, the step of treating the soil with an inoculum of one or more of the deposited bacterial strains comprises administering the deposited bacterial strains in the form of a seed coated with 1 × 10 ¹ -1 × 10 ⁸ , more preferably 1 × 10 ² -1 × 10 ⁶ colony forming units per seed.

In another embodiment, the method further includes the step of planting the plant or part of the plant. The planting step may occur before, after, or during the processing step. In one embodiment, the planting step occurs prior to the processing step. In another embodiment, the planting step occurs during the processing step (for example, the landing step occurs simultaneously with the processing step, the landing step occurs substantially simultaneously with the processing step, etc.). In another embodiment, the planting step occurs after the processing step.

In another embodiment, the method further includes the step of exposing the soil to one or more agriculturally preferred ingredients described herein. In one embodiment, the step of exposing the soil to one or more agriculturally advantageous ingredients occurs before, during, after the treatment step, or simultaneously with the treatment step. In one embodiment, the step of exposing the soil to one or more agriculturally advantageous ingredients, as described herein, occurs prior to the treatment step. In another embodiment, the step of exposing the soil to one or more agriculturally advantageous ingredients, as described herein, occurs during the treatment step. In another embodiment, the step of exposing the soil to one or more agriculturally advantageous ingredients, as described herein, occurs after the treatment step. In another embodiment, the step of exposing the soil to one or more agriculturally preferred ingredients, as described herein, occurs simultaneously with the treatment step (e.g., treating the soil with one or more of the compositions described herein, etc. )

In another embodiment, the invention includes a seed treatment method comprising applying to the inoculum seeds one or more bacterial strains selected from the group consisting of:

strain with depository access number NRRL B-50608;

strain with depository access number NRRL B-50609;

strain with accession number NRRL B-50610;

strain with depository access number NRRL B-50611;

strain with depository access number NRRL B-50612; or mixtures of two or more strains.

In a particular embodiment, the seed treatment method may include one or more of the aforementioned deposited strains (for example, including at least two of the above strains, at least three of the above strains, at least four of the above strains, all and including all of the above strains).

In one embodiment, a seed treatment method comprises applying to a seed a strain with an accession number of access NRRL B-50608. In one embodiment, a seed treatment method comprises applying to a seed a strain with an accession number of access NRRL B-50609. In one embodiment, a seed treatment method comprises applying to a seed a strain with an accession number of access NRRL B-50610. In one embodiment, a seed treatment method comprises applying to a seed a strain with an accession number of access NRRL B-50611. In one embodiment, a seed treatment method comprises applying to a seed a strain with accession number NRRL B-50612.

In another embodiment, the method further includes the step of applying to the seeds one or more agriculturally advantageous ingredients. In another embodiment, the method comprises applying to the seeds any of the compositions described herein.

In another embodiment, the method includes storing seeds with an inoculum of at least one or more of the isolated bacterial strains in a substantially anhydrous external environment for a period of time, for example at least 1 day, at least 2 days, at least 2 days 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 one aspect of the method, the seeds are leguminous plant seeds. In another aspect, the seeds of a legume plant are soybean seeds.

The methods described herein are potentially useful for improving growth conditions, resulting in increased phosphorus uptake and / or yield for any type of plant. In one specific embodiment, the plant is selected from the group consisting of non-legumes, legumes, Brassica spp., Cereals, fruits, vegetables, nuts, flowers and lawn grass. In particular, cereals are wheat, corn, rice, oats, rye, barley. In particular, legumes are lentils, chickpeas, beans, soy, peas and alfalfa.

In another specific embodiment, the plants are selected from the group consisting of alfalfa, rice, wheat, barley, rye, oats, cotton, sunflower, peanuts, corn, potatoes, sweet potatoes, beans, peas, chickpeas, lentils, chicory, lettuce, endive , cabbage, Brussels sprouts, beets, parsnips, turnips, cauliflower, broccoli, rutabaga, radish, spinach, onions, garlic, eggplant, pepper, celery, carrots, large-fruited pumpkin, pumpkin, zucchini, cucumber, apple, pear, melon, citrus fruits, strawberries, grapes, raspberries, pineapple, soy, tobacco, tomato, soy rgo and sugarcane.

SEED COATINGS

In another aspect, the seeds are coated with one or more bacterial strains selected from the group consisting of:

strain with depository access number NRRL B-50608;

strain with depository access number NRRL B-50609;

strain with accession number NRRL B-50610;

strain with depository access number NRRL B-50611;

strain with depository access number NRRL B-50612; or mixtures of two or more strains.

In a specific embodiment, the seed (s) is coated with one or more of the aforementioned deposited strains (for example, 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 from the above strains).

In one embodiment, the seed (s) is coated with a strain with accession number NRRL B-50608. In one embodiment, the seed (s) are coated with a strain with accession deposit number NRRL B-50609. In one embodiment, the seed (s) is coated with a strain with accession deposit number NRRL B-50610. In one embodiment, the seed (s) are coated with a strain with accession deposit number NRRL B-50611. In one embodiment, the seed (s) are coated with a strain with accession deposit number NRRL B-50612.

In one embodiment, the seeds can be treated with any of the composition (s) described herein in several ways, but preferably by spraying or wetting. Spraying and wetting can be accomplished by composing the compositions described herein and spraying the composition (s) onto a seed (s) or wetting the composition (s) of a seed (s) with a continuous processing system (which is calibrated to process at a predetermined frequency in proportion to the continuous flow of seeds), for example, in a drum type processing apparatus. Batch systems in which a predetermined seed lot size and composition (s), as described herein, are delivered to a mixer, can also be used. Systems and apparatuses for carrying out these processes are commercially available from a number of manufacturers, for example, Bayer CropScience (Gustafson).

In another embodiment, the treatment includes coating the seeds. One such method includes coating the inner wall of a round container with the composition (s) described herein, adding seeds, then rotating the container to make the seeds come into contact with the wall and composition (s), a method known in the art as “coating” in the container. " Seeds can be coated using a combination of coating methods. Wetting, as a rule, includes the use of liquid forms of the described compositions. For example, the seeds can be wetted for about 1 minute to about 24 hours (for example, for at least 1 minute, 5 minutes, 10 minutes, 20 minutes, 40 minutes, 80 minutes, 3 hours, 6 hours, 12 hours, 24 hours).

In particular embodiments, a seed (s) coated with one or more of the compositions described herein may contain 1 × 10 ¹ -1 × 10 ⁸ , more preferably 1 × 10 ² -1 × 10 ⁶ colony forming units of one or more from deposited bacterial strains per seed.

EXAMPLES

The following examples are provided for purposes of illustration and are not intended to limit the scope of the invention as set forth in the claims herein. Any options in the illustrated examples that are obvious to a person skilled in the art are intended to be included in the scope of the present invention.

Materials and methods

Deposited biological material

The following biological material was deposited under the terms of the Budapest Agreement in the Microbial Genomics and Bioprocessing Research Unit (NRRL) of the National Center for Agricultural Utilization Research 1815 N. University Street, Peoria, IL 61604, USA, and was assigned the following access number:

Table 1
Deposited biological material Identifier Access number Date of deposit Bradyrhizobia japonicum NRRL B-50612 November 30, 2011 Bradyrhizobia japonicum NRRL B-50611 November 30, 2011 Bradyrhizobia japonicum NRRL B-50610 November 30, 2011 Bradyrhizobia japonicum NRRL B-50609 November 30, 2011 Bradyrhizobia japonicum NRRL B-50608 November 30, 2011

The strains are deposited under conditions that ensure that access to culture is possible during the consideration of this patent application for a person identified by a member of the Patent and Trademark Commission as indicated for this purpose according to 37 C.F.R. §1.14 and 35 U.S.C. §122. The deposited biological material is a pure culture of the deposited strain. The deposited biological material is available, as required by foreign patent law in countries where equivalents of the application under consideration or its continuation have been filed. However, it should be understood that the availability of deposited biological material does not constitute a license to put the invention under consideration into practice with a decrease in patent rights granted by a public authority.

Wednesday

table 2
G16 Environment Components Components G16 (g / l distilled deionized water (DDW)) g / l Potassium Phosphate Dibasic K ₂ HPO ₄ 0.550 Magnesium Sulphate Heptahydrate MgSO ₄ · 7H ₂ O 0,200 Calcium Chloride Dihydrate CaCl ₂ · 2H ₂ O 0.130 Oxoid Yeast Extract (LP0021) 0.750 Ammonium Chloride NH ₄ Cl 0,200 Monosodium L-Glutamic Acid Monohydrate 0.250 Sucrose 1,500 Dextrose Anhydrous 4,500 Iron (III) chloride hexahydrate FeCl ₃ · 6H ₂ O. Stock solution (80 g / l DDW) stored at 4 ° C for up to 6 months 0,200

Solid Corn Extract (Sigma) 0.400 Micronutrient stock solution (see below) * 365 μl Vitamin stock solution (see below) ** 365 μl pH 6,800

Table 3
Micronutrient stock solution stored at 4 ° C for up to 6 months Component (g / l DDW) g / l Nickel (II) chloride NiCl ₂ · 6H ₂ O 0.69 Copper sulfate pentahydrate CuSO ₄ · 5H ₂ O 0.22 Boric acid H ₃ BO ₃ 7.87 Manganese Sulfate Monohydrate MnSO ₄ · H ₂ O 5.06 Zinc sulfate heptahydrate ZnSO ₄ · 7H ₂ O 0.61 Sodium molybdate Na ₂ MoO ₄ · 2H ₂ O 0.61 Cobalt (II) hexahydrate CoCl ₂ · 6H ₂ O 0.66 * Trace elements are added along with other components before sterilization.

Table 4
Vitamin stock - sterilized by filtration, followed by storage at 4 ° C for up to 6 months Component (g / l DDW) g / l Thiamine hydrochloride 1.38 Pantothenic acid 0.55

** Vitamins are added after sterilization and cooling of the medium, usually during inoculation.

Table 5
Components of the medium from yeast extract and mannitol (YEM). Agar Ingredients from Yeast Extract and Mannitol (YEMA) (g / L DDW) g / l or ml / l Mannitol 10.0 g Oxoid Yeast Extract (LP0021) 0.50 g Sodium Chloride NaCl 0.10 g STOCK SOLUTION - Potassium Phosphate Dibasic K ₂ HPO ₄ (50 g / 1000 ml) 10 ml STARTING SOLUTION - Magnesium sulfate heptahydrate MgSO ₄ · 7H ₂ O (20 g / 1000 ml) 10 ml pH 6.8

Example I: Determination of 99.99% Percent Destruction for USDA 532C

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

The parent strain USDA 532C was grown in two 10 ml disposable culture tubes with G16 (tables 2-4) and YEM (table 5) (VWR, 18-150 mm, # 47729-583) for two days and collected to obtain the highest cell concentration . This was achieved by combining both culture tubes into one tube and concentrating the cells by sedimentation to 2 ml. About fifty seeds (Stine RR 1108-4) were superficially sterilized in a 50 ml sterile disposable centrifuge tube (Fisher brand, # 06-443-18) containing 5% household bleach solution for 30 seconds and washed with sterile deionized (DI) water. The sterilization step was repeated five times. Seeds were immediately placed in a sterilized Petri dish and dried in a laminar cabinet. After the seeds have completely dried and transferred to a 250 ml measuring cup, 1.5 ml of the culture of the concentrated parental strain USDA 532C was added to the seeds. The seeds were rotated in a measuring cup to completely cover the seeds and allowed to dry under the laminar. A measuring cup containing seeds was wrapped in blue paper for sterilization and left under the laminar until the experiment was completed. Time points were taken at zero time, every two days for one week, and every week until the complete death of the cells. The results are presented in table 6.

Table 6
CFU per seed and percent kill for study 1 Number of days CFU per seed Destruction Percentage 0 3.06 × 10 ⁸ 0.00 3 3.72 × 10 ⁷ 87.86 7 4.20 × 10 ⁶ 98.63 fourteen 3.07 × 10 ⁶ 99.00 eighteen 6.43 × 10 ⁵ 99.79

24 2.87 × 10 ⁵ 99.91 37 2.64 × 10 ⁴ 99,99

As shown in Table 6, the initial CFU per seed parent strain USDA 532C was 3,0610 ^8, and on day 37 cfu was 2,6410 ^8. The percentage of destruction from time 0 to 37 days was calculated as 99.99%.

The procedure was repeated, except that G16 was used as the starting medium for growth. The results are presented in table 7.

Table 7
CFU per seed and percent kill for study 2 Number of days CFU per seed Destruction Percentage 0 2.01 × 10 ⁹ 0.00 2 3.13 × 10 ⁸ 84.41 6 3.26 × 10 ⁷ 98.38 10 9.14 × 10 ⁶ 99.55 16 3,50 × 10 ⁶ 99.83 22 1.40 × 10 ⁶ 99.93 29th 3.38 × 10 ⁵ 99.98 37 6.23 × 10 ⁴ 100.00

As shown in table 7, when G16 was used as the starting medium for growth, it took 29 to 37 days to achieve a 99.99% kill rate.

A third dehydration study was completed to determine if G16 and YEM media affected the dehydration rate of the parent strain USDA 532C. The results are presented in tables 8 and 9, respectively.

Table 8
CFU per seed and kill rate for parental strain USDA 532C grown on G16 Number of days CFU per seed Destruction Percentage 0 2.95 × 10 ⁹ 0.00 2 8.69 × 10 ⁷ 97.06 7 2.93 × 10 ⁷ 99.01 fourteen 1.68 × 10 ⁷ 99.43 21 2.11 × 10 ⁶ 99.93 28 7.50 × 10 ⁴ 100.00

Table 9
CFU per seed and kill rate for the parent strain USDA 532C grown on YEM medium. Number of days CFU per seed Destruction Percentage 0 2.90 × 10 ⁸ 0.00 2 3.46 × 10 ⁶ 98.81 7 1.81 × 10 ⁶ 99.38 fourteen 1.31 × 10 ⁶ 99.55 21 7.33 × 10 ⁵ 99.75 28 7.00 × 10 ⁴ 99.98

As shown in tables 8 and 9, there were no differences in the dehydration rate of the parent strain USDA 532C when grown on G16 or YEM. A kill rate of 99.99% was observed for the third study at approximately 28 days, which is similar to the kill rate observed in studies one (1) and two (2) above.

Example II: Determination of the percent destruction of USDA532C using ethyl methanesulfonate (EMS)

The following experiment (s) were performed to determine the level of administration of mutagen, ethyl methanesulfonate (EMS), giving a kill rate of 99.9-99.99 for the parent strain USDA 532C. This level determination may be part of a mutagenesis protocol used to produce dehydration resistant putative mutants, although the mutagenesis method can be expanded for effectiveness.

Inoculum preparation:

The parent strain USDA 532C was grown in six 10 ml disposable culture tubes with YEM for two days, and 5 ml of the culture was inoculated into four 250 ml flasks containing 50 ml of YEM medium. The flasks were incubated for two days at 30 ° C in a shaker. The flask culture was then centrifuged in 50 ml disposable sterile tubes at 8,000 rpm for ten minutes in a Sorvall RC 6 Plus® centrifuge and combined into a single tube. The pellet was resuspended in 4 ml of fresh YEM medium and separated into four 1.5 ml centrifugation tubes. Each of the tubes represents different application levels used for the mutagenesis method.

Mutagenesis Method

After placing aliquots of the culture in separate tubes and adding the EMS mutagen (Sigma, C3H803S, FW 124.16, # M0880-1G) to each tube, the tubes were vigorously shaken and placed in an empty 250-ml flask. The flask containing the reaction tubes was incubated for 30 minutes at 30 ° C. in a shaker. Immediately after the incubation period, the tubes were washed five times with a solution of 0.16 M sodium thiosulfate (STS, Fisher Chemical, Na ₂ S ₂ O ₃ * 5H ₂ O, FW 248.18, # S445-3) to inactivate the mutagen. After washing, the cells in the reaction tubes were pulverized with a 21 gauge syringe (BD 1 ml 21G1 Latex Free Syringe Precision Glide G Needle, 0.8 mm  25 mm, # 309624), and dilutions were completed and scattered into YEMA plates. Cell counting was available after five days of incubation at 30 ° C, and the percentage of killing was calculated for the level of application of EMS. The following equation was used to calculate the kill rate for each application level: ([cell number for 0 μl EMS (control) - (cell number for μl EMS (treatment)) + cell number for 0 μl EMS (control)] × 100%). In all other experiments after this experiment, this equation was used to calculate the percent kill. The results are presented in table 10.

Table 10
Baseline EMS levels for determining the upper limit of mutagenesis for the parent strain USDA 532C Application Level The number of cells (approximate CFU / ml) 0 μl EMS (control) 10 ⁸ CFU / ml 1 μl EMS similar results with 0 μl EMS 10 ⁷ CFU / ml 10 μl EMS similar results with 1 μl EMS 100 μl EMS 100% destruction rate

As shown in Table 10, the initial EMS levels used were 0 μl, 1 μl, 10 μl and 100 μl. There was no difference between 0 μl, 1 μl and 10 μl EMS, but 100 μl EMS resulted in 100% killing.

The experiments were repeated and refined to determine an acceptable percentage of destruction. See tables 11-17.

Table 11
Clarification of EMS levels to determine 99.9% percent kill for parent strain USDA 532C Application Level The number of cells (approximate CFU / ml) Destruction Percentage 0 μl EMS (control) 2.49 × 10 ⁹ CFU / ml 15 μl EMS 2.24 × 10 ⁹ CFU / ml 10.17% 25 μl EMS 1.72 × 10 ⁹ CFU / ml 30.92% 50 μl EMS 1.67 × 10 ¹ CFU / ml one hundred%

According to the initial results, the amount of EMS used was narrowed to 0 μl, 15 μl, 25 μl and 50 μl EMS. As shown in Table 11, the kill rate was 10.17% -30.92% for applying 15 μl and 25 μl of EMS and 100% for 50 μl.

Table 12
Further clarification of EMS application levels for parent strain USDA 532C Application Level The number of cells (approximate CFU / ml) Destruction Percentage 0 μl EMS (control) 4.70 × 10 ⁹ CFU / ml 25 μl EMS 1.27 × 10 ⁹ CFU / ml 73.09% 35 μl EMS 5.37 × 10 ⁸ CFU / ml 88.58% 50 μl EMS 2.93 × 10 ² CFU / ml one hundred%

From table 11, it was determined that 25 μl of EMS had an still too low kill rate. The amounts applied for Table 12 were 0 μl, 25 μl, 35 μl, and 50 μl EMS. The application of 25 μl of EMS had a higher kill rate than the results in table 11, due to a decrease in washing; thus, the percentage of destruction was higher than expected. However, the kill rate was still too low at 88.58% kill, even when using 35 μl of EMS. See table 12.

Table 13
Further clarification of EMS application levels for parent strain USDA 532C Application Level The number of cells (approximate CFU / ml) Destruction Percentage 0 μl EMS (control) 3.93 × 10 ⁹ CFU / ml 40 μl EMS 6.97 × 10 ⁸ CFU / ml 82.26% 45 μl EMS 8.57 × 10 ⁷ CFU / ml 97.82% 50 μl EMS 2.65 × 10 ⁵ CFU / ml 99.99%

The amount of EMS used was increased to 40 μl, 45 μl and 50 μl of EMS. EMS dose levels resulted in a range of kill rates of 82.26% -99.99%. See table 13.

Table 14
Repeat application levels used in table 13 Application Level The number of cells (approximate CFU / ml) Destruction Percentage 0 μl EMS (control) 7.07 × 10 ¹¹ CFU / ml 40 μl EMS 1.35 × 10 ⁹ CFU / ml 99.81% 45 μl EMS 5.27 × 10 ⁸ CFU / ml 99.93% 50 μl EMS 2.19 × 10 ⁶ CFU / ml one hundred%

The results presented in table 13 were again repeated in table 14 using the same EMS levels, and this time the kill rate was 99.81% for 40 μl EMS and 99.93% for 45 μl EMS, and 100% for 50 μl EMS. A desired kill rate of 99.9% was observed using 45 μl of EMS, so that application was repeated. See table 15.

Table 15
Repeat application level used in table 14 Application Level The number of cells (approximate CFU / ml) Destruction Percentage 0 μl EMS (control) 1.51 × 10 ⁸ CFU / ml 45 μl EMS 3.33 × 10 ⁴ CFU / ml 99.98% 50 μl EMS 4.33 × 10 ¹ CFU / ml one hundred%

The application level of 45 μl EMS was doubled to determine whether the results in table 14 are reproducible. The kill rate for 45 μl of EMS was 99.98%. See table 15.

Example III: Mutagenesis

The following experiment (s) were carried out to obtain the alleged dehydration-resistant mutants of the strains from the parent strain USDA 532C using classical, for example, chemical, mutagenesis.

Inoculum preparation:

A cell suspension of the parent strain USDA 532C was obtained by sampling a cell loop from a fresh USDA 532C plate using 10 μl of a sterile plastic loop (Fisher brand, # 22-363-600) and mixing the cells in 1 ml of sterile, deionized (DI) water in 1.5 ml disposable tube for microcentrifugation. The cell suspension was inoculated into two 250 ml flasks containing 50 ml of YEM medium to achieve a final optical density (OD) of OD _{600 nm 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 8000 rpm in a Sorvall RC 6 Plus centrifuge. The supernatant was discarded and the pellet was resuspended in 30 ml of DI water. The OD of the concentrated culture was determined and inoculated into ten 250 ml flasks containing 50 ml of YEM medium at OD = 0.05. These flasks were incubated at 30 ° C in a shaker for two days before using the culture for mutagenesis.

Mutagenesis Method:

Cultures of ten flasks were combined in a 1 liter centrifugation bottle. The absorbance of the pooled cultures was recorded and the cultures were centrifuged for 20 minutes at 8000 rpm in a Sorvall RC 6 Plus centrifuge. Most of the supernatant was discarded, leaving approximately 30 ml of the supernatant in a centrifugation bottle. The supernatant was mixed with the pellet and transferred into a 50 ml sterile disposable centrifuge tube. The OD of the concentrated culture was determined and recorded. 1 ml of concentrated culture was placed in six 1.5 ml of disposable tubes for microcentrifugation. Microcentrifuge tubes were centrifuged and the supernatant discarded. This was repeated three more times or until the pellet size reached 0.1 ml in the microcentrifuge tube. The cells were mixed well with 1 ml of fresh YEM medium using a sterile 1 ml syringe with a 21 gauge needle before adding the mutagen, ethyl methanesulfonate (EMS). The mutagen levels added to each tube included a high and a low dose with medium doses between high and low doses, as indicated in Experiment II.

Immediately after the addition of EMS, reaction tubes were placed in an empty 250-ml flask and incubated at 30 ° C for 30 minutes. After incubation, the reaction tubes were centrifuged for one minute at 13,200 rpm using an Eppendorf 5415D centrifuge. The supernatant from the reaction tubes was discarded. Mutagen in the reaction tubes was inactivated by washing five times with 1 ml of 0.16 M sodium thiosulfate (STS) and vigorous mixing by shaking the tubes. For each wash cycle, the reaction tubes were centrifuged after shaking, and the supernatant was discarded. After the fifth time, all reaction tubes were combined into one 15 ml disposable tube for use in the enrichment method.

Example IV: Enrichment and Dehydration

The following experiment (s) were performed to enrich and dehydrate the mutated cells of the parent strain USDA 532C to exclude wild-type rescue and increase the population of putative mutants and to simplify the isolation of mutants (mutants) with dehydration resistance characteristics.

The parent strain USDA 532C was subjected to the mutagenesis process mentioned in Example III. The mutated population of the parent strain USDA 532C was enriched by inoculating 0.5 ml of the reaction mixture in two 50 ml flasks with YEM and incubating the cells for two days in a shaker at 30 ° C. After two days, the cultures were dehydrated by coating the cells with soybean seeds and membrane filters and subjecting them to drying conditions. The culture from the enrichment stage was adjusted to OD _600nm 0.5 to be used to coat soybean seeds and membrane filters.

Soybean seed coating:

Forty sterilized soybean seeds were coated with 0.5 ml of culture. Seeds were placed in a 100 ml sterile measuring cup for drying in a laminar cabinet and covered with autoclaving paper. Seed samples were taken in triplicate to obtain the initial CFU of seeds. For each sample, three seeds were placed in a 15 ml disposable tube containing 5 ml of sterile DI water and allowed to swell in the tube for approximately two hours before sterile dilution of the suspension and distribution to YEMA plates. The remaining seeds remaining in the covered measuring cup were placed in a laminar for four days before seed enrichment.

To enrich the seeds, twenty seeds were placed in a 250 ml flask containing 50 ml of fresh YEM. The final CFU after seed enrichment was also determined to determine the percent killing for the cell population. The same sampling as at the starting time point was completed at the second time point. After incubating the seed containing culture for two days, the culture was harvested by removing the entire debris of the seeds, allowing the debris to settle before removing the supernatant. The culture supernatant was centrifuged and the pellet was washed with sterile DI water until the culture was used to cover new batches of soybean seeds. This process was repeated until the calculated cell culture kill percentage was less than 80%. Upon reaching 80%, the cell population was ready to isolate putative mutants for a confirmatory experiment.

Membrane Filter Coating:

For control in studies of the violation of the integrity and contamination of soybean seeds, membrane filters were used as an alternative medium for coating cells. For membrane filters, 1 ml of culture was used to coat both dupore (Millipore, 0.22 μm, PVDF, # GVWP02500) and isopore (Millipore, 0.4 μm, polycarbonate, # HTTP02500) membrane filters. For each type of filter, fifteen filters were coated using a 25 mm Easy Pressure syringe filter holder (VWR, # 28144-109), and the filters were placed in a sterile Petri dish containing two pieces of sterile, quality 125 mm Whatman paper (Whatman, # 1001125) . After the filters were dried in the laminar, the initial CFU was determined in triplicate for each type of filter by placing one filter in a 15 ml disposable tube containing 5 ml of sterile DI water and stirring by shaking. After two hours of wetting in a 15 ml tube, the filter suspension was diluted and plated on YEMA plates. After drying the filters in a laminar for three days, eight filters were added to a 250 ml flask with 50 ml of fresh YEM and incubated at 30 ° C for three days.

The final CFU was determined at the same time after enrichment on filters to obtain calculations of the percentage of destruction. The same method as for the original CFU was used for the final CFU. The coating and drying process was repeated until the destruction rate was less than 80%. After reaching 80%, isolates of individual colonies were selected for further confirmation.

Example V: Confirmation of putative mutants

The following experiment (s) were performed to confirm the alleged dehydration-resistant mutants by comparing their viability on the seed after application to the seed with the viability of the original parent strain of Bradyrhizobium japonicum strain USDA 532C.

When 80% percent kill was observed for seeds or filters, individual colonies were randomly selected at the final time point, and putative mutants from the batch after mutagenesis were analyzed for dehydration resistance characteristics. Twenty separate colonies selected for each batch obtained after mutagenesis were individually grown in a 250 ml flask containing 50 ml of YEM medium. Each suspected mutant strain was incubated in a shaker at 30 ° C. for three days, and the OD for each strain was adjusted to 0.5. Each strain was used to cover thirty non-sterile soybean seeds with 0.5 ml of culture in a 100 ml beaker covered with autoclaving paper. Time points were selected at T = 0, T = 3, and T = 7 days for the first round. Seed samples in triplicate were taken for each time point, where each sample consisted of three seeds placed in 5 ml of sterile DI water in a 15 ml disposable tube. Seeds were allowed to swell for two hours before each sample was diluted and plated on YEMA plates. After comparing the number of cells recovered at each time point with respect to the parent strain of USDA 532C, all strains with better efficiency than the parent strain were subjected to a second round of confirmation. See FIG. 1. For the second round, the strains with the best resistance to dehydration compared to the wild type were again tested for dehydration. See FIG. 2. Time points were taken at T = 0, T = 7, and T = 14 days. For a batch of putative mutants from the second round, dehydration resistance was further confirmed two more times. See FIGS. 3-6.

Twenty suspected mutants of the parent USDA 532C strain were isolated and selected for dehydration resistance. See FIG. 1-3. Of the twenty tested candidate strains, for five allegedly mutant strains, their dehydration resistance characteristics were confirmed compared to the dehydration resistance of the parent strain, Bradyrhizobium japonicum strain USDA 532C. See Fig 4-6.

Example VI: Testing in a greenhouse of confirmed mutant strains

The following experiment (s) were performed to test the suspected mutant strains in the greenhouse to test the effectiveness of the mutant strains compared to the parent strain, USDA 532C.

Mutant strains with the best resistance to dehydration compared to the parent strain USDA 532C were tested in the greenhouse for efficacy compared to the parent strain USDA 532C. Mutant and parent strains were grown in 50 ml of YEM for two days before seed coating. Each strain was seeded in three different time periods; T = 0, T = 7 and T = 14 days after seed coating. All time points were set at the same time, but the seeds were sown in the indicated time periods. To establish time points, thirty soybean seeds were coated with 0.5 ml of culture at OD _{600 nm} = 0.5 in a 100 ml measuring cup, and for a time point T = 0 days, they were allowed to remain in the laminar for 30 minutes before sowing. For two other time points, complete drying was allowed and covered with autoclaving paper. Seeds from the last two time points were sown at a later date. At each time point, two seeds were sown per pot for ten pots for the strain. The remaining seeds were used to obtain CFU for comparison with T = 0. After nine weeks of growing in a greenhouse, soybean pods were collected from each plant at each time point, and dry weight was analyzed for statistical significance.

When the mass of soybean pods of the mutant strains was compared with the parent strain at any time point, there was no statistical significance for 95% confidence. This indicates that there is no difference in efficacy between the mutant strains and the parent strain, which can affect the production of soybean pods when using mutant strains to cover soybean seeds.

SUMMARY ITEMS

The present invention is defined in the claims and the accompanying description. Additional aspects of the present invention are presented herein by numbered paragraphs.

1. A biologically pure culture of a strain of Bradyrhizobium japonicum selected from the group consisting of:

strain with depository access number NRRL B-50612;

strain with depository access number NRRL B-50611;

strain with accession number NRRL B-50610;

strain with depository access number NRRL B-50609;

strain with accession number NRRL B-50608, or a combination of at least two or more strains.

2. The strains of paragraph 1, where these strains have excellent resistance to dehydration.

3. The strains of paragraph 2, where the specified resistance to dehydration is compared with the specified resistance to dehydration of the parent strain of these isolated strains, for example, the parent strain of Bradyrhizobium japonicum USDA 532C.

4. The strains of paragraph 2, wherein said excellent dehydration resistance includes an increased percentage of bacterial survival in a substantially anhydrous external environment, when the indicated survival rate of said isolated Bradyrhizobium strain (s) is compared with said percent survival of the parent strain of said isolates, for example parent strain of Bradyrhizobium japonicum USDA 532C, for a period of time, for example at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least m Heresy 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. Strains from any of paragraphs 2-4, where the specified increased percentage of survival in the specified essentially anhydrous external environment includes an increased percentage of survival of bacteria in the external environment, which is at least 70% anhydrous, for example, at least 75%, at least 80%, at least 85%, 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%, m at least 99%, up to 100% anhydrous external environment, when the specified survival rate of the indicated isolated strain (s) of Bradyrhizobium is compared with the specified percentage of survival of the parent strain of these isolates, for example, the parent strain of Bradyrhizobium japonicum USDA 532C.

6. A composition comprising one or more of these isolated bacterial strain (s) in accordance with paragraphs 1-5 and a carrier acceptable for agriculture.

7. The composition of paragraph 6, where the composition contains at least one agriculturally advantageous ingredient.

8. The composition of paragraph 7, wherein said at least one agriculturally advantageous ingredient includes one or more plant signal molecules.

9. The composition of paragraph 8, where the signal molecule of the plant is a lipo-chitooligosaccharide (LCO).

10. The composition of paragraph 9, where the LCO is synthetic.

11. The composition of paragraph 9, where the LCO is recombinant.

12. The composition of paragraph 9, where the LCO is natural.

13. The composition of paragraph 9, wherein the LCO is derived from Rhizobia spp. Selected from Rhizobium spp., Sinorhizobium spp. and Azorhizobium spp.

14. The composition of paragraph 9, where the LCO is derived from Bradyrhizobium japonicum.

15. The composition of paragraph 9, where the LCO is obtained from a mushroom with a tree mycorrhiza.

16. The composition of paragraph 8, where the signal molecule of the plant is a chitin compound.

17. The composition of paragraph 16, wherein the chitin compound is a chito-oligomer (CO).

18. The composition of paragraph 17, where the CO is synthetic.

19. The composition of paragraph 18, where the CO is recombinant.

20. The composition of paragraph 18, where CO is natural.

21. The composition of paragraph 8, where the signal molecule of the plant is a flavonoid.

22. The composition of paragraph 21, where the flavonoid is selected from the group consisting of luteolin, apigenin, tangeritin, quercetin, kempferol, myricetin, fisetin, isoramnetin, pachipodol, ramnazine, hesperetin, naringenin, formononetin, eriodiktirodigiolifeol homo, , genistein, daidzein, glycitein, catechin, gallocatechin, 3-gallate catechin, 3-gallate gallocatechin, epicatechin, epigallocatechin, 3-gallate epicatechin, 3-gallate epigallocatechin, cyanidine, dolphinidine, malvidin, pelin Idina, peonidina, petunidin or their derivatives.

23. The composition of paragraph 8, where the signal molecule of the plant is jasmic acid or its derivative.

24. The composition of paragraph 8, where the signal molecule of the plant is linoleic acid or its derivative.

25. The composition of paragraph 8, where the signal molecule of the plant is linolenic acid or its derivative.

26. The composition of paragraph 8, where the signal molecule of the plant is carrikin.

27. The composition according to any one of paragraphs 8-26, where the composition includes at least two different plant signal molecules.

28. The composition of paragraph 27, wherein the agricultural advantageous ingredient is a herbicide, insecticide or fungicide.

29. The composition of paragraph 27, wherein the agricultural advantageous ingredient is at least one phosphate solubilizing microorganism.

30. The composition of paragraph 29, where at least one solubilizing phosphate microorganism contains a strain of the fungus Penicillium.

31. The composition of paragraph 30, where at least one solubilizing phosphate microorganism contains a strain of P. bilaiae.

32. The composition of paragraph 31, wherein the P. bilaiae strain is selected from the group consisting of NRRL 50162, NRRL 50169, ATCC 20851, ATCC 22348, and ATCC 18309.

33. The composition of paragraph 30, where at least one solubilizing phosphate microorganism contains a strain of P. gaestrivorus.

34. The composition of paragraph 33, where the strain P. gaestrivorus is NRRL 50170.

35. A method of treating seeds, comprising applying to said seeds an inoculum of one or more of an isolated bacterial strain (s) according to paragraph 1.

36. The method according to paragraph 35, where the composition according to any one of paragraphs 6-34 is applied to the seed.

37. The method according to any one of paragraphs 35-36, wherein the method further comprises the step of storing said treated seeds in a substantially anhydrous environment for a period of time, for example 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, each necks least 5 months, at least 6 months, at least 1 year or more.

38. The method according to paragraph 37, where these seeds are seeds of a legume plant.

39. The method according to paragraph 38, wherein said bean seeds are soybean seeds.

40. A method of enhancing plant growth, comprising applying to plants, plant seeds or the soil surrounding plants or plant seeds, a composition according to any one of paragraphs 6-34.

41. The method according to paragraph 40, where these seeds are seeds of a legume plant.

42. The method according to paragraph 41, wherein said bean plant seeds are soybean seeds.

43. A method of enhancing the growth of a plant or part of a plant, comprising contacting a plant or part of a plant with an inoculum of one or more strains of paragraph 1.

44. The method of paragraph 43, where the method further includes the step of exposing the soil to one or more agriculturally advantageous ingredients.

45. The method of paragraph 43, wherein the processing step comprises administering the inoculum of one or more strains of paragraph 1 in the form of a composition.

46. The method of paragraph 43, where the composition is a composition according to any one of paragraphs 6-34.

47. The method of paragraph 43, where the plant part is a plant seed.

48. The method according to paragraph 47, where these seeds are seeds of a legume plant.

49. The method according to paragraph 48, wherein said legume seeds are soybean seeds.

50. A method of enhancing the growth of a plant or part of a plant, comprising

a. soil treatment with the inoculum of one or more strains of paragraph 1; and

b. growing a plant or part of a plant in cultivated soil.

51. The method of paragraph 50, wherein the method further includes the step of planting the plant or part of the plant before, during or after the processing step.

52. The method of paragraph 50, wherein the method further includes the step of exposing the soil to one or more agriculturally preferred ingredients.

53. The method of paragraph 50, where the processing includes the introduction of one or more strains of paragraph 1 in the form of a composition.

54. The method of paragraph 50, where the composition is a composition according to any one of paragraphs 6-34.

55. A seed coated with a composition according to any one of paragraphs 6-34.

The invention described and claimed in the claims herein is not intended to be limiting in scope to the specific embodiments described herein, as these embodiments are intended to illustrate certain aspects of the invention. Any equivalent embodiments are intended to be included within the scope of this invention. Indeed, various modifications of the invention in addition to the modifications shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to be included within the scope of the appended claims. In case of conflict, the present description, including definitions, has the advantage.

Various references are cited herein, the entire contents of which are incorporated by reference.

Claims (71)

1. A biologically pure culture of a strain of Bradyrhizobium japonicum to improve plant growth, where the specified strain with a deposit access number NRRL B-50608. 2. A biologically pure culture of a strain of Bradyrhizobium japonicum to improve plant growth, where the specified strain with a depository access number NRRL B-50609. 3. A biologically pure culture of the strain Bradyrhizobium japonicum to improve plant growth, where the strain with a deposit access number NRRL B-50610. 4. A biologically pure culture of the strain Bradyrhizobium japonicum to improve plant growth, where the strain with a depository access number NRRL B-50611. 5. A biologically pure culture of the strain Bradyrhizobium japonicum to improve plant growth, where the strain with a depository access number NRRL B-50612. 6. Composition for improving plant growth, containing an agriculturally acceptable carrier and inoculum that contains a strain of Bradyrhizobium japonicum with a deposit access number NRRL B-50608, a strain of Bradyrhizobium japonicum with a deposit access number NRRL B-50609, a strain of Bradyrhizobium japonicum with a deposit access number NRRL B-50610, Bradyrhizobium japonicum strain with depository access number NRRL B-50611 and / or Bradyrhizobium japonicum strain with depository access number NRRL B-50612. 7. The composition according to claim 6, where the composition includes at least one agriculturally advantageous ingredient. 8. The composition of claim 7, wherein said at least one agriculturally advantageous ingredient comprises one or more plant signaling molecules. 9. The composition of claim 8, where the signal molecule of the plant includes a lipo-chitooligosaccharide (LCO). 10. The composition according to claim 9, where the LCO is synthetic. 11. The composition according to claim 9, where the LCO is recombinant. 12. The composition according to claim 9, where the LCO is natural. 13. The composition of claim 9, wherein the LCO is derived from a strain of Rhizobium, Sinorhizobium, Azorrhizobium, Mesorhizobium or Bradyrhizobium. 14. The composition of claim 9, wherein the LCO is derived from a Bradyrhizobium japonicum strain. 15. The composition of claim 9, wherein the LCO is derived from a Sinorhizobium meliloti strain. 16. The composition according to claim 9, where the LCO obtained from a strain of Rhizobium leguminosarum. 17. The composition according to claim 9, where the LCO obtained from mycorrhizal fungi. 18. The composition of claim 9, wherein the LCO is derived from a Glomerocycota strain. 19. The composition of claim 9, wherein the LCO is derived from a Glomus intraradicus strain. 20. The composition according to claim 9, where the LCO is represented by the formula:

wherein:
G represents hexosamine, which may be substituted by an acetyl group on a nitrogen atom, a sulfate group, an acetyl group and / or an ether group on an oxygen atom;
R ₁ , R ₂ , R ₃ , R ₅ , R ₆ and R ₇ , which may be identical or different, are H, CH ₃ CO—, C _x H _y CO—, where x is an integer between 0 and 17, and y is an integer between 1 and 35, or any other acyl group;
R ₄ represents a mono-, di- or trisaturated aliphatic chain containing at least 12 carbon atoms; and
n is an integer between 1 and 4. 21. The composition according to claim 9, where the LCO is represented by the formula:

wherein:
R represents H or CH ₃ CO—; and
n is equal to 2 or 3. 22. The composition of claim 8, wherein said plant signal molecule comprises a chitin compound. 23. The composition of claim 22, wherein the chitin compound is chitin. 24. The composition of claim 22, wherein the chitin compound is chitosan. 25. The composition of claim 8, wherein said plant signal molecule comprises a chito-oligomer (CO). 26. The composition according A.25, where the CO is synthetic. 27. The composition according A.25, where the CO is recombinant. 28. The composition according A.25, where the CO is natural. 29. The composition according A.25, where the CO is a chitooligosaccharide. 30. The composition of claim 8, where the signal molecule of the plant is a flavonoid. 31. The composition of claim 7, wherein said at least one agriculturally preferred ingredient comprises jasmic acid or a derivative thereof. 32. The composition of claim 7, wherein said at least one agriculturally preferred ingredient comprises linoleic acid or a derivative thereof. 33. The composition of claim 7, wherein said at least one agriculturally preferred ingredient comprises linolenic acid or a derivative thereof. 34. The composition according to claim 7, wherein said at least one agriculturally advantageous ingredient comprises one or more carricken. 35. The composition of claim 7, wherein said at least one agriculturally preferred ingredient comprises one or more herbicides. 36. The composition of claim 7, wherein said at least one agriculturally advantageous ingredient comprises one or more fungicides. 37. The composition of claim 7, wherein said at least one agriculturally advantageous ingredient comprises one or more insecticides. 38. The composition of claim 7, wherein said at least one agriculturally advantageous ingredient comprises one or more advantageous microorganisms. 39. The composition of claim 38, wherein said one or more advantageous microorganisms include one or more strains of Rhizobium, Sinorhizobium, Azorrhizobium, Mesorhizobium and / or Bradyrhizobium. 40. The composition according to § 38, where the specified one or more advantageous microorganisms include one or more strains of Bradyrhizobium japonicum. 41. The composition of claim 38, wherein said one or more advantageous microorganisms comprises one or more strains of Sinorhizobium meliloti. 42. The composition according to § 38, where the specified one or more advantageous microorganisms include one or more strains of Rhizobium leguminosarum. 43. The composition according to § 38, where the specified one or more advantageous microorganisms include one or more strains of Mesorhizobium ciceri. 44. The composition according to § 38, where the specified one or more advantageous microorganisms include one or more mycorrhizal fungi. 45. The composition according to § 38, where the specified one or more advantageous microorganisms include one or more strains of Glomerocycota. 46. The composition of claim 38, wherein said one or more advantageous microorganisms comprises one or more Penicillium strains. 47. The composition according to § 38, where the specified one or more advantageous microorganisms include one or more strains of P. bilaiae. 48. The composition according to § 38, where the specified one or more advantageous microorganisms include one or more strains of P. gaestrivorus. 49. The composition according to § 38, where the specified one or more advantageous microorganisms include one or more strains of Streptomyces. 50. The composition of claim 6, wherein said agriculturally acceptable carrier is in the form of a liquid. 51. The composition of claim 6, wherein said agriculturally acceptable carrier is in the form of a suspension. 52. The composition of claim 6, wherein said agriculturally acceptable carrier is in the form of a solid. 53. The composition of claim 6, wherein said agriculturally acceptable carrier is in powder form. 54. The composition of claim 6, wherein said agriculturally acceptable carrier is in the form of a seed coating. 55. The composition according to claim 6, where in the specified composition there are no anionic surfactants. 56. A seed coated with a composition according to any one of paragraphs. 6-55. 57. The seed of claim 56, wherein the composition comprises at least one agriculturally advantageous ingredient. 58. The seed of claim 57, wherein said at least one agriculturally advantageous ingredient comprises one or more plant signaling molecules. 59. Seed according to any one of paragraphs. 56-58, where the seed is a legume seed. 60. Seed according to any one of paragraphs. 56-58, where the seed is a non-legume seed. 61. Seed according to any one of paragraphs. 56-58, where the seed is a seed of wheat. 62. Seed according to any one of paragraphs. 56-58, where the seed is a seed of barley. 63. Seed according to any one of paragraphs. 56-58, where the seed is a seed of oats. 64. Seed according to any one of paragraphs. 56-58, where the seed is the seed of rye. 65. Seed according to any one of paragraphs. 56-58, where the seed is a potato seed. 66. Seed according to any one of paragraphs. 56-58, where the seed is a sunflower seed. 67. Seed according to any one of paragraphs. 56-58, where the seed is a soybean seed. 68. Seed according to any one of paragraphs. 56-58, where the seed is a seed of cabbage. 69. Seed according to any one of paragraphs. 56-58, where the seed is the seed of a tomato. 70. Seed according to any one of paragraphs. 56-58, where the seed is the seed of a carrot. 71. Seed according to any one of paragraphs. 56-58, where the seed is the seed of an apple tree.

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