BR122018069737B1 - METHOD TO INTENSE PLANT GROWTH - Google Patents

Abstract

In accordance with the present invention, novel isolates of bacterial strains have been shown to have unique properties. These bacterial strains are plant growth promoting rhizobacteria (PGPR), have a better competitive advantage in colonizing legume plants, and improve overall legume plant growth performance. Additionally, the present invention reveals an unprecedented method for evaluating and selecting bacterial strains that have the previously mentioned beneficial characteristics.

Description

REFERENCE TO SEQUENCE LISTING

[0001] This application contains a listing of sequences in machine readable form. Computer-readable form is incorporated herein by reference.

REFERENCE TO A BIOLOGICAL MATERIAL DEPOSIT

[0002] This application contains a reference to a repository of biological material, which deposit is incorporated herein by reference. For complete information, see table 1.

FIELD OF THE INVENTION

[0003] The present invention relates to isolated bacterial strains, and to a method of selecting bacterial strains with enhanced competitiveness and performance characteristics.

FUNDAMENTALS OF THE INVENTION

[0004] In order to maintain healthy growth, plants must extract a variety of elements from the soil in which they grow. These elements include nitrogen and the so-called micronutrients (e.g. copper, iron and zinc), but many soils are deficient in these elements or contain them only in forms that cannot be easily taken up by plants (it is generally believed that elements Essential oils cannot be easily taken up by plants unless they are present in dissolved form in the soil). Nitrogen is an essential element for most plants, as it plays a role in the synthesis of amino acids, proteins, nucleotides, nucleic acids, chlorophyll, coenzymes, and in overall plant growth and health. To counteract such deficiencies, sources of the deficient elements are commonly applied to soils in order to improve growth rates and yields obtained from crop plants. For example, nitrate and/or ammonium is often added to soil to offset a lack of available nitrogen.

[0005] In the field of crop science, it is well known that many cultivated crops require the soil to supply relatively large amounts of nitrogen to the plant. The notable exceptions to those plants that require nitrogen through the soil are plants in the legume family.

[0006] Specifically, legume plants are unique from non-legume plants in their ability to fix atmospheric nitrogen into ammonia. The ability to fix atmospheric nitrogen into a plant usable source of nitrogen eliminates the need for the plant to obtain nitrogen from the soil. Nitrogen fixation, however, requires a symbiotic relationship between the legume plant and native bacteria in the soil. One of the most extensively 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.

[0007] Symbiosis is usually achieved through a complex bidirectional signaling exchange between the plant and the microbe and the microbe and the plant. Typically, plant factors such as flavonoids and flavonoid-like substances induce colonization of bacteria in the root nodule of the legume plant. (Gresshoff, 1999). Once the bacteria have colonized the root nodule, the bacteria cause morphological changes in the plant, namely, curving and the development of root hair from a new root organ - the nodule. (Gresshoff, 1999). The nodule allows the establishment of a new physiological environment in the nodule, inducing bacteria to differentiate into a nitrogen-fixing endosymbiont, or bacteroid, for the colonized plant. (Gresshoff, 1999). Petition 870180134926, of 09/26/2018, page 16/105

[0008] It is well known that Rhizobial motility and chemotaxis are important attributes for strain competitiveness. For example, Althabegoiti, et al., 2008, FEMS Microbiol. Lett. 282: 115-123 discuss deriving a spontaneous mutant USDA 110 strain with higher motility that improves nodulation when compared to its wild-type strain. Additionally, Maier, et al., 1990, Appl. Environ. Microbiol. 56(8): 2341-2346 discuss the role of molybdenum during the process of biological nitrogen fixation. Still further, Alves, et al., 2003, Plant and soil 252: 1-9 discuss soybean inoculants used in Brazil and the importance of competitiveness for effective nitrogen fixation. Finally, Bloem, J.F., et al., 2001, Bio Fertil. Soils 33: 181-189 report the importance of competitiveness in strain selection. In the study, researchers used genetic engineering methods to put a reporter gene (GUS) into their index strain as a way to determine the strains' competitiveness. (Bloem, et al. 2001). As the study performed (Bloem, et al. 2001) required extensive use of chemical staining and microscopy technology, the reported method remains an impractical approach to sorting large samples of microbes.

[0009] It is an object of the present invention to provide a supercompetitive isolate(s) of bacteria of the genus Bradyrhizobia to colonize leguminous plants that perform(s) the colonization capacity of commercially available strains, for example, USDA strain 532C commercial. It is additionally an object of the present invention to provide a supercompetitive isolate(s) of bacteria of the genus Bradyrhizobia to colonize leguminous plants capable of intensifying the effectiveness in promoting leguminous plant growth, in comparison with commercially available strains. available, for example commercial USDA 532C strain.

SUMMARY OF THE INVENTION

[0010] In order to improve overall plant health and the availability of a usable nitrogen source for plants, there is a need for bacterial strains that are superior at colonizing plants and that enhance overall plant growth. The isolated strains of the present invention realize these benefits.

[0011] The present invention relates to isolated strains of bacteria having at least the following improved characteristics compared to commercially available strains, for example, commercial USDA 532C strain, wherein improved characteristics include, but are not limited to: a. heightened competitiveness to colonize a plant; and b. effectiveness in promoting enhanced plant growth.

[0012] The present invention relates to biologically pure culture(s) of strain(s) Bradyrhizobia japonicum the strain with accession number NRRL B-50592 (also filed as NRRL B-59571); the strain with deposit accession number NRRL B-50593 (also filed as NRRL B-59572); the strain with deposit accession number NRRL B-50586 (also filed as NRRL B-59565); the strain with deposit accession number NRRL B-50588 (also filed as NRRL B-59567); the strain with deposit accession number NRRL B-50587 (also filed as NRRL B-59566); the strain with deposit accession number NRRL B-50589 (also filed as NRRL B-59568); the strain with deposit accession number NRRL B-50591 (also filed as NRRL B-59570); the strain with the deposit access number NRRL B-50590 Petition 870180134926, of 09/26/2018, pg. 18/105 (also filed as NRRL B-59569); the strain with deposit accession number NRRL B-50594 (also filed as NRRL B-50493); the strain with warehouse accession number NRRL B-50726; the strain with warehouse accession number NRRL B-50727; the strain with warehouse accession number NRRL B-50728; the strain with warehouse accession number NRRL B-50729; and the strain with deposit accession number NRRL B-50730, or a combination of at least two or more of the previously deposited strains.

[0013] The present invention also relates to isolated bacterial strain(s) of the present invention which includes strain(s) which is(are) closely related to any of the above strains based on the 16S rDNA sequence identity, and which are at least 95% identical to any of the above strains based on 16S rDNA sequence identity.

[0014] The present invention additionally relates to a method of enhancing plant growth, comprising applying to plants, plant seeds, or plants involving soil or plant seeds a composition comprising at least one of the strains of the present invention or a combination of at least two or more of the previously deposited strains.

[0015] The invention additionally relates to compositions comprising one or more strains of the present invention, including an agronomically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1A is an image of a PCR gel showing a unique nucleotide primer specific for USDA 532C.

[0017] Figure 1B is an image of a PCR gel showing primer specificity of nucleotide 209 using USDA 532C and native strains.

[0018] Figure 2A is an image of a PCR gel showing USDA strain 532C Bradyrhizobia japonicum as the competitively dominant strain for soybean nodulation.

[0019] Figure 2B is an image of a PCR gel showing strains other than USDA strain 532C Bradyrhizobia japonicum as the competitively dominant strain for soybean nodulation.

[0020] Figure 3A is a DNA fingerprint dendrogram of isolated strains and USDA 532C: 138 - NRRL B-50589 (also filed as NRRL B-59568); 13 - NRRL B-50586 (also filed as NRRL B-59565); p140 - USDA 532C; 184 - NRRL B-50594 (also filed as NRRL B-50493); 142 - NRRL B-50590 (also filed as NRRL B-59569); 130 - NRRL B-50587 (also filed as NRRL B-59566); 65 - NRRL B-50588 (also filed as NRRL B-59567); 198 - NRRL B-50592 (also filed as NRRL B-59571); 135 - NRRL B-50591 (also filed as NRRL B-59570); and 48 - NRRL B-50593 (also filed as NRRL B-59572).

[0021] Figure 3B is a DNA fingerprint dendrogram of isolated strains and USDA 532C: 138 - NRRL B-50589 (also filed as NRRL B-59568); 13 - NRRL B-50586 (also filed as NRRL B-59565); 140 - USDA 532C; 184 - NRRL B-50594 (also filed as NRRL B-50493); 142 - NRRL B-50590 (also filed as NRRL B-59569); 130 - NRRL B-50587 (also filed as NRRL B-59566); 65 - NRRL B-50588 (also filed as NRRL B-59567); 198 - NRRL B-50592 (also filed as NRRL B-59571); 135 - NRRL B-50591 (also filed as NRRL B-59570); 48 - NRRL B-50593 (also filed as NRRL B-59572); 318 - NRRL B-50727, 278 - NRRL B-50726, 727 - NRRL B-50730, 370 - NRRL B-50728; and 518 - NRRL B-50729.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention relates to isolated strains of bacteria having at least the following improved characteristics compared to commercially available strains, for example, commercial USDA 532C strain, wherein improved characteristics include, but are not limited to: a. heightened competitiveness to colonize a plant; and b. effectiveness in promoting enhanced plant growth.

[0023] "Bacterial strain(s)" as used herein means bacterial strains that are diazotrophic. That is, bacteria that are symbiotic nitrogen-fixing bacteria. Non-limiting examples of bacterial strains as used herein include, but are not limited to, 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. mongolense, R. miluonense, R. sullae, R. tropici, R. undicola, and/or R. yanglingense), Bradyrhizobium spp. (for example, B. bete, B. canariense, B. elkanii, B. iriomotense, B. japonicum, B. jicamae, B. liaoningense, B. pachyrhizi, and/or B. yuanmingense), Azorhizobium spp. (eg 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. temperatum, M. tianshanense). In a particular embodiment, bacterial strain(s) of the invention further includes Bradyrhizobium japonicum strains with deposit accession numbers NRRL B-50592 (deposited also as NRRL B-59571), NRRL B-50593 (deposited also as NRRL B-59572), NRRL B-50586 (also filed as NRRL B-59565), NRRL B-50588 (also filed as NRRL B-59567), NRRL B-50587 (also filed as NRRL B-59566), NRRL B-50589 (also filed as NRRL B-59568), NRRL B-50591 (also filed as NRRL B-59570); NRRL B-50590 (also filed as NRRL B-59569); NRRL B-50594 (also filed as NRRL B-50493); NRRL B-50726; NRRL B-50727; NRRL B-50728; NRRL B-50729; NRRL B-50730, or a combination of at least two or more of the previously deposited strains, including two of the above strains, at least three of the above strains, at least four of the above strains, at least five of the above strains, at least six of the above above strains, at least seven of the above strains, at least eight of the above strains, at least nine of the above strains, at least ten of the above strains, at least eleven of the above strains, at least twelve of the above strains, at least thirteen of the above strains above, up to and including all above strains.

[0024] The terms “commercially available strain(s)” 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.

[0025] As used herein, "enhanced competitiveness" and/or "enhanced nodulation" shall mean bacterial strain(s) having a dominant percent nodule occupancy, e.g., at least 50%, at least 55%, 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 96%, at least 97%, at least 98%, at least 99%, up to 100% node occupancy. “Enhanced competitiveness” was determined according to the detailed test(s) described below (See Materials and Methods: “Primary Classification Protocol” and “Competition Study Protocol”).

[0026] As used herein, the term "nodule" is defined to include, but not be limited to, determinate nodules, indeterminate nodules, or a combination thereof. Examples of determinate nodules and indeterminate nodules are well known in the art and described in Denison, R.F., 2000, The Amer. Naturalist. 156(6): 567-576. Determinate nodules are found on Glycine, Lotus, or Phaseolus species and are round and spherical in shape. (Denison, 2000) Certain nodules only grow for a limited period of time - typically a few weeks. (Denison, 2000) Unlike determinate nodules, indeterminate nodules are found in Medicago, Trifolium, and Pisium species, have an elongated shape, and grow continuously. (Denison, 2000)

[0027] "Nodule occupancy" is a term known in the art. McDermott T.R. & Graham, P.H., Appl. and Environ. Microbiol. 55(10): 2493-2498. As used herein, "nodule occupancy" means the percentage of nodules occupied by a bacterial strain(s) other than a commercially available Bradyrhizobium strain, e.g., USDA 532C, and/or the number of nodules containing a particular bacterial strain(s) other than a commercially available Bradyrhizobium strain, eg USDA 532C, divided by the total number of nodules containing all bacterial strains. “Nodule occupancy” was determined according to the detailed test(s) described below (See Materials and Methods: “Primary Classification Protocol” and “Competition Study Protocol”) and can be determined from an analysis of plant nodules obtained from both greenhouse and field samples. By way of example, percent nodule occupancy = A/(A+B) where A is the number of nodules containing a particular bacterial strain(s) other than a commercially available Bradyrhizobium strain, for example, USDA 532C, and B is the number of nodules containing a commercially available Bradyrhizobium strain, for example, USDA 532C. It is well known in the art that, despite the rare exception, a single nodule will contain 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.

[0028] As used herein, "enhanced growth promoting effectiveness" includes at least increased plant yield measured in terms of bushels/acre, increased fruit number, increased root number, increased root length, root mass increased, increased root volume, increased foliage area, increased plant support, increased plant vigor, and/or increased nitrogen (N2) fixing capacity. “Effectiveness in enhancing growth promotion” was determined according to the detailed trial(s) described below (See Materials and Methods: “Primary Screening Protocol” and “Study Protocol Performance”) and can be determined from an analysis of plants obtained from both greenhouse and field samples.

[0029] As used herein, "increased fruit number" means an increased total number of soybean pods in a soybean plant and/or an increased total dry weight of soybean pods in a soybean plant.

[0030] As used herein, “total dry weight” means the weight of plant matter (e.g., plant fruit, plant pods, plant roots, plant nodules, total plants, partial plants, etc.) after incubation at 80°C for a specified period of time, e.g. at least 4 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 48 hours, etc., or any length of time necessary to dry the plant matter. It should be understood that drying times for the purposes of determining “total dry weight” depend on many factors. Non-limiting factors that can impact drying time include the material being dried, the mass of the material being dried, the amount of material being dried, and/or combinations thereof. Incubation can be performed in any temperature-controlled device used in the technique. For the purposes of this invention, “total dry weight” was determined with an Eppendorf Innova® 42R incubator.

[0031] The terms “increased nitrogen (N2) fixing capacity” as used herein mean that the isolated bacteria can increase nitrogen (N2) fixation. According to the “Performance Study Protocol” provided below (Materials and Methods), the relative nitrogen (N2) fixing capacity of bacteria can be quantified by measuring the total nitrogen content of the plant using standard nitrogen quantification methods known to the experts. skilled in the art (e.g. the Kjeldahl method, etc.). See Takahashi, M., et al., 2007. Uptake, Assimilation, and Novel Metabolism of Nitrogen Dioxide in Plants, p. 109-118. In N. Willey (ed.), Phytoremediation: Methods and Reviews, vol. 23. Human Press, New York; Bremner, J.M. 1965. Total nitrogen, p. 1149-1178. In CA Black (ed.), Methods of soil analysis, vol. 2. American Society for Agronomy, Madison; Schank, S.C., et al., 1981, App. and Enviro. Microbiol., 41(2): 342-345.

[0032] In yet another aspect of the present invention, the isolated bacterial strain(s) has an increased temperature tolerance. “Increased temperature tolerance” means the range of temperatures at which the isolated bacterial strain(s) is(are) able to grow, e.g. the maximum and minimum temperatures at which the strain(s) ) Isolated Bradyrhizobium(s) can grow. In one aspect, "increased temperature tolerance" was determined in accordance with the "Temperature Profile Protocol" discussed infra (Materials and Methods).

[0033] In yet another aspect of the present invention, the isolated bacterial strain(s) is(are) naturally resistant to glyphosate. In one aspect, "increased temperature tolerance" was determined in accordance with the "Glyphosate Resistance Profile Protocol" discussed infra (Materials and Methods).

[0034] In another aspect, the isolated bacterial strain(s) of the present invention include(s) strain(s) that(are) closely related to any of the above strains based on 16S rDNA sequence identity. 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 relatedness in bacteria. In one embodiment, at least one strain is at least 95% identical to any of the foregoing strains based on 16S rDNA sequence identity, at least 96% identical to any of the foregoing strains based on 16S rDNA sequence identity, at least at least 97% identical to any of the above strains based on 16S rDNA sequence identity, at least 98% identical to any of the above strains based on 16S rDNA sequence identity, at least 98.5% identical to any of the above strains strains based on 16S rDNA sequence identity, at least 99% identical to any of the above strains based on 16S rDNA sequence identity, or at least 99.5% identical to any of the above strains based on 16S sequence identity rDNA.

[0035] In another embodiment, the present invention includes a method for isolating bacterial strain(s) with enhanced competitiveness to occupy the nodules of a legume plant and effectiveness in promoting enhanced growth of the legume plant. As used here, the terms “isolate, isolate, isolating, and/or isolated, etc.” mean that the referenced material is removed from the environment in which it is normally found. The method includes, among other things: a. obtaining a bacterial strain(s) from a soil sample; B. subjecting the bacterial strain(s) and a commercially available strain to a leguminous plant; w. selecting the bacterial strain(s) that is(are) more competitive than the commercially available strain to occupy the nodules of a leguminous plant; d. analyze the selected bacterial strain(s) that is(are) more competitive than the commercially available strain(s) to occupy the nodules of a leguminous plant for the strain(s) bacterial(s) with an effectiveness in promoting enhanced leguminous plant growth; and is. isolate the bacterial strain(s) effectively in promoting enhanced leguminous plant growth.

[0036] In one aspect, the bacterial strain(s) isolated is(are) strains of the genus BradyrhizobiA. In yet another aspect, the method further includes the step of screening the Bradyrhizobium strain(s) against a specific nucleotide primer unique to a commercially available strain of Bradyrhizobia, for example commercial USDA 532C strain.

[0037] In yet another aspect, the method includes isolating a culture of Bradyrhizobia japonicum selected from the group consisting of: f. strain with deposit accession number NRRL B-50592 (also filed as NRRL B-59571); g. strain with deposit accession number NRRL B-50593 (also filed as NRRL B-59572); H. strain with deposit accession number NRRL B-50586 (also filed as NRRL B-59565); i. strain with deposit accession number NRRL B-50588 (also filed as NRRL B-59567); j. strain with deposit accession number NRRL B-50587 (also filed as NRRL B-59566); k. strain with deposit accession number NRRL B-50589 (also filed as NRRL B-59568); l. strain with deposit accession number NRRL B-50591 (also filed as NRRL B-59570); m. strain with deposit accession number NRRL B-50590 (also filed as NRRL B-59569); n. strain with deposit accession number NRRL B-50594 (also filed as NRRL B-50493); O. strain with warehouse accession number NRRL B-50726; P. strain with warehouse accession number NRRL B-50727; the strain with warehouse accession number NRRL B-50728; the strain with warehouse accession number NRRL B-50729; and the strain with deposit accession number NRRL B-50730, or a combination of at least two or more of the foregoing deposited strains, including more than two, such as at least three of the foregoing strains, at least four of the foregoing strains , at least five of the above strains, at least six of the above strains, at least seven of the above strains, at least eight of the above strains, at least nine of the above strains, at least ten of the above strains, at least eleven of the above strains, at least twelve of the above strains, at least thirteen of the above strains, up to one including all of the above strains.

[0038] In yet another aspect, the method includes isolating bacterial strain(s) with increased temperature tolerance. See Materials and Methods: “Temperature Profile Protocol”.

[0039] Still further, the method includes isolating a bacterial strain(s) with natural resistance to glyphosate. See Materials and Methods: “Glyphosate Profile Strength Protocol”.

[0040] In another preferred aspect, the method includes isolating selected bacterial strain(s) from the genus consisting of Rhizobium and Bradyrhizobium capable of increasing the nodulation of a leguminous plant. Composition

[0041] The present invention includes a composition comprising at least one of the isolated bacterial strain(s) of the present invention or a combination of at least two or more of the previously deposited strains, including more than two , such as at least three of the above strains, at least four of the above strains, at least five of the above strains, at least six of the above strains, at least seven of the above strains, at least eight of the above strains, at least nine of the above strains above strains, at least ten of the above strains, at least eleven of the above strains, at least twelve of the above strains, at least thirteen of the above strains, up to one including all of the above strains and an agronomically suitable carrier.

[0042] In some embodiments, the composition may be an inoculant composition. As used herein and in the art, the terms "inoculating composition" generally refer to compositions or materials that introduce compatible bacterial strains to both an outer surface of the seeds and into the seed furrow.

[0043] The composition may comprise one or more agronomically acceptable carriers. In examples where multiple agronomically acceptable carriers are used, the agronomically acceptable carriers may be the same or different. As used herein with "carrier", the terms "agronomically acceptable" refer to any material that can be used to deliver the actives to a seed, soil or plant, and preferably to which carrier can be added (in seed, soil or or plant) without having an adverse effect on plant growth, soil structure, soil drainage or the like. Suitable carriers include, but are not limited to, wheat straw, cereal bran, ground wheat straw, peat-based powders or granules, gypsum-based granules, and clays (eg, kaolin, bentonite, montmorillonite). Formulations such as liquid powder, peat or humectant will be suitable for seed dressing. When used to coat seeds, the material can be mixed with water, applied to the seeds and dried naturally. Yet other examples of carriers include bran that is moistened, dried, sieved and applied to seeds before coating with an adhesive, for example, acacia. In modalities that involve the formulation of actives, the agronomically acceptable carrier can be aqueous. If a liquid carrier is used, the liquid carrier (eg, water) will typically include growth medium to grow the bacterial strains. Non-limiting examples of suitable growth media for the bacterial strains include mannitol yeast extract, glycerol yeast extract, or any medium known to those skilled in the art to be compatible with and/or to provide growth nutrients for the bacterial strains.

[0044] Also encompassed by the compositions of the present invention are compositions including one or more signal molecules. Non-limiting examples of plant signal molecules include nodulation factors (i.e., lipo-chito-oligosaccharide), chito-oligosaccharides, chitinous compounds, flavonoids, jasmonic acid or its derivatives, linoleic acid or its derivatives, linoleic acid or its derivatives , carricines, or combinations thereof.

na qual: G é uma hexosamina que pode ser substituída, por exemplo, por um grupo acetila no nitrogênio, um grupo sulfato, um grupo acetila e/ou um grupo éter em um oxigênio, R1, R2, R3, R5, R6 e R7, que podem ser idênticos ou diferentes, representam H, CH3 CO--, Cx Hy CO-- onde x é um número inteiro entre 0 e 17, e y é um número inteiro entre 1 e 35, ou qualquer outro grupo acila tal como, por exemplo, um carbamila, R4 representa uma cadeia alifática mono, di ou tri insaturada contendo pelo menos 12 átomos de carbono, e n é um número inteiro entre 1 e 4.[0045] Lipo-chito-oligosaccharide compounds (LCO's), also known in the art as symbiotic Nod signals or Nodulation Factors, consist of an oligosaccharide backbone of N-acetyl-D-glucosamine ("GIcNAc") β residues -1,4- linked with an N-linked fatty acyl chain condensed at the non-reducing terminus. LCO's differ in the number of GIcNAc residues in the backbone, in the length and degree of saturation of the fatty acyl chain, and in the substitutions of reducing and non-reducing sugar residues. An example of an LCO is presented below as formula I:

in which: G is a hexosamine which may be replaced, for example, by an acetyl group on nitrogen, a sulfate group, an acetyl group and/or an ether group on an oxygen, R1, R2, R3, R5, R6 and R7 , which may be identical or different, represent H, CH3 CO--, Cx Hy CO-- where x is an integer between 0 and 17, and y is an integer between 1 and 35, or any other acyl group such as, for example, a carbamyl, R4 represents a mono-, di- or tri-unsaturated aliphatic chain containing at least 12 carbon atoms, and n is an integer between 1 and 4.

na qual R representa H ou CH3 CO-- e n é igual a 2 ou 3.[0046] LCOs 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 for each such bacterial species, and each strain can produce multiple LCO's with different structures. For example, S. meliloti specific LCOs were also described in US patent 5,549,718 having formula II:

where R represents H or CH3 CO-- and n is equal to 2 or 3.

[0047] Even more specific LCOs include NodRM, NodRM-1, NodRM-3. When acetylated (the R=CH3 is CO--), they become AcNodRM-1, and AcNodRM-3, respectively (U.S. Patent 5,545,718).

[0048] Bradyrhizobium japonicum LCOs are described in U.S. patents. 5,175,149 and 5,321,011. Generally speaking, they are pentasaccharide phytohormones comprising methylfucose. A number of such LCOs derived from B. japonicum are described: BjNod-V (C18:1); BjNod-V (AC, C18:1), BjNod-V (C16:1); and BjNod-V (AC1 C16:0), with "V" indicating the presence of five N-acetylglucosamines; "Ac" is an acetylation; the number after the "C" indicating the number of carbons in the fatty acid side chain; and the number after the ":" the number of double bonds. leguminosarum), Sinorhizobium (including S. meliloti), and bacterial strains genetically engineered to produce LCO's.

[0049] LCO's used in compositions of the invention can be obtained (i.e., isolated and/or purified) from bacterial strains that produce LCO's, such as strains of Azorhizobium, Bradyrhizobium (including B. japonicum), Mesorhizobium, Rhizobium (including R. .

[0050] Also encompassed by the present invention are compositions using LCOs obtained (ie, isolated and/or purified) from a mycorrhizal fungus, such as fungi of 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 therein also referred to as "Myc factors").

[0051] Further encompassed by the compositions of the present invention is the use of synthetic LCO compounds, such as those described in WO 2005/063784, and recombinant LCO's produced through genetic engineering. The basic naturally occurring LCO structure may contain modifications or substitutions seen in naturally occurring LCO's, such as those described in Spaink, Crit. Rev. Plant Sci. 54:257-288 (2000) and D'Haeze, et al., Glycobiology 12:79R-105R (2002). Precursor oligosaccharide molecules (COs, which as described below are also used as plant signal molecules in the present invention) for the construction of LCOs can also be synthesized by genetically engineered organisms, for example, as in Samain, et al. ., carb. Res. 302:35-42 (1997); Samain, et al., J. Biotechnol. 72:33-47 (1999).

[0052] LCO's can be used in various forms of purity and can be used alone or in the form of an LCO-producing bacterial or fungal culture. Methods for providing substantially pure LCO's include simply removing the microbial cells from a mixture of LCOs and the microbe, or continuing to isolate and purify the LCO molecules by solvent phase separation of the LCO followed by HPLC chromatography as described, for example, in the U.S. patent 5,549,718. Purification can be improved by repeated HPLC, and purified LCO molecules can be lyophilized for long-term storage.

[0053] Chitooligosaccharides (COs) are known in the art as N-acetylglucosamine β-1-4 linked structures identified as chitin oligomers, also as N-acetylchitooligosaccharides. CO's have unique and different side chain decorations that make them different from chitin molecules [(C8H13NO5)n, CAS No. 1398-61-4], and chitosan molecules [(C5H11NO4)n, CAS No. 9012-76-4]. Representative literature describing the structure and production of COs is as follows: Van der Holst, et al., Current Opinion in Structural Biology, 11:608-616 (2001); Robina, et al., Tetrahedron 58:521-530 (2002); Hanel, et al., Plant 232:787-806 (2010); Rouge, et al. Chapter 27, "T Molecular Immunology of Complex Carbohydrates" in Advances in Experimental Medicine and Biology, Springer Science; Wan, et al., Plant Cell 21:1053-69 (2009); PCT/F100/00803 (9/21/2000); and Demont-Caulet, et al., Plant Physiol. 120(1):83-92 (1999). OCs can be synthetic or recombinant. Methods for preparing recombinant COs are known in the art. See, for example, Samain, et al. (supra.); Cottaz, et al., Meth. Eng. 7(4):311-7 (2005) and Samain, et al., J. Biotechnol. 72:33-47 (1999).

[0054] Compositions of the present invention may also include chitin compounds (other than COs), flavonoids, jasmonic acid, linoleic acid and linoleic acid and their derivatives, and carricins.

[0055] Chitin and chitosans, which are major components of fungal cell walls and the exoskeletons of insects and crustaceans, are also composed of GIcNAc residues. Chitinous compounds include chitin, (IUPAC: N-[5-[[3-acetylamino-4,5-dihydroxy-6-(hydroxymethyl)oxan-2yl]methoxymethyl]-2-[[5-acetylamino-4,6 -dihydroxy-2-(hydroxymethyl)oxan-3-yl]methoxymethyl]-4-hydroxy-6-(hydroxymethyl)oxan-3-yl]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). Such compounds can be commercially obtained, for example, from Sigma-Aldrich, or prepared from insect, crustacean shells, or fungal cell walls. Methods for preparing chitin and chitosan are known in the art, and have been described, for example, in U.S. Pat. 4,536,207 (preparation of crustacean shells), Pochanavanich, et al., Lett. app. Microbiol. 35:17-21 (2002) (preparation of fungal cell walls), and U.S. patent 5,965,545 (preparation of crab shells and hydrolysis of commercial chitosan). Deacetylated chitins and chitosans can be obtained that range from less than 35 to more than 90% deacetylation, and cover a wide spectrum of molecular weights, for example, low molecular weight chitosan oligomers less than 15kD and 0.5 kD chitin oligomers. at 2kD; "practical grade" chitosan having a molecular weight of about 15kD; and high molecular weight chitosan of up to 70kD. Chitin and chitosan compositions formulated for seed treatment are also commercially available. Commercial products include, for example, ELEXA® (Plant Defense Boosters, Inc.) and BEYOND™ (Agrihouse, Inc.).

[0056] Flavonoids are phenolic compounds with the general structure of two aromatic rings connected by a three-carbon bond. Flavonoids are produced by plants and have many functions, for example as beneficial signaling molecules, and as protection against insects, animals, fungi and bacteria. Classes of flavonoids include chalcones, anthocyanidins, coumarins, flavones, flavonols, flavonones, and isoflavones. See Jain, et al., J. Plant Biochem. & Biotechnol. 11:1-10 (2002); Shaw, et al., Environmental Microbiol. 11:1867-80 (2006).

[0057] Representative flavonoids that can be used in compositions of the present invention include genistein, daidzein, formononetin, naringenin, hesperetin, luteolin, and apigenin. 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, in the manner described in U.S. patents. 5,702,752; 5,990,291; and 6,146,668. Flavonoid compounds can also be produced by genetically engineered organisms, such as yeast, in the manner described in Ralston, et al., Plant Physiology 137:1375-88 (2005).

[0058] Jasmonic acid (JA, [1R-[1α,2β(Z)]]-3-oxo-2-(pentenyl)cyclopentanoacetic acid) and its derivatives, linoleic acid ((Z,Z)-9,12 acid -octadecadienoic) and derivatives thereof, and linoleic acid ((Z,Z,Z)-9,12,15-octadecatrienoic acid) and derivatives thereof, can also be used in compositions of the present invention. Jasmonic acid and its methyl ester, methyl jasmonate (MeJA), known collectively as jasmonates, are octadecanoid-based compounds that occur naturally in plants. Jasmonic acid is produced by the roots of wheat seedlings, and by fungal microorganisms such as Botryodiplodia theobromae and Gibbrella fujikuroi, yeast (Saccharomyces cerevisiae), and pathogenic and non-pathogenic strains of Escherichia coli. Linoleic acid and linoleic acid are produced in the course of jasmonic acid biosynthesis. Jasmonates, linoleic acid and linoleic acid (and their derivatives) are reported to be 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 linoleic acid induce the expression of Nod genes in Bradyrhizobium japonicum," USDA 3, May 17, 2001.

[0059] Used derivatives of linoleic acid, linoleic acid, and jasmonic acid that can be used in compositions of the present invention include esters, starches, glycosides, and salts. Representative esters are compounds in which the carboxyl group of linoleic acid, linoleic acid, or jasmonic acid has been replaced with a --COR group, where R is a --OR1 group, where R1 is: an alkyl group, such as a group unbranched or branched C1-C8-alkyl, for example a methyl, ethyl or propyl group; an alkenyl group, such as an unbranched or branched C2-C8-alkenyl group; an alkynyl group, such as an unbranched or branched C2-C8 alkynyl group; an aryl group, for example, having 6 to 10 carbon atoms; or a heteroaryl group, for example, having 4 to 9 carbon atoms, where the heteroatoms in the heteroaryl group can be, for example, N, O, P, or S. Representative starches are compounds in which the carboxyl group of linoleic acid , linoleic acid, or jasmonic acid has been replaced with a --COR group, where R is an NR2R3 group, in which R2 and R3 are independently: hydrogen; an alkyl group, such as an unbranched or branched C1-C8-alkyl group, for example a methyl, ethyl or propyl group; an alkenyl group, such as an unbranched or branched C2-C8-alkenyl group; an alkynyl group, such as an unbranched or branched C2-C8 alkynyl group; an aryl group, for example, having 6 to 10 carbon atoms; or a heteroaryl group, for example, having 4 to 9 carbon atoms, where the heteroatoms in the heteroaryl group can be, for example, N, O, P, or S. Esters can be prepared by known methods, such as nucleophilic addition acid-catalyzed, in which the carboxylic acid is reacted with an alcohol in the presence of a catalytic amount of a mineral acid. Starches can also be prepared by known methods, such as reacting the carboxylic acid with the appropriate amine in the presence of a coupling agent such as dicyclohexyl carbodiimide (DCC), under neutral conditions. Suitable salts of linoleic acid, linoleic acid, and jasmonic acid include, for example, base addition salts. Bases that can be used as reagents to prepare metabolically acceptable base salts of these compounds include those 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 prepared by mixing together a solution of linoleic acid, linoleic acid, or jasmonic acid with a solution of the base. The salt may precipitate out of solution and be collected by filtration, or it may be recovered by other means, such as solvent evaporation.

em que; Z é O, S ou NR5; R1, R2, R3, e R4 são cada qual independentemente H, alquila, alquenila, alquinila, fenila, benzila, hidróxi, hidroxialquila, alcóxi, fenilóxi, benzilóxi, CN, COR6, COOR=, halogênio, NR6R7, ou NO2; e R5, R6, e R7 são cada qual independentemente H, alquila ou alquenila, ou um sal biologicamente aceitável destes. Exemplos de sais biologicamente aceitáveis desses compostos podem incluir sais de adição ácida formados com ácidos biologicamente aceitáveis, exemplos dos quais incluem cloridrato, bromidrato, sulfato ou bissulfato, fosfato ou hidrogeno fosfato, acetato, benzoato, succinato, fumarato, maleato, lactato, citrato, tartrato, gluconato; metanossulfonato, benzenosulfonato e ácido p- toluenosulfônico. Sais metálicos biologicamente aceitáveis adicionais podem incluir sais de metal alcalino, com bases, exemplos dos quais incluem os sais de sódio e potássio. Exemplos de compostos abrangidos pela estrutura que podem ser adequados para uso na presente invenção incluem o seguinte: 3- metil-2H-furo[2,3-c]piran-2-ona (onde R1=CH3, R2, R3, R4=H), 2H-furo[2,3- c]piran-2-ona (onde R1, R2, R3, R4=H), 7-metil-2H-furo[2,3-c]piran-2-ona (onde R1, R2, R4=H, R3=CH3), 5-metil-2H-furo[2,3-c]piran-2-ona (onde R1, R2, R3=H, R4=CH3), 3,7-dimetil-2H-furo[2,3-c]piran-2-ona (onde R1, R3=CH3, R2, R4=H), 3,5-dimetil-2H-furo[2,3-c]piran-2-ona (onde R1, R4=CH3, R2, R3=H), 3,5,7-trimetil-2H-furo[2,3-c]piran-2-ona (onde R1, R3, R4=CH3, R2=H), 5-metoximetil-3-metil-2H-furo[2,3-c]piran-2-ona (onde R1=CH3, R2, R3=H, R4=CH2OCH3), 4-bromo-3,7-dimetil-2H-furo[2,3-c]piran- 2-ona (onde R1, R3=CH3, R2=Br, R4=H), 3-metilfuro[2,3-c]piridin-2(3H)-ona (onde Z=NH, R1=CH3, R2, R3, R4=H), 3,6-dimetilfuro[2,3-c]piridin-2(6H)- ona (onde Z=N--CH3, R1=CH3, R2, R3, R4=H). Vide, patente U.S. 7.576.213. Estas moléculas são também conhecidas como carricinas. Vide Halford, supra.[0060] Carricines are vinylologous 4H-pyrones, for example, 2H-furo[2,3-c]pyran-2-ones including derivatives and analogues thereof. Examples of these compounds are represented by the following structure:

on what; Z is O, S or NR5; R1, R2, R3, and R4 are each independently H, alkyl, alkenyl, alkynyl, phenyl, benzyl, hydroxy, hydroxyalkyl, alkoxy, phenyloxy, benzyloxy, CN, COR6, COOR=, halogen, NR6R7, or NO2; and R5, R6, and R7 are each independently 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 can include alkali metal salts with bases, examples of which include the sodium and potassium salts. Examples of compounds encompassed by the framework that may be suitable for use in the present invention include the following: 3-methyl-2H-furo[2,3-c]pyran-2-one (where R1=CH3, R2, R3, R4= H), 2H-furo[2,3-c]pyran-2-one (where R1, R2, R3, R4=H), 7-methyl-2H-furo[2,3-c]pyran-2-one (where R1, R2, R4=H, R3=CH3), 5-methyl-2H-furo[2,3-c]pyran-2-one (where R1, R2, R3=H, R4=CH3), 3 ,7-dimethyl-2H-furo[2,3-c]pyran-2-one (where R1, R3=CH3, R2, R4=H), 3,5-dimethyl-2H-furo[2,3-c ]pyran-2-one (where R1, R4=CH3, R2, R3=H), 3,5,7-trimethyl-2H-furo[2,3-c]pyran-2-one (where R1, R3, R4=CH3, R2=H), 5-methoxymethyl-3-methyl-2H-furo[2,3-c]pyran-2-one (where R1=CH3, R2, R3=H, R4=CH2OCH3), 4 -bromo-3,7-dimethyl-2H-furo[2,3-c]pyran-2-one (where R1, R3=CH3, R2=Br, R4=H), 3-methylfuro[2,3-c ]pyridin-2(3H)-one (where Z=NH, R1=CH3, R2, R3, R4=H), 3,6-dimethylfuro[2,3-c]pyridin-2(6H)-one (where Z=N--CH3, R1=CH3, R2, R3, R4=H). See US Patent 7,576,213. These molecules are also known as carricins. See Halford, supra.

[0061] Compositions of the present invention may additionally include an agriculturally/agronomically beneficial agent. Non-limiting examples of such agents that can be used in the practice of the present invention include herbicides, fungicides and insecticides.

[0062] Suitable herbicides include bentazone, acifluorphen, chlorimuron, lactofen, clomazone, fluazifop, glufosinate, glyphosate, sethoxydime, imazethapyr, imazamox, fomesafe, flumiclorac, imazaquine, and clethodim. Commercial products containing each of these compounds are readily available. Herbicide concentration in the composition will generally correspond to the marked usage rate for a particular herbicide.

[0063] A "fungicide" as used herein and in the art is an agent that kills or inhibits fungal growth. As used herein, a fungicide "displays activity against" a particular species of fungi if treatment with the fungicide results in killing or inhibiting the growth of a fungal population (e.g., in soil) relative to an untreated population. Effective fungicides according to the invention will suitably exhibit activity against a wide range of pathogens, including but not limited to Phytophthora, Rhizoctonia, Fusarium, Pythium, Phomopsis or Selerotinia and Phakopsora and combinations thereof.

[0064] Commercial fungicides may be suitable for use in the present invention. Suitable commercially available fungicides include PROTÉGÉ, RIVAL or ALLEGIANCE FL or LS (Gustafson, Plano, 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 Aires, Argentina). Active ingredients in these and other commercial fungicides include, but are not limited to, fludioxonil, mefenoxam, azoxystrobin, and metalaxyl. Commercial fungicides are best used according to the manufacturer's instructions at recommended concentrations.

[0065] As used herein, an insecticide "displays activity against" a particular insect species if treatment with the insecticide results in killing or inhibition of an insect population relative to an untreated population. Effective insecticides according to the invention will suitably exhibit activity against a wide range of insects including, but not limited to, beetles, caterpillars, grubs, corn rootworms, corn seedworms, flea beetles, stink bugs, aphids, leaf, and brown bugs.

[0066] Commercial insecticides may be suitable for use in the present invention. Suitable commercially available insecticides include CRUISER (Syngenta, Wilmington, DE), GAUCHO and PONCHO (Gustafson, Plano, TX). Active ingredients in these and other commercial insecticides include thiamethoxam, clothianidin, and imidacloprid. Commercial insecticides are best used according to the manufacturer's instructions at recommended concentrations.

[0067] Compositions of the present invention are also intended to include the use of one or more phosphate solubilizing agents. As used herein, phosphate solubilizing agents include, but are not limited to, phosphate solubilizing microorganisms. As used herein, "phosphate solubilizing microorganism" is a microorganism that is capable of increasing the amount of phosphorus available to a plant. Phosphate solubilizing microorganisms include fungal and bacterial strains. In one embodiment, the phosphate solubilizing microorganism is a spore-forming microorganism.

[0068] Non-limiting examples of phosphate solubilizing microorganisms include species from a genus selected from the group consisting of Acinetobacter, Arthrobacter, Arthrobotrys, Aspergillus, Azospirillum, Bacillus, Burkholderia, Candida Chryseomonas, Enterobacter, Eupenicillium, Exiguobacterium, Klebsiella, Kluyvera , Microbacterium, Mucor, Paecilomyces, Paenibacillus, Penicillium, Pseudomonas, Serratia, Stenotrophomonas, Streptomyces, Streptosporangium, Swaminathania, Thiobacillus, Torulospora, Vibrio, Xanthobacter, and Xanthomonas.

[0069] Non-limiting examples of phosphate solubilizing microorganisms are selected from the group consisting of Acinetobacter calcoaceticus, Acinetobacter spp., Arthrobacter spp., Arthrobotrys oligospora, Aspergillus niger, Aspergillus spp., Azospirillum halopraeferans, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans, Bacillus licheniformis, Bacillus subtilis, Burkholderia cepacia, Burkholderia vietnamiensis, Candida krissii, Chryseomonas luteola, Enterobacter aerogenes, Enterobacter asburiae, Enterobacter spp., Enterobacter taylorae, Eupenicillium parvum, Exiguobacterium spp., Klebsiella spp., Kluyvera cryocrescen s, Microbacterium spp. P. Seudomonas trivialis, Serratia marcescens, Stenotrophomonas maltophilia, Streptomyces sp., Streptosporangium spp., Swaminathania salitolerans, Thiobacillus ferrooxidans, Torulospora globosa, Vibrio proteolyticus, Xanthobacter agilis, and Xanthomonas campestris.

[0070] In one embodiment, the phosphate solubilizing microorganism is a strain of the fungus Penicillium. Strains of the Penicillium fungus that can be used in the practice of the present invention include P. bilaiae (formerly known as P. bilaii), P. albidum, P. aurantiogriseum, P. chrysogenum, P. citreonigrum, P. citrinum, P. digitatum, P. frequentas, P. fuscum, P. gaestrivorus, P. glabrum, P. griseofulvum, P. implicatum, P. janthinellum, P. lilacinum, P. minioluteum, P. montanense, P. nigricans, P. oxalicum, P. pinetorum, P. pinophilum, P. purpurogenum, P. radicans, P. radicum, P. raistrickii, P. rugulosum, P. simplicissimum, P. solitum, P. variabile, P. velutinum, P. viridicatum, P. glaucum, P. fussiporus, and P. expansA.

[0071] In another embodiment, the species of Penicillium phosphate solubilizing microorganism is P. bilaiae, P. gaestrivorus, and/or a combination thereof. In yet another embodiment, the P. bilaiae strains are selected from the group consisting of ATCC 20851, NRRL 50169, ATCC 22348, ATCC 18309, NRRL 50162 (Wakelin, et al., 2004. Biol Fertil Soils 40:36-43) and the P. gaestrivorus strain is NRRL 50170 (See, Wakelin, supra.).

[0072] According to compositions of the invention, it is envisaged that more than one phosphate solubilizing microorganism can be used, such as at least two, at least three, at least four, at least five, at least 6, including any combination of Acinetobacter, Arthrobacter, Arthrobotrys, Aspergillus, Azospirillum, Bacillus, Burkholderia, Candida Chryseomonas, Enterobacter, Eupenicillium, Exiguobacterium, Klebsiella, Kluyvera, Microbacterium, Mucor, Paecilomyces, Paenibacillus, Penicillium, Pseudomonas, Serratia, Stenotrophomonas, Streptomyces , Streptosporangium , Swaminathania, Thiobacillus, Torulospora, Vibrio, Xanthobacter, and Xanthomonas, including a species selected from the following group: Acinetobacter calcoaceticus, Acinetobacter spp., Arthrobacter spp., Arthrobotrys oligospora, Aspergillus niger, Aspergillus spp., Azospirillum halopraeferans, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans, Bacillus licheniformis, Bacillus subtilis, Burkholderia cepacia, Burkholderia vietnamiensis, Candida krissii, Chryseomonas luteola, Enterobacter aerogenes, Enterobacter asburiae, Enterobacter spp., Enterobacter taylorae, Eupenicillium parvum, Exiguobacterium spp., Klebsiella spp., Kluy vera cryocrescens, Microbacterium spp., Mucor ramosissimus, Paecilomyces hepialid, Paecilomyces marquandii, Paenibacillus macerans, Paenibacillus mucilaginosus, Pantoea agglomerans, Penicillium expansum, Pseudomonas corrugato, Pseudomonas fluorescens, Pseudomonas lutea, Pseudomonas poae, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas trivialis, Serratia marcescens, Stenotrophomonas maltophilia, Streptomyces spp., Streptosporangium spp., Swaminathania salitolerans, Thiobacillus ferrooxidans, Torulospora globosa, Vibrio proteolyticus, Xanthobacter agilis, and Xanthomonas campestris.

[0073] In another embodiment, the present invention includes a method of enhancing plant growth, comprising applying to plants, plant seeds, or plants involving soil or plant seeds one or more of the bacterial strain(s) of the present invention (including a composition comprising at least one of the isolated bacterial strain(s) of the present invention and an agronomically acceptable carrier. One or more bacterial strains may comprise only a bacterial strain or a combination of at least Petition 870180134926, of 09/26/2018, page 44/105 two or more of the strains of the present invention, including more than two, such as at least three of the above strains, at least four of the above strains, at least five of the above strains, at least six of the above strains, at least seven of the above strains, at least eight of the above strains, at least nine of the above strains, at least ten of the above strains, at least eleven of the above strains, at least twelve of the above strains, at least thirteen of the above strains, up to one including all of the above strains. applications

[0074] The methods of the invention include a treatment step for applying at least one of the isolated strains and/or compositions comprising at least one of the isolated strains to seeds, seedlings, roots, plants, soils, or combinations thereof. "Treating" or "treatment", as the terms are used here and in the art, refers to any application that results in contacting seeds, seedlings, roots, or plants with an effective amount of a composition or components for treatment. to increase competitiveness to colonize a plant and effectiveness in promoting plant growth.

[0075] The effective amount and/or adequate application rates vary according to the type of soil, the type of plants, the amounts of the micronutrient source present in the soil or added to it, etc. and an adequate rate can be observed without difficulty by simple trial and error experiments for each particular case. Typically, the rate for applying at least one of the isolated strains and/or compositions comprising at least one of the isolated strains falls in the range of 1 x 102 - 1 x 108 colony forming units (cfu) per seed (when coated seeds are used ). In a specific embodiment, the application rate falls in the range of 1 x 104 - 1 x 105 colony forming units (cfu) per seed (when coated seeds are used. In a Petition 870180134926, dated 09/26/2018, page 45/105 granular carrier, the application rate falls in the range of 1 x 108 - 1 x 1013 cfu per hectare. In a specific embodiment, the application rate in a granular carrier falls in the range of 2 x 1011 - 6 x 1011 cfu per hectare. Although inoculant compositions and/or compositions used in accordance with the present invention may include a mixture/combination of at least two or more different bacterial strains, it is the total amount of colony forming units in the combined mixture that is referred to throughout the specification. The effective amount of LCO and/or CO used in a composition of the invention to directly treat a seed is expressed in units of concentration and generally ranges from about 10-5 to about 10-14 M ( molar concentration), and in some embodiments from about 10 -5 to about 10 -11 M, and in some other embodiments from about 10 -7 to about 10 -8 M. Expressed in units of weight, the amount effective range generally ranges from about 1 to about 400 µg/short quintal (cwt) of seed, and in some embodiments from about 2 to about 70 µg/cwt, and in some other embodiments from about 2.5 to about 3.0 μg/cwt of seed.

[0076] For purposes of seed treatment indirectly, that is, treatment in the furrow, the effective amount of LCO or CO generally ranges from 1 μg/acre to about 70 μg/acre, and in some embodiments, from about 50 μg /acre to about 60 µg/acre. For plant application purposes, the effective amount of LCO or CO generally ranges from 1 µg/acre to about 30 µg/acre, and in some embodiments, from about 11 µg/acre to about 20 µg/acre.

[0077] The treatment can be carried out directly, that is, by application directly to the seeds, seedlings, roots, or plants (including foliage), or it can be carried out indirectly, that is, by application to the soil (including in the furrow).

[0078] As will be understood, treatment with each component can be performed sequentially or simultaneously. For example, if a liquid carrier is used, the components can be co-suspended in a commercial treatment mix tank and subsequently applied to seeds by any suitable coating process, eg film coating. In the film coating process, a slurry is sprayed onto the seeds in a continuous coating process. Alternatively, for example if a dust or powder carrier is used, the components can be applied sequentially. Accordingly, the treatment may also encompass foliar application and/or furrow application of the compositions.

[0079] Non-limiting examples of plants to be treated by the isolated strains and/or compositions comprising at least one of the isolated strains include leguminous crops. Non-limiting examples of leguminous crops include, but are not limited to, plants such as soybeans, alfalfa, peanuts, peas, lentils, beans, and clover. As will be appreciated, the term "crop" encompasses any plant material that can be harvested. Culture

[0080] The present invention relates to a biologically pure culture of strain(s) Bradyrhizobia japonicum the strain with deposit accession number NRRL B-50592 (also filed as NRRL B-59571); the strain with deposit accession number NRRL B-50593 (also filed as NRRL B-59572); (also filed as NRRL B-59568); the strain with deposit accession number NRRL B-50591 (also filed as NRRL B-59570); the strain with deposit accession number NRRL B-50590 (also filed as NRRL B-59569); the strain with deposit accession number NRRL B-50594 (also filed as NRRL B-50493); the strain with warehouse accession number NRRL B-50726; the strain with warehouse accession number NRRL B-50727; the strain with warehouse accession number NRRL B-50728; the strain with warehouse accession number NRRL B-50729; and the strain with warehouse accession number NRRL B-50730. As used herein, the term "biologically pure culture" means a culture essentially free of biological contamination and having a genetic uniformity such that different subcultures taken from it will exhibit substantially identical genotypes and phenotypes (e.g., cultures have a purity of at least 60 %, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, up to 100% pure). Such cultures may be used in large scale fermentation or for other purposes 50589 (also filed as NRRL B-59568), NRRL B-50591 (also filed as NRRL B-59570); NRRL B-50590 (also filed as NRRL B-59569); NRRL B-50594 (also filed as NRRL B-50493); NRRL B-50726; NRRL B-50727; NRRL B-50728; NRRL B-50729; NRRL B-50730, and cultures thereof are within the scope of the invention.

[0081] In one embodiment, the culture is a strain with deposit accession number NRRL B-50592 (also filed as NRRL B-59571). In another embodiment, the culture is a strain with deposit accession number NRRL B-50593 (also filed as NRRL B-59572). In another embodiment, the culture is a strain with deposit accession number NRRL B-50586 (also filed as NRRL B-59565). In another embodiment, the culture is a strain with deposit accession number NRRL B-50588 (also filed as NRRL B-59567). In another embodiment, the culture is a strain with deposit accession number NRRL B-50587 (also filed as NRRL B-59566). In another embodiment, the culture is a strain with deposit accession number NRRL B-50589 (also filed as NRRL B-59568). In another embodiment, the culture is a strain with deposit accession number NRRL B-50591 (also filed as NRRL B-59570). In another embodiment, the culture is a strain with deposit accession number NRRL B-50590 (also filed as NRRL B-59569). In another embodiment, the culture is a strain with deposit accession number NRRL B-50594 (also filed as NRRL B-50493). In another embodiment, the culture is a strain with deposit accession number NRRL B-50726. In another embodiment, the culture is a strain with deposit accession number NRRL B-50727. In another embodiment, the culture is a strain with deposit accession number NRRL B-50728. In another embodiment, the culture is a strain with deposit accession number NRRL B-50729. In another embodiment, the culture is a strain with deposit accession number NRRL B-50730.

Deposit of Biological Material

*NRRL indica depósito com o Agricultural Research Service Culture Collection, Peoria, IL.[0082] The following biological material was deposited pursuant to the Budapest Treaty of the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20108, USA, and the Microbial Genomics and Bioprocessing Research Unit (NRRL) National Center for Agricultural Utilization Research 1815 N. University Street, Peoria, IL 61604, USA and given the following accession number: Table 1: Biological Material Deposit

*NRRL indicates deposit with the Agricultural Research Service Culture Collection, Peoria, IL.

[0083] The strain was deposited under conditions that guarantee that access to the culture will be available during the pendency of this patent application that determined by the Commissioner of Patents and Trademarks to be entitled to that under 37 C.F.R. §1.14 and 35 U.S.C. §122. The deposit represents a pure culture of the deposited strain. Filing is available as required by foreign patent laws in countries where counterparties to the subject application or its product are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the invention in question to the detriment of the patent rights secured by the governmental action.

[0084] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

EXAMPLES Materials and methods Quite

[0085] YEM Agar (YEMA): (10 g/L D-mannitol; 0.50 g/L Oxoid Yeast Extract; 0.10 g/L NaCl; 0.50 g/L K2HPO4; 0 .20 g/L of MgSOr7H2O; 12.0 g/L of Agar; pH 6.8).

[0086] YEM Liquid: (10 g/L of D-mannitol; 0.50 g/L of Oxoid Yeast Extract; 0.10 g/L of NaCl; 0.50 g/L of K2HPO4; 0.20 g/L of MgSO-7H2O; pH 6.8).

DNA Isolation Protocol

[0087] For strains grown on the plates, a 1 µL loop of each strain from the plates was added individually into 100 µL of PrepMan® Ultra DNA Isolation Solution of Applied Biosystems. The solution was heated at 100°C for 10 minutes. Isolated DNA was used for PCR analysis.

[0088] For DNA isolated from nodules, nodules were removed with forceps from soybean roots and rinsed in diH2O. Nodules were placed individually in 100 uL of PrepMan® Ultra DNA isolation solution from the applied biosystems, broken apart, and heated at 100°C for 10 minutes using a 96-well PCR plate also from the applied biosystems. Disposable toothpicks were used to break the nodules open to avoid cross-contamination. Isolated DNA was used for PCR analysis.

PCR protocol

[0089] Polymerase chain reactions (PCR) were performed using Veriti® 96-well Fast Thermal Cycler applied biosystems. PCRs were adjusted for each strain. 2 µl of DNA was added to 0.5 µl of a 3' nucleotide primer, 0.5 µl of a 5' nucleotide primer, 0.5 µl of Taq Polymerase (New England Biolabs, Inc.) and 21.5 μL of Platinum Blue PCR Supermix® (Invitrogen). The PCR mixture was heated at 94°C for 4 minutes. After denaturation, PCR was performed for 35 cycles with the following program: 94°C for 1 minute, 68°C or nucleotide primer annealing dependent temperature(s) for 1 minute, and length of reaction at 72°C for 1 minute. After completion of the PCR program, 5 µL of PCR mix was run on a Lonza® FlashGel® system.

Strain Isolation Protocol

[0090] In isolated strains, nodules were surface sterilized with 10% household bleach for 2 minutes. The nodules were rinsed in sterile water and then placed in a microcentrifuge tube with 250 uL of sterile water. Nodules were ground with sterile toothpicks and the Rhizobium strains were released into the water. Two loops of 10 uL of water were streaked for single colonies on the YEMA plates. All plates were wrapped with Parafilm® and grown at 30°C in an Eppendorf Innova® 42R incubator. Growth time differed by isolate.

Primary Classification Protocol

[0091] Strains were first classified by two distinct protocols, ie, the “Soybean Field Soil Protocol (Field Treated to USDA 532C)” and the “Untreated Field (Control) Protocol”. Each Protocol is described.

Soybean Field Soil Protocol (Treated Field with USDA 532C)

[0092] USDA 532C-coated soybean seeds have been planted in various soils in all soybean growing regions in the United States, for example, South Dakota, North Dakota, Georgia, Iowa, Nebraska, Illinois, Indiana, Texas, Kansas, Minnesota, etc. Soybean seeds treated with USDA strain 532C Bradyrhizobium japonicum were grown in these soils, harvested, and soybean nodules were analyzed using direct PCR analysis. Forty (40) nodules were collected from each soil sample. Individual soybean nodules were loaded into a single well of a 96-well microtiter plate. DNA from those individual soybean nodules was isolated directly from the nodules based on the procedure described above (See Materials and Methods: DNA Isolation Protocol).

[0093] PCR analysis using USDA-specific primer 209 nucleotide 532C was performed directly on the 96 well plate (See Materials and Methods: PCR Protocol). The 209 nucleotide primer amplification (0.9kb band) of the 40 nodes was calculated to determine the percent amplification. If the DNA amplification of 0.9kb is less than or equal to 30% (i.e., greater than or equal to 70% of the PCR was negative for amplification of the nucleotide 209 primer), then the soil sample contained Bradyrhizobium japonicum strains with higher competitiveness than the USDA 532C strain. Soybean nodules with less than or equal to 30% amplification contained native strains identified as more competitive than USDA 532C based on the described procedure (See Materials and Methods: Strain Isolation Protocol).

[0094] If more than 30% of soybean nodules were colonized by USDA strain 532C Bradyrhizobia japonicum, then the soil was considered unsuitable for isolation of the unpublished strain and the soil sample was completed.

Untreated Field Protocol (Control)

[0095] Nodules were obtained from soybean fields not treated with USDA 532C, in the following states: Arkansas, Georgia, Illinois, Indiana, Iowa, Oklahoma, Nebraska, Kansas, Missouri, and Texas. Bradyrhizobium japonicum strains were isolated directly from these nodules using the protocol described above (See Materials and Methods: Strain Isolation Protocol). Isolated strains were placed in direct competition with USDA 532C Bradyrhizobia japonicum strain by the “Competition Study Protocol” (See Materials and Methods: Competition Study Protocol). Isolated strains that occupied more than 70% of soybean nodules when compared to USDA 532C Bradyrhizobia japonicum strain were selected for performance evaluation (See Materials and Methods: Performance Study Protocol).

Competition Study Protocol

[0096] Optical densities were determined. (Nanodrop® ND1000 Spectrophotometer) A 1:1 USDA 532C inocula ratio for each isolated strain was obtained. USDA 532C was diluted or concentrated to an optical density of 0.5 to 600 nm and 0.5 mL of USDA 532C inocula was adjusted separately for each isolate. All isolated strains were either concentrated or diluted to an optical density of 0.5 to 600 nm using the following calculation: (0.5 USDA 532C optical density) x (USDA 532C 0.5 mL) = (USDA 532C optical density 0.5 mL) isolated strain) x (mL of isolated strain). 0.5 ml of USDA 532C was added to 0.5 ml of each isolate as separate treatments. Soybean seeds were coated with the inocula mixture at a rate of 0.5 mL of inocula mixture per 12 soybean seeds. The seeds were allowed to soak for 30 minutes. 5 of the 12 treated soybeans were planted in Fafard® 3B Potting Mix in a 1 gallon pot. Gloves were used to plant the seeds and were changed between treatments. After planting, the remaining 7 treated soybean seeds were discarded.

[0097] At germination, pots were reduced to 3 plants. Plants were grown for 6-7 weeks in a greenhouse and cross-contamination during the irrigation process was avoided. Temperatures ranging from approximately 23°C – 32°C. Irrigation was carried out on an “as needed” basis. Nodules were harvested from each treatment and used for DNA isolation and PCR analysis with USDA-specific 209 nucleotide primer 532C. See Materials and Methods: DNA Isolation Protocol and PCR Protocol.

Performance Study Protocol

[0098] The performance study is a direct strain-by-strain performance comparison between a single strain and commercially available isolated USDA 532C Bradyrhizobium japonicum strain. The isolated strain and the control strain (USDA 532C Bradyrhizobium japonicum strain) were streaked simultaneously on separate YEMA plates. The isolated strain and the control strain were separately inoculated into 5 mL of liquid YEM medium to obtain an initial optical density of 0.03 at 600 nm in each inoculum tube. (Nanodrop® ND1000 Spectrophotometer) the isolated strain and the control strain were incubated separately at 30°C for 3 days. After incubation, the inoculum for the isolated strain and the control strain was further diluted or concentrated to a final optical density of 0.5 to 600 nm in each inoculum tube (Nanodrop® ND1000 Spectrophotometer) to obtain a test treatment (strain isolated) and a control treatment (control strain. Bradyrhizobium japonicum USDA 532C strain).

[0099] 0.75 mL of the test treatment was added to 32 soybean seeds. The treated seeds were allowed to soak for 30 minutes. After the 30 minutes, 2 seeds were planted in 15 separate pots (4”x4”x6”) in Fafard® 3B Potting mix. Gloves were used to plant the seeds and were changed between treatments. After planting, the remaining 2 treated soybean seeds were discarded. This process was repeated for the control treatment.

[00100] At germination, the pots of the test treatment and control treatment were reduced to a single plant. Plants were grown for 8-10 weeks in a greenhouse and cross-contamination during the irrigation process was avoided. Temperatures ranging from approximately 23°C – 32°C. Irrigation was carried out on an “as needed” basis. After 8-10 weeks, pods were harvested and dried overnight at 80°C. Analysis with JMP® statistical software (SAS Institute, Inc.) was used to determine statistically significant performance improvement compared to USDA 532C Bradyrhizobium japonicum strain.

Temperature Profile Protocol

[00101] Isolated strains of Bradyrhizobium japonicum were streaked onto YEMA plates (10 g/L of D-mannitol; 0.50 g/L of oxoid yeast extract; 0.10 g/L of NaCl; 0.50 g /L of K2HPO4; 0.20 g/L of MgSOr7H2O; 12.0 g/L of Agar; pH 6.8) and incubated at 30°C and 35°C respectively for 7 days. Isolated strains were analyzed for their ability to grow single colonies.

Glyphosate Resistance Profile Protocol

[00102] Isolated strains of Bradyrhizobium japonicum were streaked on 1 mM glyphosate, and 2 mM glyphosate, YEMA plates (10 g/L of D-mannitol; 0.50 g/L of Oxoid yeast extract; 0, 10 g/L of NaCl; 0.50 g/L of K2HPO4; 0.20 g/L of MpSO-7H2O; 12.0 g/L of Agar; pH-6.8). Plates were incubated at 30°C for 7 days and strains were analyzed for their ability to grow single colonies.

Antibiotic Profiling Protocol

[00103] Isolated strains of Bradyrhizobium japonicum were T-stretched in gentamicin (50 mg/L) YEMA, chloramphenicol (50 mg/L) YEMA, polymyxin B (100 mg/L) YEMA, carbenicillin (100 mg/L) YEMA, neomycin (50 mg/L) YEMA, and nalidixic acid (50 mg/L) YEMA. Plates were incubated at 30°C for 7 days and strains were analyzed for their ability to grow single colonies.

Diversilab® PCR protocol

[00104] Diversilab® PCR was fitted using the Diversilab Pseudomonas Kit® by BioMerieux. This kit contained proprietary nucleotide primers designed to amplify various portions of the genome to produce a fingerprint of multiple DNA amplifications. Each strain has a unique fingerprint and percent similarity between strains can be determined using Diverilab software.

[00105] The PCR was consequently configured. 2 µL DNA was added to 18.0 µL Re-PCR MM1, 2.0 µL Nucleotide Primer Mix, 0.5 µL Taq Polymerase (New England Biolabs, Inc.) and 2.5 µL Buffer Taq Polymerase (New England Biolabs, Inc.) to a total volume of 25 uL. The PCR was heated to 94°C for 2 minutes and then run for 35 cycles as follows: 94°C for 0.5 minutes, then 50°C for 0.5 minutes, and finally 70°C for 1.5 minutes. After the completion of 35 cycles, the total reaction was maintained at 70°C for 3 minutes. After completion of the PCR analysis, the PCR product was run on the Agilent® 2100 Series Bioanalyzer. Diversilab® DNA Reagents & Supplie Kit by BioMerieux was used to load PCR product onto Diversilab® System chips. The case has been maintained according to the instructions. Before use, the case was set at room temperature for 30 minutes before loading the Diversilab® chip. The Diversilab® chip was loaded in full accordance with all protocols and instructions provided. Upon completion of loading the Diversilab® chip, the chip was loaded into the Agilent® 2100 Series Bioanalyzer and the analysis performed until completion.

Example 1 Unique Nucleotide Primer Design for Commercial USDA 532C Bradyrhizobium Strain

[00106] A genetic identification method was developed to evaluate the competitiveness of commercial USDA 532C Bradyrhizobium japonicum strain against native strains in the field. A specific nucleotide primer for USDA 532C was identified and PCR technology was used to efficiently assess the competitiveness of USDA 532C in the field.

[00107] Whole genome sequencing of USDA 532C was performed at Novozymes Davis. It was observed that twenty-five different DNA fragments had low homology with public sequences of Bradyrhizobium japonicum. DNA was isolated from B. japonicum strains (USDA 532C, P152, Br173, Br187, P190, and P194) grown on the plates according to the procedure described above (See Materials and Methods: DNA Isolation Protocol). Additional sequence analysis was performed on those twenty-five DNA fragments. Putative unique nucleotide primers for USDA 532C strain were chosen and used for USDA 532C specific nucleotide primer sorting by PCR against some representative Bradyrhizobium japonicum strains. After PCR evaluation, a single nucleotide primer unique to USDA 532C was identified and was designated as p209.

[00108] The sequence of the 209 nucleotide primer was as follows: SEQ ID NO: 1 - P209p5-TTGGGTTGAGCATGCCCACCCGGACGG, SEQ ID NO: 2 - P209p3-GTCTCAGTTGCCGAGCCCACGGCC

Nucleotide primer specificity

[00109] The twenty-five nucleotide primers were individually tested for positive USDA 532C identification. Nucleotide primers were further tested for USDA 532C specificity by PCR using USDA 532C and 5 different native strains of B. japonicum (P152, Br173, Br187, P190, and P194) for comparison. Genome sequencing indicated that Br187 and USDA 532C were genetically the same. Table 2: Nucleotide primer classification summary

[00110] Table 2 summarizes the observations (“+” indicates a positive identification of a PCR amplification band on the gel, “-” indicates no DNA amplification).

[00111] With reference to Figure 1A, isolated nucleotide primer 209 has been shown to be specific for USDA 532C and Br187. Additional genetic testing showed that USDA 532C and the P187 Bradyrhizobium japonicum strain are identical (results not shown). The well contents (left to right) indicate the following USDA native strains 532C, P152, Br173, Br187, Br190, Br194, and the control scale. See figure 1A.

[00112] The specificity of 209 nucleotide primer was tested against native strains P152, Br173, Br187, Br190, Br194, and USDA 532C. See figure 1B. PCR with 209 nucleotide primer was performed on 100 strains obtained from untreated seedlings in field experiments according to the above method (See Materials and Methods: Untreated Field Protocol (Control)). USDA 532C Bradyrhizobium japonicum strain was not added to these beds. Of the strains tested, only 3% of the native strains could be amplified to a 0.9kb band specific for nucleotide primer 209. These results demonstrated that primer nucleotide 209 could be used for specific detection of USDA 532C.

Example 2 Analysis and Isolation of Unpublished Strains

[00113] Nucleotide primer 209 was used as a marker to indicate the presence or absence of colonization by USDA strain 532C Bradyrhizobia japonicum in soybean root nodules. Bands (ie, a 0.9kb band) indicating the presence of nucleotide 209 primer represent a positive identification of USDA strain 532C Bradyrhizobia japonicum. See figure 2A. Figure 2A is exemplary of an example where USDA 532C Bradyrhizobia japonicum strain is the dominantly competitive strain for colonization of soybean nodules when compared to other native Rhizobial strains. In Figure 2B, the number of bands indicating positive identification for primer 209 nucleotide, and therefore the presence of USDA strain 532C Bradyrhizobia japonicum, is reduced compared to the total number of bands present for primer 209 nucleotide in Figure 2A. Figure 2A and Figure 2B demonstrate that the presence or absence of nucleotide primer 209 can be used to determine whether USDA 532C is the dominant competitive strain in soybean nodules.

[00114] Strains isolated from Bradyrhizobium japonicum were selected from nodules according to both classification protocols (See Materials and Methods: Primary Classification Protocol). Selected strains were isolated from nodules according to the isolation procedure (See Materials and Methods: Strain Isolation Protocol).

Example 3 Head-to-Head Competition Evaluation

[00115] All isolated strains were placed into a classification program designed to directly challenge the competitiveness of the isolate against USDA strain 532C Bradyrhizobium japonicum in terms of nodule colonization ability (See Materials and Methods: Competition Study Protocol).

[00116] Isolated nucleotide 209 primer specific for USDA 532C was used as a marker to indicate the presence or absence of colonization by USDA 532C Bradyrhizobia japonicum strain on soybean root nodules. Positive identification for 209 nucleotide primer indicated colonization of USDA strain 532C Bradyrhizobia japonicum in soybean root nodules. In contrast, the absence of bands indicating positive identification for 209 nucleotide primer indicated colonization of a native strain in soybean root nodules other than USDA 532C Bradyrhizobia japonicum strain. Isolated strains that showed a colonization greater than 70% of the analyzed nodules were chosen for a second evaluation for confirmation. Strains were subjected to at least two rounds of competition evaluation. Through this procedure, more than 1,000 isolates were classified for enhanced competitiveness.

Example 4 Performance Assessment

[00117] The performance study is a direct strain-by-strain comparison of strains isolated to USDA 532C. Best performance was measured as a function of Pod Dry Weight (g). See Materials and Methods: Performance Study Protocol. Results are provided in Tables 3A - 3D Table 3A: Enhanced Performance as a function of Pod Dry Weight (g) I Strain I Percent Colonization | Pod dry weight (g) |

[00118] Percent colonization was confirmed in triplicate studies and increased pod dry weight was confirmed in duplicate studies for all strains. Table 3B: Enhanced Performance as a Function of Pod Dry Weight (g)

[00119] Percent colonization was confirmed in triplicate studies and increased pod dry weight was confirmed in duplicate studies for all strains. Table 3C: Enhanced Performance as a Function of Pod Dry Weight (g)

[00120] Percent colonization was confirmed in triplicate studies and increased pod dry weight was confirmed in duplicate studies for all strains. Table 3D: Intensified Performance as a function of Pod Dry Weight (g)

[00121] Percent colonization and increased pod dry weight were confirmed in duplicate studies for all strains except NRRL B-50730* which had only one test run.

[00122] Commercially available USDA 532C strain was used as a control for each evaluation. Results from Tables 3A-3D indicate that all but one of the isolated strains performed better when compared to the control, USDA 532C.

Example 5 Characterization Study

[00123] Isolated Bradyrhizobium japonicum strains were further characterized based on temperature, glyphosate resistance, and antibiotic profiles (See Materials and Methods: Temperature Profile, Glyphosate Resistance Profile, and Antibiotic Profile Protocols). Results provided in Table 4. Table 4: Characterization of Isolated Strains as a Function of Temperature Profile, Glyphosate Resistance, and Antibiotic Resistance

[00124] Table 4 summarizes the results (“+” indicates growth with isolated colonies, “-” indicates no growth, and “+-” indicates few isolated colonies/minimal and sporadic growth). The results indicate that strains NRRL B-59570, NRRL B-59568, NRRL B-59565, NRRL B-59566 and NRRL B-50493 are tolerant of temperatures of substantially 35°C. The results further indicate that isolated strains NRRL B-59569, NRRL B-59568, NRRL B-59565, and NRRL B-50493 are naturally resistant to glyphosate. NRRL B-59566 and NRRL B-59569 strains have been observed to have resistance to nalidixic acid.

Example 5 DNA fingerprint development

[00125] Superior performance isolates were placed through Diversilab® DNA fingerprint analysis (See Materials and Methods: Diversilab® PCR Protocol). DNA was isolated from each strain according to the methods discussed (See Materials and Methods: DNA Isolation Protocol). Isolated DNA was used for PCR analysis.

[00126] Results for the Diversilab DNA fingerprint analysis are indicated in Figures 3A - 3B. Results demonstrate that the isolated strains are unique strains and different strains than USDA 532C.

[00127] The present invention is described, at least in part, by the following numbered paragraphs: 1. A biologically pure culture of Bradyrhizobia japonicum selected from the group consisting of: the strain with accession number NRRL B-50592; the strain with warehouse accession number NRRL B-50593; the strain with warehouse accession number NRRL B-50586; the strain with warehouse accession number NRRL B-50588; the strain with warehouse accession number NRRL B-50587; the strain with warehouse accession number NRRL B-50589; the strain with warehouse accession number NRRL B-50591; the strain with warehouse accession number NRRL B-50590; the strain with warehouse accession number NRRL B-50594; the strain with warehouse accession number NRRL B-50726; the strain with warehouse accession number NRRL B-50727; the strain with warehouse accession number NRRL B-50728; the strain with warehouse accession number NRRL B-50729; and the strain with warehouse accession number NRRL B-50730. 2. The Bradyrhizobium strains of paragraph 1, wherein said strains are capable of promoting nitrogen fixation in a plant. 3. The Bradyrhizobium strains of any one of paragraphs 1-2, wherein said strains are tolerant to growth at a temperature of substantially 35°C. 4. The Bradyrhizobium strains of any one of paragraphs 1-3, wherein said strains are selected from the group consisting of: the strain with deposit accession number NRRL B-50591; the strain with warehouse accession number NRRL B-50589; the strain with warehouse accession number NRRL B-50586; the strain with the accession number NRRL 50594; and a combination of at least two or more of the strains. 5. The Bradyrhizobium strains of any one of paragraphs 1-4, wherein said Bradyrhizobium strains are naturally resistant to glyphosate. 6. The Bradyrhizobium strains of paragraph 5, wherein said strains are selected from the group consisting of: the strain with deposit accession number NRRL B-50590; the strain with warehouse accession number NRRL B-50594; and a combination of at least two or more of the strains. 7. The Bradyrhizobium strains of any one of paragraphs 1-6, wherein said strains have enhanced competitiveness to colonize a plant. 8. The Bradyrhizobium strains of any one of paragraphs 1-7, wherein said strains are effective in promoting enhanced plant growth. 9. The Bradyrhizobium strains of any of paragraphs 1-8, where enhanced competitiveness includes at least 51% nodule occupancy, eg, at least 55%, 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 96%, at least 97%, at least 98%, at least 99%, or 100% node occupancy. 10. The Bradyrhizobium strains of any of paragraphs 1-9, where effectiveness in promoting enhanced plant growth includes at least increased plant yield measured in terms of bushels/acre, increased fruit number, increased root number , increased root length, increased root mass, increased root volume, increased foliage area, increased plant support, increased plant vigor, and/or increased nitrogen (N2) fixing capacity when compared to a commercially available strain , for example, USDA 532C. 11. The Bradyrhizobium strains of any one of paragraphs 1-10, wherein the enhanced effectiveness in promoting soybean growth includes an increase in the total dry weight of soybean pods in said soybean plant when said total dry weight of soybean pods is compared as the total dry weight of soybean pods in a soybean plant subjected to a commercially available strain, for example, commercial USDA 532C strain. 12. A composition comprising a strain of Bradyrhizobia japonicum selected from the group consisting of: the strain having deposit accession number NRRL B-50592; the strain with warehouse accession number NRRL B-50593; the strain with warehouse accession number NRRL B-50586; the strain with warehouse accession number NRRL B-50588; the strain with warehouse accession number NRRL B-50587; the strain with warehouse accession number NRRL B-50589; the strain with warehouse accession number NRRL B-50591; the strain with warehouse accession number NRRL B-50590; the strain with warehouse accession number NRRL B-50594; the strain with warehouse accession number NRRL B-50726; the strain with warehouse accession number NRRL B-50727; the strain with warehouse accession number NRRL B-50728; Petition 870180134926, of 09/26/2018, page 66/105 the strain with deposit access number NRRL B-50729; the strain with warehouse accession number NRRL B-50730; and a combination of at least two or more of the strains and an agronomically suitable carrier. 13. The composition of paragraph 12, wherein said composition includes one or more signal molecules. 14. The composition of paragraph 13, wherein the plant signal molecule is a lipo-chito-oligosaccharide (LCO). 15. The composition of any of paragraphs 13-14, where the LCO is summary. 16. The composition of any one of paragraphs 13-15, wherein the LCO is recombinant. 17. The composition of any of paragraphs 13-16 where the LCO is naturally occurring. 18. The composition of any one of paragraphs 13-17, in which the LCO is obtained from a species of Rhizobia selected from Rhizobium spp., Bradyrhizobium spp., Sinorhizobium spp. and Azorhizobium spp. 19. The composition of any of paragraphs 13-18, where LCO is obtained from Bradyrhizobium japonicum. 20. The composition of any one of paragraphs 13-19, wherein the LCO is obtained from an arbuscular mycorrhizal fungus. 21. The composition of paragraph 13, wherein the plant signal molecule is a chitinous compound. 22. The composition of paragraph 22, wherein the chitinous compound is a chito-oligomer (CO). 23. The composition of any of paragraphs 21-22 where CO is synthetic. 24. The composition of any one of paragraphs 21-23, wherein the CO is recombinant. 25. The composition of any of paragraphs 21-24 where CO is naturally occurring. 26. The composition of paragraph 13, where the plant signal molecule is a flavonoid. 27. The composition of paragraph 13, where the plant signal molecule is jasmonic acid or a derivative thereof. 28. The composition of paragraph 13, wherein the plant signal molecule is linoleic acid or a derivative thereof. 29. The composition of paragraph 13, wherein the plant signal molecule is linoleic acid or a derivative thereof. 30. The composition of paragraph 13, where the plant signal molecule is a carricin. 31. The composition of any one of paragraphs 12-30, wherein the composition includes at least two different plant signal molecules. 32. The composition of any one of paragraphs 12-31, wherein the composition includes at least one agronomically beneficial agent. 33. The composition of paragraph 32, wherein the agronomically beneficial agent is a herbicide, insecticide or a fungicide. 34. The composition of paragraph 33, wherein the agronomically beneficial agent is at least one phosphate solubilizing microorganism. 35. The composition of paragraph 34, wherein the at least one phosphate solubilizing microorganism comprises a strain of the fungus Penicillium. 36. The composition of paragraph 35, wherein the at least one phosphate solubilizing microorganism comprises a strain of P. bilaiae. 37. The wording of paragraph 36, in which the strain of P. bilaiae is selected from the group consisting of NRRL 50162, NRRL 50169, ATCC 20851, ATCC 22348, and ATCC 18309. 38. The wording of paragraph 34, in which at least at least one phosphate-solubilizing microorganism comprises a strain of P. gaestrivorus. 39. The wording of paragraph 38, where the strain of P. gaestrivorus is NRRL 50170. 40. A method for isolating selected bacterial strain(s) from the genus consisting of Rhizobium and Bradyrhizobium with enhanced competitiveness for colonize a leguminous plant and effectiveness in promoting enhanced leguminous plant growth comprising: a. obtaining a bacterial strain(s) from a soil sample; B. subjecting said bacterial strain(s) and a commercially available strain(s), for example commercial USDA 532C strain, to a leguminous plant; w. selecting said bacterial strain(s) which is(are) more competitive than the commercially available strain to occupy the nodules of a leguminous plant; f. analyze the selected bacterial strain(s) that is(are) more competitive than the commercially available strain(s) to occupy the nodes of a leguminous plant for those bacterial strain(s) ) with an effectiveness in promoting intensified growth of leguminous plant; and d. isolating said bacterial strain(s) effectively in promoting enhanced leguminous plant growth. 41. The isolated bacterial strain of paragraph 40, wherein said bacterial strain is a Bradyrhizobium spp. strain. 42. The isolated Bradyrhizobium strain of paragraph 41, wherein said Bradyrhizobium spp. is a Bradyrhizobium japonicum strain. 43. The isolated Bradyrhizobium strain of any one of paragraphs 41-42, wherein said strain is a Bradyrhizobia japonicum strain selected from the group consisting of: the strain with deposit accession number NRRL B-50592; the strain with warehouse accession number NRRL B-50593; the strain with warehouse accession number NRRL B-50586; the strain with warehouse accession number NRRL B-50588; the strain with warehouse accession number NRRL B-50587; the strain with warehouse accession number NRRL B-50589; the strain with warehouse accession number NRRL B-50591; the strain with warehouse accession number NRRL B-50590; the strain with warehouse accession number NRRL B-50594; the strain with warehouse accession number NRRL B-50726; the strain with warehouse accession number NRRL B-50727; the strain with warehouse accession number NRRL B-50728; the strain with warehouse accession number NRRL B-50729; the strain with warehouse accession number NRRL B-50730; and a combination of at least two or more of the strains. 44. The method of any one of paragraphs 40-43, wherein the isolated Bradyrhizobia japonicum strain is capable of fixing nitrogen in a plant. 45. The method of any one of paragraphs 40-44, wherein the isolated Bradyrhizobia japonicum strain is tolerant of a growth temperature of substantially 35°C. 46. The method of paragraph 45, wherein the isolated Bradyrhizobia japonicum strain is selected from the group consisting of: the strain with deposit accession number NRRL B-50591; the strain with warehouse accession number NRRL B-50589; the strain with warehouse accession number NRRL B-50586; the strain with the accession number NRRL 50594; and a combination of at least two or more of the strains. 47. The method of any one of paragraphs 40-46, wherein the Bradyrhizobia japonicum strain has a natural resistance to glyphosate. 48. The method of paragraph 47, wherein the isolated Bradyrhizobia japonicum strain is selected from the group consisting of: the strain with deposit accession number NRRL B-50590; the strain with warehouse accession number NRRL B-50594; and a combination of at least two or more of the strains. 49. A method of enhancing plant growth, comprising treating a seed, seedling, root, plant, soil, or combinations thereof with a composition as defined in any of paragraphs 1-39. 50. The method in accordance with paragraph 49, wherein the seed, seedling, root, or plant is a legume. 51. The method according to paragraph 50, wherein the seed, seedling, root or plant is a soybean seed, seedling, root or plant. 52. The method of any one of paragraphs 49-51, wherein said composition is added to the soil in an amount of 1 x 108 to 1 x 1013 colony forming units per hectare, preferably 2 x 1011 to 6 x 1011 colony formation units per hectare. 53. The method according to any of paragraphs 49-52, wherein said composition is introduced as a seed coat comprising 1 x 102 to 1 x 108, preferably 1 x 104 to 1 x 105 colony forming units per seed .

[00128] The invention described and claimed herein should not be limited in scope by the specific embodiments disclosed herein, as these embodiments are intended to illustrate various aspects of the invention. Any equivalent modality should be within the scope of this invention. However, various modifications of the invention in addition to those shown and described herein will become apparent to those in the art from the foregoing description. Such modifications shall also fall within the scope of the appended claims. In case of conflict, this disclosure including the definitions will control.

[00129] Various references are cited herein, the disclosures of which are incorporated by the reference in their entirety.

Claims (50)

1. Method for enhancing plant growth, characterized in that it comprises applying an inoculant composition to a plant seed, to the soil in which said seed will be planted, to the soil in which said seed is being planted, to the soil in which said seed has been planted and/or to a plant which germinates from said seed, said inoculant composition comprising: one or more strains of Bradyrhizobium japonicum selected from the group consisting of: the strain of Bradyrhizobium japonicum with accession number to warehouse NRRL B-50592; the Bradyrhizobium japonicum strain with deposit accession number NRRL B-50593; the Bradyrhizobium japonicum strain with deposit accession number NRRL B-50586; the Bradyrhizobium japonicum strain with deposit accession number NRRL B-50588; the Bradyrhizobium japonicum strain with deposit accession number NRRL B-50587; the Bradyrhizobium japonicum strain with deposit accession number NRRL B-50589; the Bradyrhizobium japonicum strain with deposit accession number NRRL B-50591; the Bradyrhizobium japonicum strain with deposit accession number NRRL B-50727; the Bradyrhizobium japonicum strain with deposit accession number NRRL B-50728; the Bradyrhizobium japonicum strain with deposit accession number NRRL B-50729, and the Bradyrhizobium japonicum strain with deposit accession number NRRL B-50730; and a liquid carrier, wherein the liquid carrier is water, and at least one chitin, at least one chitosan, at least one lipochitooligosaccharide (LCO), and at least one chitin oligomer (CO) and/or at least one least one flavonoid, and a culture medium that supplies one or more nutrients to the Bradyrhizobium japonicum strain(s). 2. Method according to claim 1, characterized in that said inoculant composition comprises the strain of Bradyrhizobium japonicum with accession number NRRL B-50586. 3. Method according to claim 1 or 2, characterized in that said inoculant composition comprises the strain of Bradyrhizobium japonicum with the deposit accession number NRRL B-50587. 4. Method according to any one of claims 1 to 3, characterized in that said inoculant composition comprises the strain of Bradyrhizobium japonicum with the deposit accession number NRRL B-50588. 5. Method according to any one of claims 1 to 4, characterized in that said inoculant composition comprises the strain of Bradyrhizobium japonicum with accession number NRRL B-50589. 6. Method according to any one of claims 1 to 5, characterized in that said inoculant composition comprises the strain of Bradyrhizobium japonicum with the deposit accession number NRRL B-50590. 7. Method according to any one of claims 1 to 6, characterized in that said inoculant composition comprises the strain of Bradyrhizobium japonicum with accession number NRRL B-50591. 8. Method according to any one of claims 1 to 7, characterized in that said inoculant composition comprises the strain of Bradyrhizobium japonicum with accession number NRRL B-50592. 9. Method according to any one of claims 1 to 8, characterized in that said inoculant composition comprises the strain of Bradyrhizobium japonicum with accession number NRRL B-50593. 10. Method according to any one of claims 1 to 9, characterized in that said inoculant composition comprises the strain of Bradyrhizobium japonicum with accession number NRRL B-50594. 11. Method according to any one of claims 1 to 10, characterized in that said inoculant composition comprises the strain of Bradyrhizobium japonicum with accession number NRRL B-50726. 12. Method according to any one of claims 1 to 11, characterized in that said inoculant composition comprises the strain of Bradyrhizobium japonicum with accession number NRRL B-50727. 13. Method according to any one of claims 1 to 12, characterized in that said inoculant composition comprises the strain of Bradyrhizobium japonicum with accession number NRRL B-50728. 14. Method according to any one of claims 1 to 13, characterized in that said inoculant composition comprises the strain of Bradyrhizobium japonicum with accession number NRRL B-50729. 15. Method according to any one of claims 1 to 14, characterized in that said inoculant composition comprises the strain of Bradyrhizobium japonicum with accession number NRRL B-50730. 16. Method according to claim 1, characterized in that said inoculant composition comprises: the strain of Bradyrhizobium japonicum with accession number NRRL B-50586; the Bradyrhizobium japonicum strain with deposit accession number NRRL B-50587; the Bradyrhizobium japonicum strain with deposit accession number NRRL B-50589; the Bradyrhizobium japonicum strain with deposit accession number NRRL B-50591; and the Bradyrhizobium japonicum strain with deposit accession number NRRL B-50594. 17. Method according to claim 1, characterized in that said inoculant composition comprises: the strain of Bradyrhizobium japonicum with accession number NRRL B-50586; the Bradyrhizobium japonicum strain with deposit accession number NRRL B-50589; the Bradyrhizobium japonicum strain with deposit accession number NRRL B-50590; and the Bradyrhizobium japonicum strain with deposit accession number NRRL B-50594. 18. Method according to claim 1, characterized in that said inoculant composition comprises: the strain of Bradyrhizobium japonicum with the deposit accession number NRRL B-50590; and the Bradyrhizobium japonicum strain with deposit accession number NRRL B-50594. 19. Method according to claim 1, characterized in that said at least one LCO comprises at least one synthetic LCO. 20. Method according to claim 1, characterized in that said at least one LCO comprises at least one recombinant LCO. 21. Method according to claim 1, characterized in that said at least one LCO comprises at least one naturally occurring LCO. 22. Method according to claim 1, characterized in that said at least one LCO comprises an LCO obtained from a strain of Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium and/or Sinorhizobium. 23. Method according to claim 1, characterized in that said at least one LCO comprises an LCO obtained from a strain of mycorrhizal fungus. 24. Method according to any one of claims 1 to 23, characterized in that said at least one LCO comprises at least one LCO represented by the formula:

where R represents H or CH3CO- and n is equal to 2 or 3. 25. Method according to any one of claims 1 to 23, characterized in that at least one LCO is present in a concentration ranging from about 10-14 to about 10-5 Molar. 26. Method according to any one of claims 1 to 23, characterized in that at least one LCO is present in a concentration ranging from about 10-11 to about 10-5 Molar. 27. Method according to any one of claims 1 to 23, characterized in that at least one LCO is present in a concentration ranging from about 10-8 to about 10-7 Molar. 28. Method according to claim 1, characterized in that said at least one CO is present in a concentration ranging from about 10-14 to about 10-5 Molar. 29. Method according to claim 1, characterized in that said at least one CO is present in a concentration ranging from about 10-11 to about 10-5 Molar. 30. Method according to claim 1, characterized in that said at least one CO is present in a concentration ranging from about 10-8 to about 10-7 Molar. 31. Method according to any one of claims 1 to 30, characterized in that said inoculant composition comprises apigenin, daidzein, formononetin, genistein, hesperetin, luteolin and/or naringenin. 32. Method according to any one of claims 1 to 31, characterized in that said composition comprises at least one carricine. 33. Method according to any one of claims 1 to 32, characterized in that said inoculant composition comprises jasmonic acid or an ester, starch, glycoside or salts thereof; linoleic acid or an ester, starch, glycoside or salts thereof; and/or linoleic acid or an ester, starch, glycoside or salts thereof. 34. Method according to any one of claims 1 to 33, characterized in that said inoculant composition comprises one or more fungicides, herbicides and/or insecticides. 35. Method according to any one of claims 1 to 34, characterized in that said inoculant composition comprises at least one phosphate solubilizing microorganism. 36. Method according to any one of claims 1 to 35, characterized in that said inoculant composition comprises at least one strain of Penicillium. 37. Method according to any one of claims 1 to 36, characterized in that said inoculant composition comprises at least one strain of Penicillium bilaiae. 38. Method according to any one of claims 1 to 37, characterized in that said inoculant composition comprises at least one strain of Penicillium gaestrivorus. 39. Method according to any one of claims 1 to 38, characterized in that said inoculant composition comprises the strain of Penicillium gaestrivorus with the accession number to the deposit NRRL 50170, the strain of Penicillium bilaiae with the accession number to deposit NRRL 50162, the strain of Penicillium bilaiae with deposit accession number NRRL 50169, the strain of Penicillium bilaiae with deposit accession number ATCC 20851, the strain of Penicillium bilaiae with deposit accession number ATCC 22348, and /or the Penicillium bilaiae strain with deposit accession number ATCC 18309. 40. Method according to any one of claims 1 to 39, characterized in that said inoculant composition comprises at least one strain of Streptomyces. 41. Method according to any one of claims 1 to 40, characterized in that said inoculant composition comprises peat-based powder or granules. 42. Method according to any one of claims 1 to 41, characterized in that said inoculant composition comprises a gypsum granule. 43. Method according to any one of claims 1 to 42, characterized in that said inoculant composition comprises a clay. 44. Method according to any one of claims 1 to 43, characterized in that said seed is leguminous. 45. Method according to any one of claims 1 to 44, characterized in that said seed is a soybean seed. 46. Method according to any one of claims 1 to 45, characterized in that said seed is an alfalfa seed. 47. Method according to any one of claims 1 to 46, characterized in that said seed is a pea seed. 48. Method according to any one of claims 1 to 47, characterized in that said seed is a lentil seed. 49. Method according to any one of claims 1 to 48, characterized in that said seed is a bean seed. 50. Method according to any one of claims 1 to 49, characterized in that said seed is a clover seed.

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