Biologically pure culture, composition, method for treating seeds and seeds

专利摘要:
BIOLOGICALLY PURE CULTURE, COMPOSITION, METHOD TO TREAT SEEDS, AND, SEEDS. In accordance with the present invention new isolates of Bradyrhizobium japonicum have been isolated and have unique properties. These Bradyrhizobia are plant growth promoting rhizobacteria (PGPR), have superior desiccation tolerance/resistance, and improve the general growth performance of leguminous plants.
公开号:BR112014014526B1
申请号:R112014014526-1
申请日:2012-12-17
公开日:2022-01-25
发明作者:Yaowei Kang；Anh Tran；Shawn Semones
申请人:Novozymes Bioag A/S；Novozymes Biologicals, Inc；
IPC主号:

专利说明:

REFERENCE TO A BIOLOGICAL MATERIAL DEPOSIT
[0001] This application contains a reference to a repository of biological material, which repository is incorporated herein by reference. For complete information, see Table 1. FIELD OF THE INVENTION
[0002] The present invention relates to isolated Bradyrhizobium bacteria having improved characteristics, including, but not limited to, improved desiccation resistance. FUNDAMENTALS OF THE INVENTION
[0003] 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 so-called micronutrients (e.g. copper, iron and zinc), but many soils are either deficient in these elements or that they contain them only in forms that cannot be readily absorbed by plants (it is generally believed that essential elements cannot be easily absorbed 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 overall plant growth and health. To counteract such deficiencies, sources of the deficient elements are generally applied to the soil in order to improve growth rates and yields obtained from crop plants. For example, nitrates and/or ammonium are often added to the soil to combat the lack of available nitrogen.
[0004] In the field of crop science, it is well known that many cultivated species require the soil to provide relatively high amounts of nitrogen to the plant. Notable exceptions to those plants that require nitrogen via the soil are plants in the legume family.
[0005] Specifically, leguminous plants are unique from non leguminous plants because of their ability to fix atmospheric nitrogen into ammonia. The ability to fix atmospheric nitrogen into a usable nitrogen source for the plant eliminates the need for the plant to obtain nitrogen from the soil. Nitrogen fixation, however, requires a symbiotic relationship between the leguminous plant and the native bacteria within the soil. One of the most extensively studied partners in this symbiotic relationship are bacteria belonging to the genera 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.
[0006] Symbiosis is generally 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 bacterial colonization within the root nodule of the leguminous plant. (Gresshoff, 1999). Once the bacteria have colonized the root nodule, the bacteria effect morphological changes in the plant, i.e. curling of the hair root and development of a new root organ - the nodule. (Gresshoff, 1999). The nodule allows the establishment of a new physiological environment for the nodule-inducing bacteria to differentiate into a nitrogen-fixing endosymbiont, or bacteriode, for the colonized plant. (Gresshoff, 1999).
[0007] In order to help with the symbiotic exchange of bidirectional signaling between the plant and microbe, bacteria, such as Bradyrhizobia sp., are often coated onto a seed. To prolong the viability of the microbe on the seed, it is desirable for the microbe to be tolerant of desiccation and dry environmental conditions in general.
[0008] So there remains a need for microbes with improved desiccation resistance. SUMMARY OF THE INVENTION
[0009] Described here are new bacterial strains having improved resistance to desiccation, especially when the new strains are compared with their parental strain, eg Bradyrhizobium sp., parental strain USDA 532C. The inventors isolated and tested a significant number of bacterial strains for their desiccation resistance properties.
[0010] As described throughout the document, the strains isolated are strains of the genus Bradyrhizobium spp. In particular, the strains isolated are strains of Bradyrhizobium japonicum. Even more particularly, the isolated strains are isolated strains of Bradyrhizobium japonicum selected from the group consisting of: the strain having deposit accession number NRRL B-50608; the strain having deposit accession number NRRL B-50609; the strain having deposit accession number NRRL B-50610; the strain having deposit accession number NRRL B-50611; the strain having deposit accession number NRRL B-50612, or a combination of at least two or more of the strains deposited above.
[0011] Also as described herein are compositions comprising a carrier and one or more of the bacterial strains described herein. In one embodiment, the composition comprises one or more plant signaling molecules. In one embodiment, the composition comprises at least one lipo-chitooligosaccharide (LCO). In another embodiment the composition comprises at least one chitooligosaccharide (CO). In yet another embodiment, the composition comprises at least one flavonoid. In yet another embodiment, the composition comprises jasmonic acid or a derivative thereof. In another embodiment, the composition comprises linoleic acid or a derivative thereof. In yet another embodiment, the composition comprises linolenic acid or a derivative thereof. In yet another embodiment, the composition comprises a carriquin.
[0012] Further described herein is a method for enhancing the growth of a plant or plant part comprising contacting a plant or plant part with one or more with one or more of the bacterial strains described herein. The method comprises introducing into the soil an inoculum of one or more of the bacterial strains described herein. In another embodiment, the method comprises introducing into the soil an inoculum of one or more of the bacterial strains as a seed coat.
[0013] Also as described herein is a method for improving nitrogen fixation in a plant(s) comprising growing the plant(s) in a soil that contains one or more of the bacterial strains described herein. In one embodiment, the plant(s) is a leguminous plant(s), non-legume plant(s), or combinations thereof. In another embodiment, the plant is a plant selected from the group consisting of soybeans, beans, alfalfa, clover, corn, lettuce, tomatoes, potatoes, cucumbers, and combinations thereof.
[0014] Seeds coated with the bacterial strains described here are still described here. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 is a graphical representation of the second preliminary of desiccation resistant mutants compared when compared to the desiccation resistance of the USDA 532C parental strain.
[0016] Figure 2 is a graphical representation of the second screen of desiccation resistant mutants compared when compared to the desiccation resistance of the USDA parental strain 532C.
[0017] Figure 3 is a graphical representation of the third screen of desiccation resistant mutants compared when compared to the desiccation resistance of the parental strain USDA 532C.
[0018] Figure 4 is a graphical representation of the fourth screen of desiccation resistant mutants compared when compared to the desiccation resistance of the USDA 532C parental strain.
[0019] Figure 5 is a bar graph representation of the desiccation resistance of selected desiccation resistant mutants compared when compared to the desiccation resistance of the parental strain USDA 532C at zero (0) and fourteen (14) days.
[0020] Figure 6 is a bar graph representation of desiccation resistance of selected desiccation resistant mutants compared when compared to desiccation resistance of the parental strain USDA 532C at seven (7) and fourteen (14) days. DETAILED DESCRIPTION OF THE INVENTION
[0021] The bacterial strains described were isolated and tested for their ability to solubilize phosphorus. This is described in detail in section fg “Gzgorlou” fcfc cdckzqo Cu fotoau fg tgcnkzc>«q fguetkVcu ckpfc fg refer to compositions, seed coatings, methods to increase the availability of phosphorus for plant uptake from the soil, and methods for increasing phosphorus uptake in plants comprising growing the plants in a soil containing a source of phosphorus. Definitions:
[0022] As used here, cu fotocu ukpiwlctgu "wo", $woa$ g $o$ “a” are meant to include the plural forms as well, unless the context clearly indicates otherwise.
[0023] As used herein, the term "biologically pure culture" is intended to mean a culture essentially free from biological contamination and having a genetic uniformity such that different subcultures taken from the same will demonstrate substantially identical genotypes and phenotypes (e.g. 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).
[0024] How used cswk. q Vgtoq “kuqnct, kuqnc. kuqncpfq. glqw kuqncfq. gVeo” fi fguVkpcfq c ukipkfiect swg q ocVgtkcn fg tghgtêpekc fi tgoqxkfq of the environment in which it is normally found
[0025] As used herein, the term "inoculum" is intended to mean any form of bacterial cells, or spores, which is capable of propagating in or within soil, when conditions of temperature, humidity, etc., are favorable for the bacterial growth.
[0026] Eqoq wucfq cswk, q Vgtoqu “gurqtq” Vgo its normal meaning which is well known and understood by those skilled in the art and generally refers to a microorganism in its dormant, protected state.
[0027] As used herein, the term "source" of a particular element is intended to mean a compound of that element which, at least under soil conditions, does not take the element fully available for plant uptake.
[0028] Eqoq wucfq cswk, q Vgtoqu “swanVkfcfg gfiecz”, “eqnegnVtc>«q gfiecz”, qw “fqucigo gfiecz” fi fguVkncfq c ukinkfiect c amount, concentration, or dosage of one or more bacterial isolates sufficient to cause an increase in growth of a plant or plant part or an increase in nitrogen fixation. The actual effective dosage in absolute value depends on factors including, but not limited to, the size (e.g. area, total acre area, etc.) of land for application with the bacterial isolates, synergistic or antagonistic interactions between the other active or inert ingredients that can increase or reduce the activity of bacterial isolates, and the stability of bacterial isolates in seed compositions and/or treatments. The "effective amount", "effective concentration" or "effective dose" of the bacterial composition can be determined, for example, by a routine dose-response experiment.
[0029] As used herein, the terms "carrier" or "agronomically acceptable carrier" are intended to refer to any material that can be used to deliver the actives (e.g., a bacterial strain) to a seed, soil, plant, or part of the plant.
[0030] As used herein, the term "soil compatible carrier" is intended to refer to any material that can be added to a soil without causing/having an adverse effect on plant growth, soil structure , soil drainage, or others.
[0031] As used herein, the term "seed compatible carrier" is intended to refer to any material that can be added to a seed without causing/having an adverse effect on the seed, the plant that grows from the seed. seed, seed germination, or the like.
[0032] As used herein, the term "agriculturally beneficial ingredient(s)" is intended to mean any agent or combination of agents capable of causing or providing a beneficial effect and/or usable in agriculture.
[0033] As used herein, "at least one biologically active ingredient" is intended to mean biologically active ingredients (e.g., signaling molecules, other microorganisms, etc.) other than one or more bacterial isolates described herein.
[0034] As used herein, the term "desiccation" is intended to mean a state of extreme dryness, for example, conditions without moisture and/or water. The terms "resistance to desiccation" and/or "tolerance to desiccation" are intended to encompass the ability of an organism to withstand and/or survive and/or maintain itself in conditions of extreme dryness.
[0035] As used herein, the terms "nitrogen fixation", "atmospheric nitrogen fixation", or "nitrogen fixation", etc., are intended to encompass the biological processes in which molecular nitrogen or nitrogen in the atmosphere is converted to one or more nitrogenous (N) compounds, including but not limited to ammonia, ammonium salts, urea, and nitrates.
[0036] As used herein, the term "nitrogen fixing organism" is intended to refer to any organism (e.g. diazotrophs) capable of converting molecular nitrogen or nitrogen in the atmosphere into one or more nitrogenous (N) compounds , including, but not limited to, ammonia, ammonium salts, urea and nitrate.
[0037] As used herein, the terms "phosphate solubilizing", or "phosphate solubilizing", etc. are intended to mean the conversion of insoluble phosphate (eg rock phosphate, etc.) to a soluble phosphate form.
[0038] As used herein, the term "phosphate solubilizing organism" is intended to refer to any organism capable of converting insoluble phosphate to a soluble phosphate form.
[0039] As used herein, the terms "plant(s)" and "plant part(s)" are intended to refer to all plants and plant populations, such as desired and unwanted wild plants or crop plants (including of naturally occurring crops). Cultivated plants can be plants, which can be obtained by conventional plant breeding and optimization methods or by biotechnological and genetic engineering methods or by combinations of these methods, including transgenic plants and including plant cultivars that can be protected or not. for the rights of plant breeders. Plant parts shall be understood to mean all plant parts and organs above and below ground, such as stem, leaf, flower and root, examples of which may be mentioned being leaves, needles, stalks, stems, flowers, bodies of fruits, fruits, seeds, roots, nodules, tubers, rhizomes. Plant parts also include harvested material and vegetative and generative propagation material (e.g. cuttings, tubers, rhizomes, offshoots of shoots and seeds, etc.)
[0040] As used herein, the term "nodule" is intended to include, but not be limited to, certain nodules, indeterminate nodules, or a combination thereof. Examples of determinate nodules and indeterminate nodules are well known in the art and described in Denison, RF, 2000, The Amer. Naturalist 156 (6): 567-576. Determined nodules are found in 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) In contrast to determinate nodules, indeterminate nodules are found in Medicago, Trifolium, and Pisium species, and have an elongated shape and grow continuously. (Denison, 2000) The term “qewrc>«q fg p„fwnq” fi wo Vgtoq eqpjgekfq pc technique. McDermott T.R. & Graham, P.H., Appl. And Environ. Microbiol. 55(10): 2493-2498. It is well known in the art that, although a 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.
[0041] As used herein, term "improved plant growth" is intended to refer to increased plant productivity (e.g., increased biomass, increased number of fruits, or a combination thereof as measured by 1 bushel per 0, 4047 hectare), increased root number, increased root mass, increased root volume, increased leaf area, increased plant population, increased plant vigor, increased weight of a plant (e.g. total dry weight of a plant or part of the plant, weight of total fresh mass, or part of the plant or plants, etc.), or combinations thereof.
[0042] As used herein, "improved competitiveness" and/or "increased nodulation" is defined to mean bacterial strain(s) having a percent nodule occupancy, e.g. at least 50%, at least 55%, at least at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 91%, at least 92%, at least at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, up to 100% nodule occupancy.
[0043] As used herein, the term "temperature tolerance" is intended to mean the range of temperatures at which the bacterial strain(s) are able to grow, e.g. the maximum and minimum temperatures at which a strain(s) bacteria can grow.
[0044] Eqoq wucfq cswk. q Vgtoq “egrc*u+ eqogtekcnogpVg fkurqpixgn*u+” fi fguVkncfq c ukipkfieat egrcu dceVgtkcpcu eqogtekcnogpVg available eg 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.
[0045] As used here, the term “mietqpwVtkgpVg*u+” rtgVgpfg means nutrients that are necessary for plant growth, plant health and/or plant development.
[0046] Eqoq wucfq cswk. q Vgtoq “dkquVkowncnVg*u+” rtgVgpfg means any substance capable of enhancing metabolic or physiological processes within plants and soils.
[0047] Eqoq wucfq cswk. q Vgtoq “cigntg*u+ wogeVcnVg*u+” rtgVgpfg means any substance capable of lowering and/or reducing the surface tension of water. CEPAS
[0048] In one embodiment, the isolated strain(s) described herein is(are) a nitrogen-fixing bacterial strain(s). In another embodiment, the strain(s) is(are) a strain(s) of Bradyrhizobum sp. In another aspect, the strain is derived from a strain of Bradyrhizobium, including, but not limited to, a selected strain from the group consisting of Bradyrhizobium bete, Bradyrhizobium canariense, Bradyrhizobium elkanii, Bradyrhizobium iriomotense, Bradyrhizobium japonicum, Bradyrhizobium jicamae, Bradyrhizobium liaoningense, Bradyrhizobium pachyrhizi, and Bradyrhizobium yuanmingense. In yet another embodiment, the strain(s) is a strain(s) of Bradyrhizobum japonicum.
[0049] In yet another embodiment, the isolated strain(s) is(are) a strain(s) of Bradyrhizobium sp. having improved/increased bacterial survival rate in a substantially moisture-free medium when the survival rate of the isolated Bradyrhizobium strain(s) is compared to a survival rate of the parental strain(s) , for example, parental strain of Bradyrhizobium japonicum USDA 532C, over a period of time, for example, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days , at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months , at least 1 year or more.
[0050] In yet another embodiment, the isolated strain(s) is(are) a strain(s) of Bradyrhizobium sp. having an improved/increased survival rate in a substantially moisture-free medium, wherein an increased survival rate in a substantially moisture-free medium includes an increased bacterial survival rate in an environment that is at least 70% moisture-free , for example, at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least least 95%, at least 96%, at least 97%, at least 98%, at least 99%, up to 100% moisture-free medium, when the survival rate of the isolated strain(s) of Bradyrhizobium is compared to a survival rate of parental strain(s), for example, parental strain of Bradyrhizobium japonicum USDA 532C.
[0051] In another embodiment the isolated strain(s) is(are) a strain(s) of Bradyrhizobium sp. having the following improved/superior characteristics when compared to commercially available strains, for example, the commercial strain of Bradyrhizobium japonicum USDA 532C, where the improved/superior characteristics include, but are not limited to: a. improved competitiveness to colonize a soybean plant; and b. improved effectiveness to provide soybean plant growth.
[0052] In yet another embodiment, the isolated strain(s) is(are) a strain(s) of Bradyrhizobium sp. having improved/superior competitiveness to colonize a plant. In yet another embodiment, the isolated strain(s) is(are) a strain(s) of Bradyrhizobium sp. having improved/superior effectiveness to promote plant growth. In yet another embodiment, the isolated strain(s) is(are) a strain(s) of Bradyrhizobium sp. having improved/superior competitiveness to colonize a plant and improved/superior effectiveness to promote plant growth.
[0053] In yet another aspect of the present invention, the isolated strain(s) is(are) a strain(s) of Bradyrhizobium sp. having improved/higher temperature tolerance.
[0054] In yet another aspect of the present invention, the isolated strain(s) is(are) a strain(s) of Bradyrhizobium sp. having natural resistance to glyphosate.
[0055] In yet another embodiment, the strains are strains of Bradyrhizobum japonicum selected from the group consisting of: the strain having deposit accession number NRRL B-50608; the strain having deposit accession number NRRL B-50609; the strain having deposit accession number NRRL B-50610; the strain having deposit accession number NRRL B-50611; and the strain having deposit accession number NRRL B-50612.
[0056] In a particular embodiment, the strain(s) may be one or more of the aforementioned deposited strains (e.g. including at least two of the above strains, at least three of the above strains, at least four of the above strains, up to and including all of the above strains).
[0057] In one embodiment, the strain is the strain having access to the NRRL B-50608 deposit. In one embodiment, the strain is the strain having deposit accession number NRRL B-50609. In one embodiment, the strain is the strain having deposit accession number NRRL B-50610. In one embodiment, the strain is the strain having deposit accession number NRRL B-50611. In one embodiment, the strain is the strain having deposit accession number NRRL B-50612.
[0058] In another embodiment, the bacterial culture(s) have identical properties/characteristics to at least one of the deposited strains or a combination of at least two of the above deposited strains, including more than two , such as at least three of the above strains, at least four of the above strains, up to and including all of the above strains. Bacterial culture properties/characteristics include, but are not limited to, bacterial strains having improved and/or superior desiccation resistance. In yet another embodiment, the strain(s) is(are) a strain(s) of Bradyrhizobium having improved and/or superior desiccation resistance when desiccation resistance is compared to desiccation resistance and/or tolerance of a parental strain(s) of bacteria, for example, parental strain of Bradyrhizobium japonicum USDA 532C.
[0059] In another aspect, the isolated bacterial culture(s) of the present invention include(s) strain(s) that are closely related to any of the above strains based on sequence identity of 16S rDNA. See Stackebrandt E, et al., “Tgrort qh Vjg cf jqe eqookVVgg hot Vjg tg- gxcnwcViqp oh tjg urgeigu fgfipitiop ip dcetgrionoi{,”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, the at least one strain is at least 95% identical to any of the above strains based on 16S rDNA sequence identity, at least 96% identical to any of the above strains based on sequence identity of 16S rDNA, at least 97% identical to any of the above strains based on 16S rDNA sequence identity, at least 98% to any of the above strains based on 16S rDNA sequence identity, at least 98.5 % identical to any of the above 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% to any of the strains above based on 16S rDNA sequence identity.
[0060] The Bradyrhizobium bacterium described herein, and in particular, strains having deposit accession numbers NRRL B-50608, NRRL B-50609, NRRL B-50610, NRRL B-50611, and NRRL B-50612, can grow according to methods known in the art.
[0061] The resulting material can be used directly in the composition, as a seed treatment, or the spores can be harvested, centrifuged, formulated, and then dried using air-drying, freeze-drying, or fluidized bed drying (Friesen T., Hill G., Pugsley T., Holloway G., and Zimmerman D. 2005, Experimental determination of viability loss of Penicillium bilaiae conidia for convective air-drying Appl Microbiol Biotechnol 68: 397- 404) to produce a wettable powder.
[0062] The aforementioned deposited strains were deposited on 1st. October 2012, as indicated in more detail below in section fg “OcVgtkcku (OfiVqfqu”, uqd qu Vgtoqu fq VtcVcfq fg Dwfcrgste on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (ATCC) ), PO Box 1549, Manassas, VA 20108, USA. COMPOSITIONS:
[0063] In another aspect, the invention relates to a composition comprising a carrier and an inoculum of one or more of the deposited strains (either in the form of spores or of strains in a vegetative state) described herein. In certain embodiments, the composition may be in the form of a liquid, a suspension, a solid, or a powder (wettable powder or dry powder). In another embodiment, the composition may be in the form of a seed coat. Liquid, paste or powder compositions (e.g. wettable powder) may be suitable for coating seeds. When used to coat seeds, the composition can be applied to seeds and allowed to dry. In embodiments where the composition is a powder (e.g., a wettable powder), a liquid, such as water, may need to be added to the powder prior to application to a seed. Examples of still other carriers include bran moistened, dried, sieved and applied to seeds previously coated with an adhesive, for example, gum arabic. Carriers:
[0064] The carriers described herein will allow the deposited bacterial strain(s) to remain effective (eg capable of nitrogen fixation) and viable once formulated. Non-limiting examples of carriers described herein include liquids, pastes or solids (including wettable powders or dry powders). In one embodiment, the carrier is a soil compatible carrier as described herein.
[0065] In one embodiment, the carrier is a liquid carrier. Non-limiting examples of liquids useful as carriers for the compositions described herein include water, an aqueous solution, or a non-aqueous solution. In one embodiment, the carrier is water. In another embodiment, the carrier is an aqueous solution, such as sugar water. In another embodiment, the carrier is a non-aqueous solution. If a liquid carrier is used, the liquid (eg, water) carrier may also include growth media for culturing the deposited bacterial strains. Non-limiting examples of suitable growth media for the bacterial strains deposited include arabinose gluconate (AG), mannitol yeast extract (YEM), G16 media, or any media known to those skilled in the art to be compatible with and/or provide nutrients of growth for the deposited bacterial strains.
[0066] In another embodiment, the carrier is a suspension. In one embodiment, the suspension may comprise an adhesive, a liquid, or a combination thereof. It is envisaged that the adhesive may be any agent capable of adhering the inoculum (e.g., one or more of the deposited strains) to a substrate of interest (e.g., a seed). Non-limiting examples of adhesive agents include alginate, mineral oil, syrup, acacia, honey, methyl cellulose, milk, wallpaper paste, and combinations thereof. Non-limiting examples of suitable liquids for a suspension include water or sugar water.
[0067] In another embodiment, the carrier is a solid. In a particular embodiment, the solid is a powder. In one embodiment, the powder is a wettable powder. In another embodiment, the powder is a dry powder. In another embodiment, the solid is a granule. Non-limiting examples of solids useful as carriers for the compositions described herein include peat, wheat, wheat bran, shredded wheat straw, bran (e.g., moistened bran, unwetted bran), vermiculite, cellulose, starch, soil (pasteurized or unpasteurized), gypsum, talc, clays (eg, kaolin, bentonite, montmorillonite), and silica gels. Agriculturally Beneficial Optional Ingredients:
[0068] The compositions described herein may comprise one or more optional ingredients. Non-limiting examples of optional ingredients include one or more biologically active ingredients, micronutrients, biostimulants, preservatives, polymers, wetting agents, surfactants, or combinations thereof. Biologically active ingredient(s)
[0069] The compositions described herein may optionally include one or more biologically active ingredients as described herein. Non-limiting examples of biologically active ingredients include signaling molecules (e.g. lipo-chitooligosaccharides (LCO), chitooligosaccharides (CO), chitin compounds, flavonoids, jasmonic acid or derivatives thereof, linoleic acid or derivatives thereof, linolenic acid or derivatives thereof thereof, kerrikines, etc.) and beneficial microorganisms (e.g. Rhizobium spp., Bradyrhizobium spp., Sinorhizobium spp., Azorhizobium spp., etc.). Signaling molecules:
[0070] In one embodiment, the compositions described herein include one or more signaling molecules. In one embodiment, the one or more signaling molecules are one or more LCOs. In another embodiment, the one or more signaling molecules are one or more chitinous compounds. In yet another embodiment, the one or more signaling molecules are one or more COs. In yet another embodiment, the one or more signaling molecules are one or more flavonoids or derivatives thereof. In yet another embodiment, the one or more signaling molecules are one or more inducers of the non-flavonoid nod gene (e.g., jasmonic acid, linoleic acid, linolenic acid, and derivatives thereof). In yet another embodiment, the one or more signaling molecules are one or more karrikins or derivatives thereof. In yet another embodiment, the one or more signaling molecules are one or more LCOs, one or more chitin compounds, one or more COs, one or more flavonoids and derivatives thereof, one or more non-flavonoid nod gene inducers, and derivatives thereof, one or more karrikins and derivatives thereof, or any combination of signaling molecules thereof. LCOs:
[0071] Lipo-chitooligosaccharide compounds (LCOs), also known in the art as symbiotic Nod signals or Nod factors, consist of an oligosaccharide backbone of β-1,4-linked N-acetyl-D-glucosamine residues ("GIcNAc") with an N-linked grease acyl chain condensed at a non-reducing end. L CO'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:
wherein: G is a hexosamine which can be substituted, for example, by an acetyl group on nitrogen, a sulfate group, an acetyl group and/or an ether group or an oxygen, R1, R2, R3, R5, R6 and R7, which may be the same 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 acyl group such as 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.
[0072] LCOs can be obtained (isolated and/or purified) from bacteria such as Rhizobia, eg Rhizobium spp., Bradyrhizobium spp., Sinorhizobium spp. and Azorhizobium spp. The LCO structure is characteristic for each of these bacterial species, and each strain can produce multiple LCO's with different structures. For example, S. meliloti-specific LCOs have also been described in US Patent 5,549,718 as having formula II:
where R represents H or CH3 CO-- en is equal to 2 or 3.
[0073] Even more specific LCOs include NodRM, NodRM-1, NodRM-3. When acylated (the R=CH3 CO--), they become AcNodRM-1, and AcNodRM-3, respectively (US Patent 5,545,718).
[0074] Bradyrhizobium japonicum LCOs are described in US patents 5,175,149 and 5,321,011. Broadly, they are phytohormones of rgpVcuucecrífgq eqortggpfgpfq ogVüfucqug. Xátkqu fguVgu NEQÓU fgtkxcfqu from B. japonicum are described: BjNod-V (C18:1); BjNod-V (AC, C18:1), BjNod-V (C16:1); and BjNod-V (AC, 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 number of double bonds.
[0075] LCOs used in the 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 . leguminosarum), Sinorhizobium (including S. meliloti), and bacterial strains genetically engineered to produce LCO's.
[0076] Also encompassed by the present invention are compositions using LCOs obtained (i.e., 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 fguetkVqu pqu oguoqu Vcodfio ugpfq ejcocfqu eqoq “faVqtgu $Oyc$).
[0077] Still 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 uwduVkVwk>õgu gpcqpVtcfcu pqu NEQÓU fg qcqttêpckc PcVwtcn. cqoq qu fguetkVqu in Spaink, Crit. Rev. Plant Sci. 54:257-288 (2000) and D'Haeze, et al., Glycobiology 12:79R-105R (2002 ). Oligosaccharide precursor molecules (COs, which as described below, are also usable as plant signaling 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).
[0078] LCO's can be used in various purity forms and can be used alone or in the form of a bacterial or fungal culture producing LCO. Methods of 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 through LCO solvent phase separation followed by HPLC chromatography as described, for example, in US Patent 5,549,718. Purification can be improved by repeated HPLCs, and purified LCO molecules can be freeze-dried for long-term storage. Waistband:
[0079] Chitooligosaccharides (COs) are known in the art as guVtwVwtcu β--4 linked N-actyl glucosamine identified as chitin oligomers, also as N-acetylchitooligosaccharides. CO's have unique and different side chain decorations which 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., Planta 232:787-806 (2010 ); Rouge, et al. Chapter 27, "The Molecular Immunology of Complex Carbohydrates" in Advances in Experimental Medicine and Biology, Springer Science; Wan, et al., Plant Cell 21:1053-69 (2009 ); PCT/F100/00803 (9/21/2000); and Demont-Caulet, et al., Plant Physiol. 120(1):83-92 (1999). COs 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). Chitinous Compounds
[0080] Chitins and chitosans, which are the main components of fungal cell walls and insect and crustacean exoskeletons, are also composed of GIcNAc residues. Chitinous compounds include chitin, (IUPAC: N-[5-[[3-acetylamino-4,5-dihydroxy-6-(hydroxy methyl)oxan-2yl]methoxymethyl]-2-[[5-acetylamino-4,6 -dihydroxy-2-(hydroxy methyl)oxan-3-yl]methoxymethyl]-4-hydroxy-6-(hydroxy methyl)oxan-3-yl]ethanamide), and chitosan, (IUPAC: 5-amino-6-[ 5-amino-6-[5-amino-4,6-dihydroxy-2(hydroxy methyl)oxan-3-yl]oxy-4-hydroxy-2-(hydroxy methyl)oxan-3-yl]oxy-2( hydroxy methyl)oxane-3,4-diol).
[0081] These compounds can be obtained commercially, for example, from Sigma-Aldrich, or prepared from insects, crustacean shells or from fungal cell walls. Methods for preparing chitin and chitosan are known in the art, and have been described, for example, in US Patent 4,536,207 (preparation of crustacean shells), Pochanavanich, et al., Lett. App. Microbiol. 35:17-21 (2002) (preparation of fungal cell walls), and US Patent 5,965,545 (preparation of crab shells and hydrolysis of commercial chitosan). Deacetylated chitins and chitosans can be obtained that are in the range of less than 35% to more than 90% deacetylation, and cover a broad spectrum of molecular weights, e.g. low molecular weight chitosan oligomers of less than 15kD and chitin oligomers from 0.5 to 2kD; "practical type" chitosan having a molecular weight of about 15kD; and high molecular weight chitosan up to 70kD. Chitin and chitosan compositions formulated for seed treatment are also commercially available. Commercial products include, for example, ELEXA® (Plant Fgfgpug DqquVgtu. Kpeo+ g DG[QPF™ *Citkjqwug, Inc.). Flavonoids:
[0082] Flavonoids are phenolic compounds having the general structure of two aromatic rings connected by a three-carbon bridge. Flavonoids are produced by plants and have many functions, for example, beneficial signaling molecules, and protection against insects, animals, fungi and bacteria. Classes of flavonoids include chalcones, anthocyanidins, coumarins, flavones, flavanols, flavonols, flavanones, and isoflavones. See, Jain, et al., J. Plant Biochem. & Biotechnol. 11:1-10 (2002); Shaw, et al., Environmental Microbiol. 11:1867-80 (2006 ).
[0083] Representative flavonoids that can be used in compositions of the present invention include luteolin, apigenin, tangeritin, quercetin, kaempferol, myricetin, fisetin, isorhamnetin, pacquipodol, rhamnazine, hesperetin, naringenin, formononetin, eriodictiol, homoeriodictiol, taxifolin, di- hydroquercetin, dihydrokaempferol, genistein, daidzein, glycitein, catechin, gallocatechin, catechin 3-gallate, gallocatechin 3-gallate, epicatechin, epigallocatechin, epicatechin 3-gallate, epigallocatechin 3-gallate, cyanidin, delphinidine, malvidin, pelargonidin, peonidin, petunidin, or derivatives thereof. Flavonoid compounds are commercially available, for example, from Natland International Corp., Research Triangle Park, NC; MP Biomedicals, Irvine, CA; LC Laboratories, Woburn MA. Flavonoid compounds can be isolated from plants or seeds, for example, as described in US Patents 5,702,752; 5,990,291; and 6,146,668. Flavonoid compounds can also be produced by genetically engineered organisms, such as yeast, as described in Ralston, et al., Plant Physiology 137:1375-88 (2005). Non-flavonoid Nod gene inducer(s)
[0084] Jasmonic acid (JA, [1R-[1g.4β*¥+__-3-oxo-2-(pentenyl)cyclopentane acetic acid) and derivatives thereof, linoleic acid ((Z,Z)-9 ,12-octadecadienoic acid) and derivatives thereof, and linolenic acid ((Z,Z,Z)-9,12,15-octadecatrienoic acid) and derivatives thereof, can also be used in compositions of the present invention. methyl ester, methyl jasmonate (MeJA), collectively known as jasmonates, are octadecanoid-based compounds that occur naturally in plants. Jasmonic acid is produced by the roots of wheat seedlings, and fungal microorganisms such as Botryodiplodia theobromae and Gibbrella fujikuroi, yeast (Saccharomyces cerevisiae), and pathogenic and non-pathogenic strains of Escherichia coli. Linoleic acid and linolenic acid are produced in the course of jasmonic acid biosynthesis. Jasmonates, linoleic acid, and linoleic acid (and derivatives thereof) are reported to be inducing expression of the nod gene or in the production of L CO by rhizobacteria. See, for example, Mabood, Fazli, Jasmonates induce the expression of nod genes in Bradyrhizobium japonicum, May 17, 2001; and Mabood, Fazli, "Linoleic and linolenic acid induce the expression of nod genes in Bradyrhizobium japonicum," USDA 3, May 17, 2001.
[0085] Usable derivatives of linoleic acid, linolenic acid, and jasmonic acid which may be usable in compositions of the present invention include esters, amides, glycosides, and salts. Representative esters are compounds in which the carboxyl group of linoleic acid, linolenic acid, or jasmonic acid has been replaced with a --COR group, where R is an --OR1 group, where R1 is: 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 unbranched or branched C2-C8 alkynyl group; an aryl group, having, for example, 6 to 10 carbon atoms; or a heteroaryl group having, for example, 4 to 9 carbon atoms, wherein the heteroatoms in the heteroaryl group may be, for example, N, O, P, or S. Representative amides are compounds wherein the carboxyl group of acid linoleic, linolenic acid, or jasmonic acid has been substituted with a --COR group, where R is an NR2R3 group, where R2 and R3 are independently: hydrogen; an alkyl group, such as 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 having, for example, 6 to 10 carbon atoms; or a heteroaryl group having, for example, 4 to 9 carbon atoms, wherein the heteroatoms in the heteroaryl group can be, for example, N, O, P, or S. Esters can be prepared by known methods, such as catalyzed nucleophilic addition by acid, wherein the carboxylic acid is reacted with an alcohol in the presence of a catalytic amount of mineral acid. Amides can also be prepared by known methods such as by 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, linolenic 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 readily prepared by mixing together a solution of linoleic acid, linolenic acid, or jasmonic acid with a solution of the base. The salt can be precipitated from solution and collected by filtration or it can be recovered by other means such as solvent evaporation. Karriquina(s):
[0086] Karriquinas are 4H-pyrones vinyl analogues eg 2H-furo[2,3-c]pyran-2-ones including derivatives thereof and analogues. Examples of these compounds are represented by the following structure:
on what; Z is O, S or NR5; each of R1 , R2 , R3 , and R4 are independently H, alkyl, alkenyl, alkynyl, phenyl, benzyl, hydroxy, hydroxyalkyl, alkoxy, phenyloxy, benzyloxy, CN, COR6, COOR=, halogen, NR6R7, or NO2 ; and each of R5, R6, and R7 is independently H, alkyl or alkenyl, or a biologically acceptable salt thereof. Examples of biologically acceptable salts of these compounds include acid addition salts formed with biologically acceptable acids, examples of which include hydrochloride, hydrobromide, sulfate or bisulfate, hydrogen phosphate or phosphate, acetate, benzoate, succinate, fumarate, maleate, lactate, citrate, tartrate, gluconate; methanesulfonate, benzenesulfonate and p-toluenesulfonic acid. Additional biologically acceptable metal salts may include alkali metal salts with bases, examples of which include the sodium and potassium salts. Examples of compounds encompassed by the structure that may be suitable for use in the present invention include the following: 3-methyl-2H-furo[2,3-c]pyran-2-one (wherein R1=CH3, R2, R3, R4=H), 2H-furo[2,3-c]pyran-2-one (wherein R1, R2, R3, R4=H), 7-methyl-2H-furo[2,3-c]pyran- 2-one (wherein R1, R2, R4=H, R3=CH3), 5-methyl-2H-furo[2,3-c]pyran-2-one (wherein R1, R2, R3=H, R4 =CH3), 3,7-dimethyl-2H-furo[2,3-c]pyran-2-one (wherein R1, R3=CH3, R2, R4=H), 3,5-dimethyl-2H-furo [2,3-c]pyran-2-one (wherein R1, R4=CH3, R2, R3=H), 3,5,7-trimethyl-2H-furo[2,3-c]pyran-2- ona (wherein R1, R3, R4=CH3, R2=H), 5-methoxymethyl-3-methyl-2H-furo[2,3-c]pyran-2-one (wherein R1=CH3, R2, R3 =H, R4=CH2OCH3), 4-bromo-3,7-dimethyl-2H-furo[2,3-c]pyran-2-one (wherein R1, R3=CH3, R2=Br, R4=H) , 3-methylfuro[2,3-c]pyridin-2(3H)-one (wherein Z=NH, R1=CH3, R2, R3, R4=H), 3,6-dimethylfuro[2,3-c ]pyridin-2(6H)-one (wherein Z=N--CH3, R1=CH3, R2, R3, R4=H). See, US Patent 7,576,213. These molecules are also known as karrikins. See, Halford, "Smoke Signals," in Chem. Eng. News (April 12, 2010), on pages 37-38 (with respect to karriquinas or butenolides that are contained in smoke act as growth stimulants and induce seed germination after a forest fire, and can invigorate seeds such as corn, tomatoes, lettuce and onions that were stored). These molecules are the subject of US Patent 7,576,213. Beneficial microorganism(s)
[0087] In one embodiment, the compositions described herein may comprise one or more beneficial microorganisms. The one or more beneficial microorganisms may have one or more beneficial properties (e.g., produce one or more of the signaling molecules described here, improve nutrient and water uptake, improve growth, improve seed germination, improve seed emergence). seedlings, breaking dormancy or quiescence of a plant, etc.).
[0088] In one embodiment, the beneficial microorganism(s) comprise one or more bacteria that produce one or more of the signaling molecules described herein. In yet another embodiment, the bacteria are bacteria of the genera Rhizobium spp. (e.g., R. cellulosilyticum, R. daejeonense, R. etli, R. galegae, R. gallicum, R. giardinii, R. hainanense, R. huautlense, R. indigoferae, R. leguminosarum, R. loessense, R. lupini, R. lusitanum, R. meliloti, R. mongolense, R. miluonense, R. sullae, R. tropici, R. undicola, and/or R. yanglingense), Azorhizobium spp. (e.g. A. caulinodans and/or A. doebereinerae), Sinorhizobium spp. (e.g. S. abri, S. adhaerens, S. americanum, S. aboris, S. fredii, S. indiaense, S. kostiense, S. kummerowiae, S. medicae, S. meliloti, S. mexicanus, S. morelense, S. saheli, S. terangae, and/or S. xinjiangense), Mesorhizobium spp., (M. albiziae, M. amorphae, M. chacoense, M. ciceri, M. huakuii, M. loti, M. mediterraneum , M. pluifarium, M. septentrionale, M. temperatum, and/or M. tianshanense), and combinations thereof. In a particular embodiment, the beneficial microorganism is selected from the group consisting of R leguminosarum, R meliloti, S. meliloti, and combinations thereof. In another embodiment, the beneficial microorganism is R leguminosarum. In another embodiment, the beneficial microorganism is R meliloti. In another embodiment, the beneficial microorganism is S. meliloti.
[0089] In another embodiment, the one or more beneficial microorganisms comprise one or more phosphate-solubilizing microorganisms. Phosphate solubilizing microorganisms include fungal strains and bacterial strains. In one embodiment, the phosphate-solubilizing microorganism includes species of a genus selected from the group consisting of Acinetobacter spp. (e.g. Acinetobacter calcoaceticus, etc.), Arthrobacter spp, Arthrobotrys spp. (e.g. Arthrobotrys oligospora, etc.), Aspergillus spp. (e.g. Aspergillus niger, etc.), Azospirillum spp. (e.g. Azospirillum halopraeferans, etc.), Bacillus spp. (e.g. Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans, Bacillus licheniformis, Bacillus subtilis, etc.), Burkholderia spp. (eg, Burkholderia cepacia, Burkholderia vietnamiensis, etc.), Candida spp. (e.g. Candida krissii, etc.), Chryseomonas spp. (eg, Chryseomonas luteola, etc.), Enterobacter spp. (eg, Enterobacter aerogenes, Enterobacter asburiae, Enterobacter spp., Enterobacter taylorae, etc.), Eupenicillium spp. (e.g. Eupenicillium parvum, etc.), Exiguobacterium spp., Klebsiella spp., Kluyvera spp. (e.g. Kluyvera cryocrescens, etc.), Microbacterium spp., Mucor spp. (eg, Mucor ramosissimus, etc.), Paecilomyces spp. (e.g. Paecilomyces hepialid, Paecilomyces marquandii, etc.), Paenibacillus spp. (e.g. Paenibacillus macerans, Paenibacillus mucilaginosus, etc.), Penicillium spp. (e.g. Penicillium bilaiae (formerly known as Penicillium bilaii), Penicillium albidum, Penicillium aurantiogriseum, Penicillium chrysogenum, Penicillium citreonigrum, Penicillium citrinum, Penicillium digitatum, Penicillium frequentas, Penicillium fuscum, Penicillium gaestrivorus, Penicillium glabrum, Penicillium griseofull Penicillium janthinellum, Penicillium lilacinum, Penicillium minioluteum, montanense Penicillium nigricans Penicillium, Penicillium oxalicum, Penicillium pinetorum, Penicillium pinophilum, Penicillium purpurogenum, Penicillium radicans, Penicillium radicum, Penicillium raistrickii, Penicillium rugulosum, Penicillium simplicissimum, Penicillium solitum, Penicillium variabile, Penicillium velutinum , Penicillium viridicatum, Penicillium glaucum, Penicillium fussiporus, and Penicillium expansum, etc.), Pseudomonas spp. (eg, Pseudomonas corrugate, Pseudomonas fluorescens, Pseudomonas lutea, Pseudomonas poae, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas trivialis, etc.), Serratia spp. (eg, Serratia marcescens, etc.), Stenotrophomonas spp. (eg, Stenotrophomonas maltophilia, etc.), Streptomyces spp., Streptosporangium spp., Swaminathania spp. (eg, Swaminathania salitolerans, etc.), Thiobacillus spp. (e.g. Thiobacillus ferrooxidans, etc.), Torulospora spp. (eg Torulospora globosa, etc.), Vibrio spp. (e.g. Vibrio proteolyticus, etc.), Xanthobacter spp. (e.g. Xanthobacter agilis, etc.), Xanthomonas spp. (eg, Xanthomonas campestris, etc.), and combinations thereof.
[0090] In a particular embodiment, the one or more phosphate-solubilizing microorganisms is a strain of the Penicillium fungus. In another embodiment, the one or more Penicillium species is P. bilaiae, P. gaestrivorus, or combinations thereof.
[0091] In another embodiment the beneficial microorganism is one or more mycorrhizae. In particular, the one or more mycorrhiza is an endomycorrhiza (also called vesicular arbuscular mycorrhizae, VAMs, arbuscular mycorrhizae, or AMs), an ectomycorrhiza, or a combination thereof.
[0092] In one embodiment, the one or more mycorrhiza is an endomycorrhiza of the phylum Glomeromycota and genera Glomus and Gigaspora. In yet another embodiment, the endomycorrhiza is a strain of Glomus aggregatum, Glomus brasilianum, Glomus clarum, Glomus deserticola, Glomus etunicatum, Glomus fasciculatum, Glomus intraradices, Glomus monosporum, or Glomus mosseae, Gigaspora margarita, or a combination thereof.
[0093] In another embodiment, the one or more mycorrhiza is an ectomycorrhiza of the phylum Basidiomycota, Ascomycota, and Zygomycota. In yet another embodiment, the ectomycorrhiza is a strain of Laccaria bicolor, Laccaria laccata, Pisolithus tinctorius, Rhizopogon amylopogon, Rhizopogon fulvigleba, Rhizopogon luteolus, Rhizopogon villosuli, Scleroderma cepa, Scleroderma citrinum, or a combination thereof.
[0094] In yet another embodiment, the one or more mycorrhizae is an echroid mycorrhiza, an arbutoid mycorrhiza, or a monotropoid mycorrhiza. Arbuscular and ectomycorrhizas form ericoid mycorrhizae with many plants belonging to the order Ericales, while some Ericales form arbutoid and monotropoid mycorrhizae. All orchids are mycoheterotrophs at some stage during their life cycle and form orchid mycorrhizas with a range of basidiomycete fungi. In one embodiment, the mycorrhiza may be an ericoid mycorrhiza, preferably from the phylum Ascomycota, such as Hymenoscyphous ericae or Oidiodendron sp. In another embodiment, the mycorrhiza may also be an arbutoid mycorrhiza, preferably from the phylum Basidiomycota. In yet another embodiment, the mycorrhiza may be a monotriploid mycorrhiza, preferably from the Basidiomycota. In yet another embodiment, the mycorrhiza may be an orchid mycorrhiza, preferably of the genus Rhizoctonia. Micronutrient(s):
[0095] In yet another embodiment, the compositions described herein may comprise one or more beneficial micronutrients. Non-limiting examples of micronutrients for use in the compositions described herein include vitamins, (e.g., vitamin A, vitamin B complex (i.e., vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B8, vitamin B9, vitamin B12, choline) vitamin C, vitamin D, vitamin E, vitamin K, carotenoids (g-ectqVgpq. β-carotene, cryptoxanthin, lutein, lycopene, zeaxanthin, etc.), macrominerals (e.g. phosphorus, calcium , magnesium, potassium, sodium, iron, etc.), trace minerals (e.g. boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, zinc, etc.), organic acids ( e.g. acetic acid, citric acid, lactic acid, malic acid, taurine, etc.), etc.), and combinations thereof. In a particular embodiment, the compositions may comprise phosphorus, boron, chlorine, copper, iron, manganese, molybdenum, zinc or combinations thereof.
[0096] In certain embodiments, where the compositions described herein may comprise phosphorus, it is envisaged that any suitable source of phosphorus may be provided. In one embodiment, the phosphorus may be derived from a source. In another embodiment, suitable sources of phosphorus include sources of phosphorus capable of solubilization by one or more microorganisms (e.g., Penicillium bilaiae, etc.).
[0097] In one embodiment, the phosphorus may be derived from a rock phosphate source. In another embodiment the phosphorus can be derived from fertilizers comprising one or more sources of phosphorus. Commercially available manufactured phosphate fertilizers are of many types. Some of the most common are those containing rock phosphate, monoammonium phosphate, diammonium phosphate, monocalcium phosphate, super phosphate, triple super phosphate, and/or ammonium polyphosphate. All these fertilizers are produced by chemically processing insoluble natural rock phosphates in large-scale fertilizer manufacturing facilities and the product is expensive. By means of the present invention it is possible to reduce the amount of these fertilizers applied to the soil while still maintaining the same amount of phosphorus uptake from the soil.
[0098] In yet another embodiment, the phosphorus may be derived from an organic phosphorus source. In another particular embodiment, the source of phosphorus may include an organic fertilizer. An organic fertilizer refers to a soil improver derived from natural sources that ensures at least minimum percentages of nitrogen, phosphate, and potash. Non-limiting examples of organic fertilizers include by-products of plant and animal origin, rock dust, seaweed, inoculants, and conditioners. These are often available at garden centers and through horticultural supply companies. In particular, the organic source of phosphorus is bone meal, meat meal, animal manure, compost, sewage sludge or guano manure, or combinations thereof.
[0099] In yet another embodiment, phosphorus can be derived from a combination of phosphorus sources including, but not limited to, rock phosphate, fertilizers comprising one or more sources of phosphorus (e.g., monoammonium phosphate, phosphate of diammonium, monocalcium phosphate, super phosphate, triple super phosphate, ammonium polyphosphate, etc.) one or more organic sources of phosphorus, and combinations thereof. Biostimulant(s):
[00100] In one embodiment, the compositions described herein may comprise one or more beneficial biostimulants. Biostimulants can enhance metabolic or physiological processes such as respiration, photosynthesis, nucleic acid uptake, ion uptake, nutrient delivery, or a combination thereof. Non-limiting examples of biostimulants include extracts of algae (e.g., ascophyllum nodosum), humic acids (e.g., potassium humate), fulvic acids, myo-inositol, glycine, and combinations thereof. In another embodiment, the compositions comprise seaweed extracts, humic acids, fulvic acids, myo-inositol, glycine, and combinations thereof. Polymer(s):
[00101] In one embodiment, the compositions described herein may further comprise one or more polymers. Non-limiting uses of polymers in the agricultural sector include release of agrochemicals, removal of heavy metals, water retention and/or water distribution, and combinations thereof. Pouci, et al., Am. J. Agri. & Biol. Sci., 3(1):299-314 (2008 ). In one embodiment, the one or more polymers is a natural polymer (e.g., agar, starch, alginate, pectin, cellulose, etc.), a synthetic polymer, a biodegradable polymer (e.g., polycaprolactone, polylactide, poly( vinyl alcohol), etc.), or a combination thereof.
[00102] For a non-limiting list of usable polymers for the compositions described herein, see Pouci, et al., Am. J. Agri. & Biol. Sci., 3(1):299-314 (2008). In one embodiment, the compositions described herein comprise cellulose, cellulose derivatives, methylcellulose, methylcellulose derivatives, starch, agar, alginate, pectin, polyvinylpyrrolidone, and combinations thereof. Wetting agent(s):
[00103] In one embodiment, the compositions described herein may further comprise one or more wetting agents. Wetting agents are commonly used in soils, particularly hydrophobic soils, to improve infiltration and/or penetration of water into a soil. The wetting agent can be an adjuvant, oil, surfactant, buffer, acidifier, or combination thereof. In one embodiment, the wetting agent is a surfactant. In one embodiment, the wetting agent is one or more nonionic surfactants, one or more anionic surfactants, or a combination thereof. In yet another embodiment, the wetting agent is one or more non-ionic surfactants.
[00104] Suitable surfactants for the compositions described here u«q rtqxkfqu pc ug>«q “VgpuqcVkxqu” Surfactant(s):
[00105] Suitable surfactants for the compositions described herein may be non-ionic surfactants (eg semipolar and/or anionic and/or cationic and/or zwitterionic). It is intended that the surfactant(s) will (will) cause the least possible damage to the activity of one or more deposited strains and/or of one or more beneficial microorganisms. Surfactants can wet and emulsify soil(s) and/or dirt(s). It is intended that the surfactants used in the described composition have low toxicity to the microorganisms contained within the formulation. It is further intended that the surfactants used in the described composition have a low phytotoxicity (i.e. the degree of toxicity that a substance or combination of substances has on a plant). A single surfactant or a mixture of several surfactants can be used. anionic surfactants
[00106] Anionic surfactants or mixtures of anionic and nonionic surfactants can also be used in the compositions. Anionic surfactants are surfactants having a hydrophilic moiety in an anionic or negatively charged state in the aqueous solution. The compositions described herein may comprise one or more anionic surfactants. The anionic surfactant(s) may be either water soluble anionic surfactants, water insoluble anionic surfactants, or a combination of water soluble anionic surfactants and water insoluble anionic surfactants. Non-limiting examples of anionic surfactants include sulfonic acids, sulfuric acid esters, carboxylic acids, and salts thereof. Non-limiting examples of water-soluble anionic surfactants include alkyl sulfates, alkyl ether sulfates, alkyl starch ether sulfates, alkyl aryl polyether sulfates, alkyl aryl sulfates, alkyl aryl sulfonates, monoglyceride sulfates, alkyl sulfonates, sulfonates alkyl amide sulfonates, alkyl aryl sulfonates, benzene sulfonates, toluene sulfonates, xylene sulfonates, cumene sulfonates, alkyl benzene sulfonates, alkyl diphenyloxide sulfonates, alpha olefin sulfonates, alkyl naphthalene sulfonates, paraffin sulfonates, sulfonates of lignin, alkyl sulfosuccinates, ethoxylated sulfosuccinates, alkyl ether sulfosuccinates, alkylamide sulfosuccinates, alkyl sulfosuccinamate, alkyl sulfoacetates, alkyl phosphates, phosphate ether, alkyl ether phosphates, acyl sarconsinates, acyl isethionates, N-acyl, N-acyl-N-alkyltaurates, alkyl carboxylates, or a combination thereof. non-ionic surfactants
[00107] Nonionic surfactants are surfactants having no electrical charge when dissolved or dispersed in an aqueous medium. In at least one embodiment of the composition described herein, one or more nonionic surfactants are used as they provide desired wetting and emulsifying actions and do not significantly inhibit spore stability and activity. The nonionic surfactant(s) may be either water-soluble nonionic surfactants, water-insoluble nonionic surfactants, or a combination of water-soluble nonionic surfactants and water-insoluble nonionic surfactants. . Water-insoluble non-ionic surfactants
[00108] Non-limiting examples of water-insoluble nonionic surfactants include alkyl and aryl: glycerol ethers, glycol ethers, ethanolamides, sulfoanilamides, alcohols, amides, alcohol ethoxylates, glycerol esters, glycol esters, glycerol ester ethoxylates and glycol esters, sugar-based alkyl polyglycosides, polyoxyethylenated fatty acids, alkanolamine condensates, alkanolamides, tertiary acetylenic glycols, polyoxyethylenated mercaptans, carboxylic acid esters, polyoxyethylenated polyoxypropylene glycols, sorbitan fatty esters, or combinations thereof. Also included are EO/PO block copolymers (EO is ethylene oxide, PO is propylene oxide), EO polymers and copolymers, polyamines, and polyvinylpinolidones. Water-soluble non-ionic surfactants
[00109] Non-limiting examples of water-soluble nonionic surfactants include sorbitan fatty acid alcohol ethoxylates and sorbitan fatty acid ester ethoxylates. Combination of non-ionic surfactants
[00110] In one embodiment, the compositions described herein comprise at least one or more non-ionic surfactants. In one embodiment, the compositions comprise at least one water-insoluble nonionic surfactant and at least one water-soluble nonionic surfactant. In yet another embodiment, the compositions comprise a combination of nonionic surfactants having hydrocarbon chains of substantially the same length. Other surfactants
[00111] In another embodiment, the compositions described herein may also comprise organosilicone surfactants, silicone-based defoamers used as surfactants in silicone-based and mineral oil-based defoamers. In yet another embodiment, the compositions described herein may also comprise alkali metal salts of fatty acids (e.g., water-soluble alkali metal salts of fatty acids and/or water-insoluble alkali metal salts of fatty acids). Herbicide(s):
[00112] In one embodiment, the compositions described herein may further comprise one or more herbicides. In a particular embodiment, the herbicide can be a pre-emergence herbicide, a post-emergence herbicide, or a combination thereof.
[00113] Suitable herbicides include chemical herbicides, natural herbicides (eg, bio-herbicides, organic herbicides, etc.), or combinations thereof. Non-limiting examples of suitable herbicides include bentazon, acifluorfen, clorimuron, lactofen, clomazone, fluazifop, glufosinate, glyphosate, sethoxydim, imazethapyr, imazamox, fomesafe, flumiclorac, imazaquin, clethodim, pendimethalin; 3,4-dimethyl-2,6-dinitro-N-pentan-3-yl-aniline; N-(1-ethylpropyl)-2,6-dinitro-3,4-xylidine; pronamide; propyzamide; 3,5-dichloro-N-(1,1-dimethylpropynyl)benzamide; 3,5-dichloro-N-(1,1-dimethyl-2-propynyl)benzamide; N-(1,1-dimethylpropynyl)-3,5-dichlorobenzamide; S-ethyl N-ethylthiocyclohexanecarbamate; trifluralin; 2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)aniline; glyphosate; N-(phosphonomethyl)glycine; and derivatives thereof. In one embodiment, the one or more herbicides for use in accordance with this description include pronamide (commercially referred to as Kerb®); propyzamide; 3,5-dichloro-N-(1,1-dimethylpropynyl)benzamide; 3,5-dichloro-N-(1,1-dimethyl-2-propynyl)benzamide; N-(1,1-dimethylpropynyl)-3,5-dichlorobenzamide; cycloate, S-ethyl N-ethylthiocyclohexanecarbamate (commercially referred to as Ro-Neet®); trifluralin; 2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)aniline; glyphosate; N-(phosphonomethyl)glycine; and derivatives thereof. Commercial products containing each of these compounds are readily available. The herbicide concentration in the composition will generally correspond to the labeled usage rate for a particular herbicide. Fungicide(s):
[00114] In one embodiment, the compositions described herein may further comprise one or more fungicides. Fungicides usable as the compositions described herein will suitably exhibit against a wide range of pathogens, including, but not limited to, Phytophthora, Rhizoctonia, Fusarium, Pythium, Phomopsis or Selerotinia and Phakopsora and combinations thereof.
[00115] Non-limiting examples of commercial fungicides that may be suitable for the compositions described herein 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 Ares, Argentina). Active ingredients in the same and other commercial fungicides include, but are not limited to, fludioxonil, mefenoxam, azoxystrobin and metalaxyl. Commercial fungicides are most appropriately used according to the manufacturer's instructions at recommended concentrations. Insecticide(s):
[00116] In one embodiment, the compositions described herein may further comprise one or more insecticides. Insecticides usable as the compositions described herein will suitably exhibit activity against a wide range of insects including, but not limited to, maggots, borers, beetle larvae, corn rootworms, seed corn larvae, flea beetles, bed bugs, aphids, leaf beetles, stink bugs and combinations thereof.
[00117] Non-limiting examples of commercial insecticides that may be suitable for the compositions described herein 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 most appropriately used according to the manufacturer's instructions at recommended concentrations. METHODS
[00118] In another aspect, methods of using the deposited strains and compositions described here are presented.
[00119] In one embodiment a method for improving plant growth is described. The method comprises contacting a plant or plant part with an inoculum of one or more bacterial strains selected from the group consisting of: the strain having deposit accession number NRRL B-50608; the strain having deposit accession number NRRL B-50609; the strain having deposit accession number NRRL B-50610; the strain having deposit accession number NRRL B-50611; the strain having deposit accession number NRRL B-50612; or a mixture of two or more of the strains.
[00120] In a particular embodiment, the inoculum may comprise one or more of the aforementioned deposited strains (e.g., including at least two of the above strains, at least three of the above strains, at least four of the above strains, up to and including including all strains above).
[00121] In one embodiment, the inoculum comprises the strain having deposit accession number NRRL B-50608. In one embodiment, the inoculum comprises the strain having deposit accession number NRRL B-50609. In one embodiment, the inoculum comprises the strain having deposit accession number NRRL B-50610. In one embodiment, the inoculum comprises the strain having deposit accession number NRRL B-50611. In one embodiment, the inoculum comprises the strain having deposit accession number NRRL B-50612.
[00122] In yet another embodiment, the step of contacting a plant or plant part with an inoculum of one or more of the deposited bacterial strains comprises contacting a plant or plant part with one or more of the compositions described herein. The inoculum(s) or compositions may be made to contact the plant or part of the plant in accordance with methods known to those skilled in the art. Non-limiting examples include in-furrow introduction, seed coating, etc. In a particular embodiment, the contacting step comprises introducing into the furrow the inoculum or compositions described herein. In a particular embodiment, the contacting step comprises introducing into the seeds (seed coating) the inoculum or compositions described herein.
[00123] In certain embodiments, the step of contacting a plant or plant part with one or more of the deposited bacterial strains comprises introducing the inoculum into the soil in an amount of 1 x 10 1 - 1 x 10 8 , more preferably 1 x 106 - 1 x 1012 colony forming units per hectare. In certain other embodiments, the step of contacting a plant or plant part with an inoculum of one or more of the deposited bacterial strains comprises introducing the deposited bacterial strains as a seed coated with 1 x 10 1 - 1 x 10 8 , more preferably 1 x 102 - 1 x 106 colony forming units per seed.
[00124] In another aspect, the method comprises growing the plants in a soil comprising one or more of the bacterial strains. The method comprises: a) treating the soil with an inoculum of one or more bacterial strains selected from the group consisting of: the strain having deposit accession number NRRL B-50608; the strain having deposit accession number NRRL B-50609; the strain having deposit accession number NRRL B-50610; the strain having deposit accession number NRRL B-50611; the strain having deposit accession number NRRL B-50612; or a mixture of two or more of the strains; and b) growing a plant in the treated soil.
[00125] In a particular embodiment, the inoculum may comprise one or more of the aforementioned deposited strains (e.g., including at least two of the above strains, at least three of the above strains, at least four of the above strains, up to and including including all strains above).
[00126] In one embodiment, the inoculum comprises the strain having deposit accession number NRRL B-50608. In one embodiment, the inoculum comprises the strain having deposit accession number NRRL B-50609. In one embodiment, the inoculum comprises the strain having deposit accession number NRRL B-50610. In one embodiment, the inoculum comprises the strain having deposit accession number NRRL B-50611. In one embodiment, the inoculum comprises the strain having deposit accession number NRRL B-50612.
[00127] The step of treating the soil with an inoculum of one or more of the deposited bacterial strains comprises treating the soil with one or more of the compositions described herein. The inoculum(s) or compositions may be introduced into the soil according to methods known to those skilled in the art. Non-limiting examples include in-furrow treatment, seed coating, etc. In a particular embodiment, the treatment step comprises treatment within the furrow of the inoculum or compositions described herein. In a particular embodiment, the treatment step comprises treating the seeds (seed coating) of the inoculum or compositions described herein.
[00128] In a particular embodiment, the step of treating the soil with a soil having one or more of the compositions described herein. In certain embodiments, the step of treating the soil with an inoculum of one or more of the deposited bacterial strains comprises treating the soil with an inoculum in an amount of 1 x 10 1 - 1 x 10 8 , more preferably 1 x 10 6 - 1 x 1012 colony forming units per hectare. In certain other embodiments, the step of treating the soil with an inoculum of one or more of the deposited bacterial strains comprises introducing the deposited bacterial strains as a seed coated with 1 x 10 1 - 1 x 10 8 , more preferably 1 x 10 2 - 1 x 106 colony forming units per seed.
[00129] In another embodiment, the method further comprises the step of planting a plant or plant part. A planting step can occur before, after or during the treatment step. In one embodiment, a planting step occurs prior to the treatment step. In another embodiment, a planting step occurs during the treatment step (e.g., a planting step occurs simultaneously with the treatment step, a planting step occurs substantially simultaneously with the treatment step, etc.). In yet another embodiment, a planting step occurs after the treatment step.
[00130] In another embodiment, the method further comprises the step of subjecting the soil to one or more agriculturally beneficial ingredients described herein. In one embodiment, the step of subjecting the soil to one or more agriculturally beneficial ingredients occurs before, during, after, or simultaneously with the treatment step. In one embodiment, the step of subjecting the soil to one or more agriculturally beneficial ingredients as described herein occurs prior to the treatment step. In another embodiment, the step of subjecting the soil to one or more agriculturally beneficial ingredients as described herein occurs during the treatment step. In yet another embodiment, the step of subjecting the soil to one or more agriculturally beneficial ingredients as described herein occurs after the treatment step. In yet another embodiment, the step of subjecting the soil to one or more agriculturally beneficial ingredients as described herein occurs concurrently with the treatment step (e.g., treating the soil with one or more of the compositions described herein, etc.).
[00131] In yet another embodiment, the invention includes a method for treating seeds comprising applying to the seeds an inoculum of one or more bacterial strains selected from the group consisting of: the strain having deposit accession number NRRL B-50608 ; the strain having deposit accession number NRRL B-50609; the strain having deposit accession number NRRL B-50610; the strain having deposit accession number NRRL B-50611; the strain having deposit accession number NRRL B-50612; or a mixture of two or more of the strains.
[00132] In a particular embodiment, the method for treating seeds may comprise one or more of the aforementioned deposited strains (e.g. including at least two of the above strains, at least three of the above strains, at least four of the above strains , up to and including all strains above).
[00133] In one embodiment, the method of treating seeds comprises applying to the seeds the strain having deposit accession number NRRL B-50608. In one embodiment, the method of treating seeds comprises applying to the seeds the strain having deposit accession number NRRL B-50609. In one embodiment, the method of treating seeds comprises applying to the seeds the strain having deposit accession number NRRL B-50610. In one embodiment, the method of treating seeds comprises applying to the seeds the strain having deposit accession number NRRL B-50611. In one embodiment, the method of treating seeds comprises applying to the seeds the strain having deposit accession number NRRL B-50612.
[00134] In yet another embodiment, the method further comprises the step of applying to the seed one or more agriculturally beneficial ingredients to the seed. In another embodiment, the method comprises applying to seeds any of the compositions described herein for seeds.
[00135] In yet another embodiment, the method comprises storing the seeds with an inoculum of at least one or more of the isolated bacterial strains in a substantially moisture-free medium for a period of time, for example, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year or more. In one aspect of the method, the seeds are the seeds of leguminous plants. In another aspect, the seeds of leguminous plants are soybean seeds.
[00136] The methods described here are potentially useful for improving growing conditions, resulting in increased phosphorus uptake and/or yield for any type of plant. In a particular embodiment, the plant is selected from the group consisting of non-legumes, legumes, Brassica spp., cereals, fruits, vegetables, nuts, flowers and grass. Particularly cereals are wheat, corn, rice, oats, rye, barley. Particularly vegetables are lentils, chickpeas, beans, soybeans, peas and alfalfa.
[00137] In another particular embodiment, the plants are selected from the group consisting of alfalfa, rice, wheat, barley, rye, oats, cotton, sunflower, peanuts, corn, potato, sweet potato, beans, peas, chickpeas, lentils, chicory, lettuce, endive, cabbage, brussels sprouts, beets, parsnips, turnips, cauliflower, broccoli, turnips, radishes, spinach, onions, garlic, eggplant, peppers, celery, carrots, zucchini, squash, zucchini squash, cucumber, apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum and sugar cane. SEED COATINGS
[00138] In another aspect, seeds are coated with one or more bacterial strains selected from the group consisting of: the strain having deposit accession number NRRL B-50608; the strain having deposit accession number NRRL B-50609; the strain having deposit accession number NRRL B-50610; the strain having deposit accession number NRRL B-50611; the strain having deposit accession number NRRL B-50612; or a mixture of two or more of the strains.
[00139] In a particular embodiment, the seed(s) are coated with one or more of the aforementioned deposited strains (e.g., including at least two of the above strains, at least three of the above strains, at least four of the above strains, up to and including all of the above strains).
[00140] In one embodiment, the seed(s) are coated with the strain having deposit accession number NRRL B-50608. In one embodiment, the seed(s) are coated with the strain having deposit accession number NRRL B-50609. In one embodiment, the seed(s) are coated with the strain having deposit accession number NRRL B-50610. In one embodiment, the seed(s) are coated with the strain having deposit accession number NRRL B-50611. In one embodiment, the seed(s) are coated with the strain having deposit accession number NRRL B-50612.
[00141] In one embodiment, the seeds can be treated with any of the composition(s) described herein in various ways, but preferably by spraying or dripping. Spray or drip treatment can be conducted by formulating compositions described herein and spraying or dripping the composition(s) onto a seed(s) through a continuous treatment system (which is calibrated to deliver the treatment at a preset in proportion to the continuous flow of seed), such as a handler-type drum. Batch systems, in which a predetermined batch size of seeds and composition(s) as described herein are supplied to a mixer, may also be employed. Systems and apparatus for carrying out these processes are commercially available from various suppliers, for example from Bayer CropScience (Gustafson).
[00142] In another embodiment, the treatment involves coating the seeds. Such a process involves coating the inside wall of a round container with the composition(s) described herein, adding seeds, then rotating the container to cause the seeds to contact the wall and the composition(s), a process known in the art as "container lining". Seeds can be coated using combinations of coating methods. Immersion typically involves the use of liquid forms of the described compositions. For example, seeds can be soaked for about 1 minute to about 24 hours (e.g. for at least 1 min, 5 min, 10 min, 20 min, 40 min, 80 min, 3 h, 6 h, 12 h, 24 h).
[00143] In certain embodiments, the seed(s) coated with one or more of the compositions described herein will comprise 1 x 10 1 - 1 x 10 8 , more preferably 1 x 10 2 - 1 x 10 6 forming units of colonies of one or more of the bacterial strains deposited per seed. EXAMPLES
[00144] The following examples are given for illustrative purposes and are not intended to limit the scope of the invention as claimed herein. Any variations in the exemplified examples that occur to the verse are intended to be within the scope of the present invention. Materials and methods Biological Material Deposit
[00145] The following biological materials were deposited under the terms of the Budapest Treaty with the Microbial Genomics and Bioprocessing Research Unit (NRRL) National Center for Agricultural Utilization Research 1815 N. University Street, Peoria, IL 61604, USA and received the following numbers access: Table 1: Deposit of Biological Material

[00146] The strains were deposited under conditions assuring that access to the culture would be available during the pendency of this patent application to a person determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR §1.14 and 35 USC §122. The deposit represents a pure culture of the deposited strain. Deposit is available as required by foreign industrial property laws in countries where counterparties to the relevant order or products thereof are deposited. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject of the invention in derogation of patent rights granted by governmental action.Media Table 2: Components of medium G16.
Table 3: Trace element charge stored at 4°C for up to 6 months.
*Trace elements are added with all other components before sterilization. Table 4: Loading of vitamin-sterilized filter followed by storage at 4°C for up to 6 months.
**Vitamins are added after the medium has been sterilized and cooled, typically at the time of inoculation. Table 5: Components of Yeast Extract Mannitol Medium (YEM).
Example I: Determine 99.99% extermination rate for USDA 532C
[00147] The following experiment(s), consisting of three (3) studies, was(were) performed to determine the 99.99% kill rate for parental strain Bradyrhizobia japonicum USDA 532C .
[00148] The USDA parental strain 532C was grown in two disposable culture tubes of 10ml G16 (Tables 2-4) and YEM (Table 5) (VWR, 18x150mm, #47729-583) for two days and collected to obtain a concentration of larger cell. This was achieved by combining both culture tubes into one tube and concentrating the cells down to 2L. Approximately fifty soybean seeds (variety Stine RR 1108-4) were surface sterilized in a 50 ml sterile disposable centrifuge tube (Fisherbrand, #06-443-18) containing 5% household bleach solution for 30 seconds and rinsed with sterile deionized (DI) water. The sterilization step was repeated for five times. The seeds were immediately placed in a sterilized Petri dish and dried under a laminar hood. Once the seeds were completely dried and transferred to a 250 ml beaker, 1.5 ml of the concentrated USDA parental strain 532C culture was added to the seeds. The seeds were shaken in the beaker to evenly coat the seeds and allowed to dry under the hood. The beaker, containing the seeds, was wrapped with blue sterilization paper, and left in the hood until the experiment was completed. Time points were taken at time zero, every other day for a week, and every week until complete cell death occurred. The results are given in table 6.Table 6: CFU per seed and percentage extermination rate for study 1.

[00149] As shown in Table 6 the initial CFU per seed for USDA 532C parental strain was 3.06 x 108 and on day 37 the CFU was 2.64 x 108. The time percent kill rate from 0 to 37 days was calculated to be 99.99%.
[00150] The procedure was repeated except G16 was used as the initial growth medium. The results are given in table 7. Table 7: CFU per seed and percentage extermination rate for study 2.

[00151] As shown in Table 7, when G16 was used as the initial growth medium, it took 29 to 37 days for the kill rate to reach 99.99%.
[00152] A third desiccation study was completed to determine whether G16 and YEM media affected the desiccation rate of the USDA 532C parental strain. The results are given in tables 8 and 9 respectively.Table 8: CFU per seed and percentage kill rate for USDA parental strain 532C grown in G16 medium.
Table 9: CFU per seed and percentage kill rate for USDA 532C parental strain grown in YEM medium.

[00153] As shown in Tables 8 and 9, there is no difference in desiccation rate for USDA 532C parental strain when grown in G16 or YEM. A kill rate of 99.99% was observed for the third study at approximately 28 days which is similar to what was observed in studies one (1) and two (2) above. Example II: Determining the kill rate of USDA 532C using ethyl methanesulfonate (EMS)
[00154] The following experiment(s) was(were) performed to determine the rate of application of the mutagen, ethyl methanesulfonate (EMS) which would have given a percentage kill rate of 99.9 -99.99 for the USDA 532C parental strain. This rate determination will become a part of the mutagenesis protocol used to generate putative desiccation resistant mutants although the mutagenesis method may evolve to greater efficiency. Inoculum preparation:
[00155] Parental strain USDA 532C was grown in six disposable 10ml YEM culture tubes for two days and 5ml of the culture was inoculated into four 250 ml flasks containing 50 ml of YEM medium. The flasks were incubated for two days with a shaker at 30°C. Culture vials were subsequently centrifuged in sterile 50 ml disposable tubes at 8000 rpm for ten minutes in a Sorvall RC 6 Plus® centrifuge and combined into one tube. The pellet was resuspended in 4 ml of fresh YEM medium and separated into four 1.5 ml microcentrifuge tubes. The tubes should each represent different application rates used for the mutagenesis process. Mutagenesis process:
[00156] Once the culture was aliquoted into separate tubes and the mutagen EMS (Sigma, C3H8O3S, FW 124.16, #M0880-1G) added to each tube, the tubes were vortexed vigorously and placed in an empty 250 ml bottle. The flask containing the reaction tubes was incubated for 30 minutes at 30°C on a shaker. Immediately following the incubation period, the tubes were washed five times with 0.16 M sodium thiosulfate solution (STS, FisherChemical, Na2S2O3*5H2O, FW 248.18, #S445-3)) to inactivate the mutagen. After washing, the cells in the reaction tubes were sheared with a 21 gauge syringe needle (BD 1ml 21G1 Latex Free Syringe PrecisionGlide® Needle, 0.8mmx25mm, #309624) and the dilutions were completed and placed on YEMA plates. Cell counts were available after five days of incubation at 30°C and the percent kill rate of EMS application was calculated. To calculate the percentage kill rate for each application rate, the following equation was used: ([cell count of 0μl EMS (control) - (cell count of μl EMS (treatment)) +■ cell count of 0μl EMS (control)] x 100%). All other experiments following this experiment used this equation to calculate the percent kill rate. Results are given in Table 10. Table 10: Initial EMS rates to determine upper limit of mutagenesis for parental strain USDA 532C

[00157] As shown in table 10, the initial EMS rates used are forcm 2 μl, 3 μl, 10μl g 100μL P«q already differ>c fgpVtg 0μl. 1μl. and 10μl GOU, the cu 322μn GOU tguwltou at 100% kill.
[00158] The experiments were repeated and refined to determine the acceptable kill rate. See tables 11-17. Table 11: Refine EMS rates to determine 99.9% kill rate for USDA 532C parental strain

[00159] From the initial discovery, the amount of EMS used was nkokVcfc rctc 2μn. 37μl. 27μl g 72μn GOUo Eqoq oquVtcfq nc Vcdgnc 33. c Vczc fg gzVgtminko rgtegpVwal fok fg 32.39' c 32.;2% rctc 37μl g 27μl crlkec>õgu fg GOU g 322' rctc 72μl0 Additional refinement of EMS rates Table 12 for parental strain USDA 532C

[00160] Hok fgvgtokpcfo c rctvkt fc vcdglc 33 swg 47μl GOU ckpfc gtc too low for a kill rate. Application quantities for Vcdglc 34 fotcm fg 2μl. 47μl. 57μl. g 72μl GOUo C crlkec>«qc 47μl GOU Vgo a higher kill rate than the results in table 11 due to decreased washing; thus, the extermination rate was higher than expected. However, the kill rate was still very low at 88.58% gzVgtminko mgumo swcpfo 57μl GOU fotcm wucfoUo See Table 12. Table 13: Further refinement of EMS application rates for USDA 532C parental strain

[00161] C swcpvkfcfg fg GOU wucfc fok cwmgpvcfc rctc 62μl. 67μl. G 72μl GOUo Cu Vczcu fg foug GOU tguwlVctcm gm hckzcu fg gzVgtmipko percentage of 82.26%-99.99%.See Table 13. Table 14: Repetition application rate used in table 13.

[00162] The results given in table 13 were repeated again in table 14 using the same EMS rates and at this time the rgtegpvwcn hqk extermination of ;;,83' rctc 42μl GOU and ;;.;5' rctc 47μl GOU g 322' rctc 72μn GOUo C Vczc fe ezVetoínkq feuejcfc of 99.9% was observed Swcpfq 67μn GOU hqk wucfq fe oqfq swe c crnkec>«quetá tereVkfCo See Table 15.Table 15: Repetition application rate used in table 14.

[00163] C Vczc fe crnkec>«q fe 67μn GOU hqk fwrnkecfc rctc determine if the results in table 14 were repeatable. The rate of ezVetoínkq rctc 67μn GOU hqk fe 99.98%. See Table 15. Example III: Mutagenesis
[00164] The following experiment(s) was(were) performed to generate putative desiccation resistant mutants of USDA 532C parental strains using classical chemistry, eg mutagenesis. Inoculum preparation:
[00165] A cell suspension of the USDA 532C parental strain was made by taking a loop of cells from a fresh plate of USDA 532C using a 10ul sterile plastic loop (Fisherbrand, #22-363-600) and mixing the cells. in 1 ml of sterile deionized (DI) water in a 1.5 ml disposable microcentrifuge tube. The cell suspension was inoculated into two 250ml flasks containing 50ml of YEM medium to obtain a final optical density (OD) OD600nm of 0.01. The flasks were incubated at 30°C for three days and the cultures from the two flasks were combined. The culture was centrifuged for twenty minutes at 8,000 rpm in a Sorvall RC 6 Plus centrifuge. The supernatant was discarded and the pellet was replaced in 30ml of DI water. OD was taken from the concentrated culture and inoculated into ten 250ml flasks containing 50ml of YEM medium at OD=0.05. These flasks were incubated at 30°C with a shaker for two days before the culture was used for mutagenesis. Mutagenesis process:
[00166] The cultures from the ten flasks were combined in a 1L centrifuge bottle. The optical density of the combined cultures was recorded and the cultures were centrifuged for 20 minutes at 8000 rpm in the Sorvall RC 6 Plus ® centrifuge. Most of the supernatant was discarded leaving approximately 30ml of the supernatant in the centrifuge flask. The supernatant was mixed with the pellet and transferred into a 50 ml sterile disposable centrifuge tube. The OD of the concentrated culture was taken and recorded. 1ml of the concentrated culture was placed in six 1.5ml disposable microcentrifuge tubes. The microcentrifuge tubes were centrifuged and the supernatant was discarded. This was repeated three more times or until the bead size had reached the 0.1ml mark in the microcentrifuge tube. Cells were mixed well with 1ml of fresh YEM medium using a sterile 21 gauge syringe needle with 1ml before addition of the mutagen, ethyl methanesulfonate (EMS). The mutagen rates added to each tube contained a high dose and a low dose with medium dosages between the high and low doses as indicated in experiment II.
[00167] Immediately after the addition of EMS, the reaction tubes were placed in an empty 250 ml bottle and incubated at 30°C for 30 minutes. After incubation, the reaction tubes were centrifuged for one minute at 13,200 rpm using the Eppendorf 5415D centrifuge. The supernatant from the reaction tubes was discarded. The mutagen in the reaction tubes was inactivated by washing five times with 1ml of 0.16M sodium thiosulfate (STS) and vigorously vortexing the tubes. For each wash cycle, the reaction tubes were centrifuged after vortexing and the supernatant was discarded. After the fifth time, the reaction tubes were all combined into a 15ml disposable tube for use in the enrichment process. Example IV: Enrichment and Desiccation
[00168] The following experiment(s) was(were performed) to enrich and desiccate the mutated cells from the USDA parental strain 532C to eliminate wild-type escapes and increase the putative mutant population and make it easier isolate the mutant(s) that show(s) characteristics resistant to desiccation.
[00169] The USDA parental strain 532C was subjected to the mutagenesis process mentioned in example III. The mutated population of the USDA 532C parental strain was enriched by inoculating 0.5ml of the reaction into two 50ml YEM flasks and incubating the cells for two days at 30°C with shaking. After two days, the cultures were desiccated by coating the cells on soybean seeds and membrane filters and subjected to drying conditions. The crop from the enrichment step was adjusted to OD600nm of 0.5 before it was used to coat both soybean seeds and membrane filters. Soybean seed coating:
[00170] Forty sterilized soybean seeds were coated with 0.5ml of the culture. The seeds were placed in a 100 ml sterile beaker to dry under the laminar hood and covered with autoclave paper. Triplicate seed samples were taken to obtain an initial CFU of the seeds. For each sample, three seeds were placed in a 15ml disposable tube containing 5ml of sterile DI water and allowed to expand in the tube for approximately two hours before the suspension was serially diluted and sprinkled onto the YEMA plates. The remaining seeds left in the covered beaker were placed under the hood for four days before the seeds were enriched.
[00171] To enrich the seeds, twenty seeds were placed in a 250ml bottle containing 50ml of fresh YEM. A final CFU was also taken when the seeds were enriched to determine the percent kill rate for the cell population. The same sampling was completed for the second time point as the initial time point. After the culture containing the seeds was incubated for two days, the culture was harvested by removing any seed residue by allowing the residue to settle before removing the supernatant. The culture supernatant was centrifuged and the pellet washed with sterile DI water before the culture was used to coat new sets of soybean seeds. This process was repeated until the calculated percentage kill rate of the cell culture was less than 80%. Once 80% was obtained, the cell population was ready for isolation of the putative mutants for confirmation experiment. Membrane filter coating:
[00172] To control soybean seed inconsistency and contamination problems, membrane filters were used as an alternative means for cell coating. For membrane filters, 1ml of the culture was used to coat both durapore membrane filters (Millipore, 0.22 μm, PVDF, #GVWP02500) and isopore (Millipore, 0.4 μm, polyocarbonate, # HTTP02500). For each filter type, fifteen filters were coated using a 25mm Easy Pressure Syringe Filter Holder (VWR, #28144-109) and the filters were placed in a sterile Petri dish containing two pieces of Whatman qualitative 125mm sterile paper. (Whatman, #1001125). Once the filters were dried under the laminar hood, triplicates of starting CFU were taken for each filter type by placing one filter in a 15ml disposable tube containing 5ml of sterile DI water and vortexing. After two hours of soaking in the 15 ml tube, the filter suspension was diluted and placed on the YEMA plates. After the filters had dried under the hood for three days, eight filters were added to a 250ml flask with 50ml of fresh YEM and incubated at 30°C for three days.
[00173] A final CFU was taken at the same time the filters were enriched to obtain the percentage kill rate calculation. The same method was used for the final CFU as the initial CFU. The coating and drying process was repeated until the percentage kill rate was less than 80%. After 80% were obtained, single colony isolates were selected for further confirmation. Example V: Confirmation of Putative Mutants
[00174] The following experiment(s) was(were) to confirm putative desiccation resistant mutants by comparing their ability to survive on seeds after seed application with the original parental strain Bradyrhizobium strain japonicum USDA 532C.
[00175] When 80% kill rate was observed for seeds or filters, single colonies were taken randomly from the final time point and putative mutants from a mutagenesis set were analyzed for desiccation tolerance trait. The twenty single colonies taken from each set of mutagenesis results were individually grown in a 250ml flask containing 50ml of YEM medium. Each putative mutant strain was incubated at 30°C with shaking for three days and the OD for each strain was adjusted to 0.5. Each strain was used to coat thirty non-sterile soybean seeds with 0.5ml of culture in a 100ml beaker covered by autoclave paper. Time points were taken at T=0, T=3, and T=7 days for the first cycle. Triplicate seed samples were taken for each time point where each sample consisted of three seeds placed in 5ml of sterile DI water in a 15ml disposable tube. The seeds were allowed to expand for two hours before each sample was diluted and placed on YEMA plates. After comparing the amount of cells recovered from each time point to the USDA 532C parental strain, any strain that performed better than the parental strain was subjected to a second confirmation cycle. See figure 1. For the second cycle, the strains that had the best desiccation tolerance compared to the wild type were tested for desiccation again. See Figure 2. Time points were taken at T=0, T=7, and T=14 days. The pool of putative mutants from the second cycle was further confirmed for desiccation tolerance two more times. See Figs. 3-6.
[00176] Twenty putative mutants of the USDA 532C parental strain were isolated and screened for desiccation resistance. See Figs. 1-3. Of the twenty putative strains tested, five putative mutant strains were confirmed for their desiccation tolerance traits when compared to the desiccation tolerance of the parental strain, Bradyrhizobiumjaponicum USDA 532C. See Figs 4-6. Example VI: Greenhouse Test of Confirmed Mutant Strains
[00177] The following experiment(s) was(were performed) to test the putative mutant strains in the greenhouse to test the performance of the mutant strains against the performance of the parental strain, USDA 532C.
[00178] The mutant strains having the best desiccation tolerance compared to the USDA 532C parental strain were tested in the greenhouse for performance against the USDA 532C parental strain. Mutant and parental strains were cultured in 50ml of YEM for two days prior to seed coating. Each strain was planted three different times; T=0, T=7, and T=14 days after seed coating. All time points were set at the same time, but the seeds were planted at the specified times. To set the time points, thirty soybean seeds were coated with 0.5ml of culture at OD600nm = 0.5 in a 100ml beaker and the day time point T=0 was allowed to sit under the hood for 30 minutes before planting. The other two time points were allowed to dry completely and covered with autoclave paper. The seeds of the last two time points were planted at a later date. At each time point, two seeds were planted per pot for ten pots per strain. The seeds left were used to make a CFU for comparison with T=0. After nine weeks of greenhouse growth, soybean pods were harvested for each plant from each time point and dry weights were analyzed for statistical significance.
[00179] When the soybean pod weights of the mutant strains were compared with the parental strain at any time points, no statistical significance was noted at 95% confidence. This indicates that a performance difference was not noticed between the mutant strains and the parental strain that should affect the production of soybean pods when the mutant strains were used to coat soybean seeds. SUMMARY PARAGRAPHS
[00180] The present invention is defined in the claims and accompanying description. Additional aspects of the present invention are presented herein by way of numbered paragraphs. 1. A biologically pure culture of a strain of Bradyrhizobium japonicum selected from the group consisting of: the strain having deposit accession number NRRL B-50612; the strain having deposit accession number NRRL B-50611; the strain having deposit accession number NRRL B-50610; the strain having deposit accession number NRRL B-50609; the strain having deposit accession number NRRL B-50608, or a combination of at least two or more of the strains. 2. The strains of paragraph 1, wherein said strains have a superior resistance to desiccation. 3. The strains of paragraph 2 wherein said resistance to desiccation are compared with said resistance to desiccation of a parental strain of said isolated strains, eg parental strain Bradyrhizobium japonicum USDA 532C. 4. The strains of paragraph 2, wherein said superior resistance to desiccation includes an increased bacterial survival rate in a substantially moisture-free medium when said survival rate of said isolated strains of Bradyrhizobium is compared with said survival rate of a parental strain of said isolates, e.g., parental strain Bradyrhizobium japonicum USDA 532C, over a period of time, e.g. at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year or more. 5. The strains of any one of paragraphs 2-4, wherein said increased survival rate in said substantially moisture-free medium includes an increased bacterial survival rate in an environment that is at least 70% moisture-free, for example, at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, up to 100% moisture-free medium, when said survival rate of said isolated strain(s) of Bradyrhizobium is compared to said survival rate of a parental strain of said isolates, for example, parental strain Bradyrhizobium japonicum USDA 532C. 6. A composition comprising one or more of said bacterial strain(s) isolated according to paragraphs 1-5 and an agriculturally appropriate carrier. 7. The composition of paragraph 6, wherein the composition includes at least one agriculturally beneficial ingredient. 8. The composition of paragraph 7, wherein said at least one agriculturally beneficial ingredient includes one or more plant signaling molecules. 9. The composition of paragraph 8, wherein the plant signaling molecule is a lipo-chitooligosaccharide (LCO). 10. The composition of paragraph 9, where the LCO is synthetic. 11. The composition of paragraph 9, wherein the LCO is recombinant. 12. The composition of paragraph 9, where the LCO is naturally occurring. 13. The composition of paragraph 9, wherein the LCO is obtained from a species of Rhizobia selected from among Rhizobium spp., Sinorhizobium spp. and Azorhizobium spp. 14. The composition of paragraph 9, wherein the LCO is obtained from Bradyrhizobium japonicum. 15. The composition of paragraph 9, wherein the LCO is obtained from an arbuscular mycorrhizal fungus. 16. The composition of paragraph 8, wherein the plant signaling molecule is a chitinous compound. 17. The composition of paragraph 16, wherein the chitinous compound is a chito-oligomer (CO). 18. The composition of paragraph 17, where the CO is synthetic. 19. The composition of paragraph 18, wherein the CO is recombinant. 20. The composition of paragraph 18, where the CO is naturally occurring. 21. The composition of paragraph 8, wherein the plant signaling molecule is a flavonoid. 22. The composition of paragraph 21, wherein the flavonoid is selected from the group consisting of luteolin, apigenin, tangerine, quercetin, kaempferol, myricetin, fisetin, isorhamnetin, pachipodol, rhamnazine, hesperetin, naringenin, formononetin, eriodictiol, homoeriodictiol, taxifolin , dihydroquercetin, dihydrokaempferol, genistein, daidzein, glycitein, catechin, gallocatechin, catechin 3-gallate, gallocatechin 3-gallate, epicatechin, epigallocatechin, epicatechin 3-gallate, epigallocatechin 3-gallate, cyanidin, delphinidine, malvidin, pelargonidin , peonidin, petunidin, or derivatives thereof. 23. The composition of paragraph 8, wherein the plant signaling molecule is jasmonic acid or a derivative thereof. 24. The composition of paragraph 8, wherein the plant signaling molecule is linoleic acid or a derivative thereof. 25. The composition of paragraph 8, wherein the plant signaling molecule is linolenic acid or a derivative thereof. 26. The composition of paragraph 8, wherein the plant signaling molecule is a carrikin. 27. The composition of any one of paragraphs 8-26, wherein the composition includes at least two different plant signaling molecules. 28. The composition of paragraph 27, wherein the agriculturally beneficial ingredient is a herbicide, insecticide or a fungicide. 29. The composition of paragraph 27, wherein the agriculturally beneficial ingredient is at least one phosphate-solubilizing microorganism. 30. The composition of paragraph 29, wherein the at least one phosphate-solubilizing microorganism comprises a strain of the Penicillium fungus. 31. The composition of paragraph 30, wherein the at least one phosphate-solubilizing microorganism comprises a strain of P. bilaiae. 32. The composition of paragraph 31, wherein the P. bilaiae strain is selected from the group consisting of NRRL 50162, NRRL 50169, ATCC 20851, ATCC 22348, and ATCC 18309. 33. The composition of paragraph 30, wherein the at least one phosphate solubilizing microorganism comprises a strain of P. gaestrivorus. 34. The composition of paragraph 33, wherein the P. gaestrivorus strain is NRRL 50170. 35. A method for treating seeds comprising applying to said seeds an inoculum of one or more of the bacterial strain(s) isolated(s) according to paragraph 1. 36. Method according to paragraph 35, wherein a composition according to any one of paragraphs 6-34 is applied to a seed. 37. The method of any one of paragraphs 35-36, wherein the method further comprises the step of storing said treated seeds in a substantially moisture-free medium for a period of time, e.g., at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year or more. 38. Method according to paragraph 37, wherein said seeds are seeds of leguminous plants. 39. Method according to paragraph 38, wherein said seeds of leguminous plants are soybean seeds. 40. A method of improving plant growth, comprising applying to the plants, plant seeds or soil surrounding the plants or plant seeds a composition according to any one of paragraphs 6-34. 41. Method according to paragraph 40, wherein said seeds are seeds of leguminous plants. 42. Method according to paragraph 41, wherein said seeds of leguminous plants are soybean seeds. 43. A method for enhancing the growth of a plant or part of a plant comprising contacting a plant or part of a plant with an inoculum of one or more of the strains of paragraph 1. 44. The method of paragraph 43, wherein the method further comprises the step of subjecting the soil to one or more agriculturally beneficial ingredients. 45. The method of paragraph 43, wherein the treatment step comprises introducing the inoculum of one or more strains of paragraph 1 as a composition. 46. The method of paragraph 43, where the composition is the composition of any one of paragraphs 6-34. 47. The method of paragraph 43, in which the plant part is a plant seed. 48. Method according to paragraph 47, wherein said seeds are seeds of leguminous plants. 49. Method according to paragraph 48, wherein said seeds of leguminous plants are soybean seeds. 50. A method of improving the growth of a plant or plant part comprising treating a soil with an inoculum of one or more of the strains of paragraph 1; and growing a plant or part of the plant in the treated soil. 51. The method of paragraph 50, wherein the method further comprises the step of planting a plant or part of the plant before, during or after the treatment step. 52. The method of paragraph 50, wherein the method further comprises the step of subjecting the soil to one or more agriculturally beneficial ingredients. 53. The method of paragraph 50, wherein the treatment step comprises introducing the one or more strains of paragraph 1 with a composition. 54. The method of paragraph 50, where the composition is a composition of any one of paragraphs 6-34. 55. Seeds coated with a composition of any one of paragraphs 6-34.
[00181] The invention described and claimed herein should not be limited in scope by the specific embodiments disclosed herein, as these embodiments are intended to be illustrations of various aspects of the invention. Any equivalent embodiments are intended to be within the scope of the present invention. Indeed, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the event of conflict, the present description, including definitions, will serve as a control.
[00182] Various references are cited herein, the disclosures of which are incorporated herein by reference in their entirety.

权利要求:
Claims (19)
[0001]
1. Biologically pure culture of a mutant strain of Bradyrhizobium japonicum, characterized in that it is selected from the group consisting of: the strain having deposit accession number NRRL B-50612; the strain having deposit accession number NRRL B-50611; the strain having deposit accession number NRRL B-50610; the strain having deposit accession number NRRL B-50609; the strain having deposit accession number NRRL B-50608, or a combination of at least two or more of the strains.
[0002]
2. Composition, characterized in that it comprises one or more of said isolated bacterial strain(s) as defined in claim 1 and an agriculturally appropriate carrier.
[0003]
A composition according to claim 2, characterized in that the composition includes at least one agriculturally beneficial ingredient.
[0004]
A composition according to claim 3, characterized in that said at least one agriculturally beneficial ingredient includes one or more plant signaling molecules.
[0005]
5. Composition according to claim 4, characterized in that the plant signaling molecule is a lipochitooligosaccharide (LCO).
[0006]
6. Composition according to claim 5, characterized in that the LCO is synthetic.
[0007]
7. Composition according to claim 5, characterized in that the LCO is recombinant.
[0008]
8. Composition according to claim 5, characterized in that the LCO is naturally occurring.
[0009]
9. Composition according to claim 4, characterized in that the plant signaling molecule is a chitinous compound.
[0010]
10. Composition according to claim 9, characterized in that the chitinous compound is a chito-oligomer (CO).
[0011]
11. Composition according to claim 9, characterized in that the CO is synthetic.
[0012]
12. Composition according to claim 9, characterized in that the CO is recombinant.
[0013]
13. Composition according to claim 9, characterized in that the CO is naturally occurring.
[0014]
14. Composition according to claim 4, characterized in that the plant signaling molecule is a flavonoid.
[0015]
15. Seeds, characterized in that they are coated with a composition comprising one or more mutant strains of Bradyrhizobium japonicum as defined in claim 1.
[0016]
16. Method for treating seeds, characterized in that it comprises applying to said seeds an inoculum of one or more of the isolated bacterial strain(s) according to claim 1 or a composition as defined in any one of claims 1 to 15.
[0017]
17. Method according to claim 16, characterized in that a composition as defined in any one of claims 2 to 15 is applied to a seed.
[0018]
18. Method according to claim 16, characterized in that said seeds are seeds of leguminous plants.
[0019]
19. Method according to claim 18, characterized in that the seeds of leguminous plants are soybean seeds.

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法律状态:
2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-08-06| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-10-05| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2022-01-11| B09W| Correction of the decision to grant [chapter 9.1.4 patent gazette]|Free format text: A NOTIFICACAO DE DEFERIMENTO FOI EFETUADA COM INCORRECAO NO QUADRO 1. |

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2022-01-25| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 17/12/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161576470P| true| 2011-12-16|2011-12-16|

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US61/576,470|2011-12-16|

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PCT/US2012/070036|WO2013090884A1|2011-12-16|2012-12-17|Bradyrhizobium strains|

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