NZ622044B2 - Use of lipochito-oligosaccharides and/or chito-oligosaccharides in combination with phosphate-solubilizing microorganisms to enhance plant growth - Google Patents

Abstract

Disclosed are methods of enhancing plant growth, comprising a) treating plant seed with an effective amount of at least one phosphate solubilizing microorganism such as a strain of the fungus Penicillium, and b) treating the seed or plant that germinates from the seed with an effective amount of at least one lipo chitooligosaccharide (LCO) and/or at least one chitoogliosaccharide (CO), wherein upon harvesting the plant exhibits at least one of increased plant yield measured in terms of bushels/acre, increased root number, increased root length, increased root mass, increased root volume and increased leaf area, compared to untreated plants or plants harvested from untreated seed. least one lipo chitooligosaccharide (LCO) and/or at least one chitoogliosaccharide (CO), wherein upon harvesting the plant exhibits at least one of increased plant yield measured in terms of bushels/acre, increased root number, increased root length, increased root mass, increased root volume and increased leaf area, compared to untreated plants or plants harvested from untreated seed.

Description

USE OF LIPOCHITO-OLIGOSACCHARIDES AND/OR CHITO-OLIGOSACCHARIDES IN COMBINATION WITH PHOSPHATE-SOLUBILIZING MICROORGANISMS TO ENHANCE PLANT GROWTH BACKGROUND OF THE INVENTION The symbiosis between the gram—negative soil bacteria, Rhizobiaceae and Bradyrhizobiaceae, and legumes such as soybean, is well nted. The biochemical basis for these relationships includes an ge of molecular signaling, wherein the plant-to-bacteria signal compounds include flavones, isoflavones and flavanones, and the bacteria—to—plant signal nds, which include the end products of the expression of the Bradyrhizobial and Rhizobial nod genes, known as |ipo-chitooligosaccharides (LCOs). The symbiosis between these bacteria and the legumes enables the legume to fix atmospheric nitrogen for plant growth, thus obviating a need for nitrogen fertilizers. Since nitrogen fertilizers can significantly increase the cost of crops and are associated with a number of polluting effects, the agricultural industry continues its efforts to exploit this biological onship and develop new agents and s for improving plant yield without increasing the use of en-based fertilizers.

US. Patent 6,979,664 teaches a method for enhancing seed germination or seedling emergence of a plant crop, comprising the steps of providing a composition that comprises an effective amount of at least one lipo-chitooligosaccharide and an agriculturally suitable carrier and applying the composition in the immediate vicinity of a seed or seedling in an effective amount for enhancing seed ation of seedling emergence in comparison to an untreated seed or seedling.

Further development on this concept is taught in , directed to combinations of at least one plant r, namely an LCD, in combination with a fungicide, insecticide, or combination thereof, to enhance a plant characteristic such as plant stand, growth, vigor and/or yield. The compositions and methods are taught to be applicable to both legumes and gumes, and may be used to treat a seed (just prior to planting), seedling, root or plant.

Similarly, teaches itions for ing plant growth and crop yield in both legumes and non-legumes, and which contain LCOs in combination with another active agent such as a chitin or chitosan, a flavonoid compound, or an herbicide, and which can be applied to seeds and/or plants concomitantly or sequentially. As in the case of the '899 Publication, the '958 Publication teaches treatment of seeds just prior to planting.

More recently, Halford, "Smoke Signals," in Chem. Eng. News (April 12, 2010), at pages 37-38, reports that karrikins or butenolides which are contained in smoke act as growth stimulants and spur seed germination after a forest fire, and can invigorate seeds such as corn, tomatoes, lettuce and onions that had been stored. These molecules are the subject of US. Patent 7,576,213.

In order to maintain healthy growth, plants must also extract a variety of elements from the soil in which they grow. These elements e phosphorus and the so-called micro-nutrients (e.g., copper, iron and zinc), but many soils are deficient in such elements or they contain them only in forms which cannot be readily taken up by plants (it is generally believed that essential elements cannot be readily taken up by plants unless they are present in dissolved form in the soil).

To counteract such deficiencies, sources of the deficient ts are commonly d to soils in order to improve growth rates and yields obtained from crop plants. For example, phosphates are often added to soil to counteract a lack of ble phosphorus. Phosphate added to the soil as a cial fertilizer (e.g., mmonium phosphate or triple-superphosphate) is y plant available, but is rapidly converted in soil to relatively unavailable forms. It has been ted that only 10 to 30% of ate fertilizer is used by the plant in the year it is applied, and one-third to one-half of the phosphate fertilizer applied may never be recovered by the plant.

U.S. Patent 5,026,417 describes an isolated strain of Penicillium bi/aiae which is capable of improving the uptake of phosphorous by plants when applied to the soil.

There is, however, still a need for systems for improving or enhancing plant growth.

BRIEF SUMMARY OF THE INVENTION A first aspect of the present invention is directed to a method of enhancing plant growth, comprising: a) ng plant seed with an ive amount of at least one phosphate solubilizing microorganism, and b) treating the seed and/or the plant that germinates from the seed with an effective amount of at least one lipo-chitooligosaccharide (LCO) and/or at least one ligosaccharide (CO), wherein, upon harvesting, the plant exhibits at least one of increased plant yield measured in terms of bushels/acre, increased root number, increased root length, increased root mass, increased root volume and increased leaf area, ed to untreated plants or plants harvested from untreated seed. [0001A] A second aspect of the present invention is directed to a plant seed treated in accordance with the method of the first aspect. [0001B] A third aspect of the present invention is directed to a package comprising the plant seed of the second aspect. [0001C] One aspect of the present invention is directed to a e, comprising a first container and a second container, wherein the first container comprises at least one phosphate solubilizing microorganism and a first agronomically able carrier, and wherein the second container comprises at least one lipo-chitooligosaccharide (LCO) and/or at least one chitooligosaccharide (CO) and a second agronomically acceptable carrier, wherein the first and second agronomically acceptable carriers may be the same or different, and wherein the at least one phosphate lizing microorganism and the at least one LCO and/or at least one CO are each t in the first and second containers respectively, in an amount effective to enhance plant growth when applied to a plant or seed thereof as ed to an untreated plant or seed thereof. As used herein, the term "untreated" refers to seed or plants that are not treated with either active (i.e., the phosphate solubilizing microorganism, the LCO, or the CO).

Another aspect of the present invention is directed to a e, comprising a first container and a second container, wherein the first container comprises at least one phosphate solubilizing microorganism sing a strain of the fungus Penicillium and a first agronomically acceptable carrier, and wherein the second container comprises at least one lipo-chito-oligosaccharide (LCO) and/or at least one chito-oligosaccharide (CO) and a second mically acceptable carrier, 11245245_1 wherein the first and second agronomically acceptable carriers may be the same or different, and wherein the at least one phosphate solubilizing microorganism and the at least one LCO and/or at least one CO are each present in the first and second containers tively, in an amount effective to enhance plant growth when applied to a plant or seed thereof as compared to an untreated plant or seed thereof.

Another aspect of the present invention is directed to an agronomical composition for treatment of a plant or seed thereof, comprising (a) at least one phosphate solubilizing microorganism and (b) at least one lipo-chitooligosaccharide (LCO) and/or at least one a chitooligosaccharide (CO), each present in an amount effective to e plant growth when applied to a plant or seed f as compared to an untreated plant or seed thereof. 11245245_1 A related aspect of the invention is directed to an agronomical composition for treatment of a plant or seed thereof, comprising (a) at least one phosphate solubilizing microorganism sing a strain of the fungus Panic/Ilium, and (b) at least one LCO and/or at least one CO, and (c) an agronomically acceptable carrier, wherein at least one phosphate solubilizing microorganism and the at least one LCO and/or at least one CO are each t in an amount effective to enhance plant growth when applied to a plant or seed thereof as compared to an untreated plant or seed thereof.

Another related aspect of the present ion is ed to plant seed treated with (e.g., having coated or disposed thereon) (a) at least one phosphate solubilizing microorganism, and (b) at least one LCO and/or at least one CO, each in an amount to enhance plant growth when applied to a the seed as compared to seed thereof. The phosphate solubilizing microorganism and the LCO and/or the CO may be applied to the seed via the same or ent compositions.

Packages containing the plant seed are also provided.

Another related aspect of the present invention is directed to plant seed treated with (e.g., having coated or disposed thereon) (a) at least one phosphate solubilizing microorganism comprising a strain of the fungus Ilium, and (b) at least one LCO and/or at least one CO, each in an amount ive to enhance plant growth when applied to the seed compared to untreated seed. The phosphate solubilizing microorganism and the LCD and/or the CO may be applied to the seed via the same or different compositions. Packages ning the plant seed are also provided.

A further aspect of the present invention is directed to a method of enhancing plant growth, comprising a) treating (e.g., applying to) plant seed with an effective amount of at least one phosphate lizing microorganism, and b) treating the seed or treating (e.g., applying to) the plant that germinates from the seed with an effective amount of at least one LCO and/or at least one CO, wherein upon harvesting the plant exhibits at least one of increased plant yield ed in terms of bushels/acre, increased root number, increased root length, increased root mass, increased root volume and increased leaf area, compared to untreated plants or plants harvested from untreated seed.

A further aspect of the present invention is directed to a method of enhancing plant growth, comprising a) treating (e.g., applying to) plant seed with an effective amount of at least one phosphate solubilizing microorganism comprising a strain of the fungus Penicillium, and b) treating the seed or treating (e.g., applying to) the plant that germinates from the seed with an effective amount of at least one LCO and/or at least one CO, wherein upon harvesting the plant exhibits at least one of increased plant yield measured in terms of bushels/acre, increased root number, increased root length, sed root mass, increased root volume and increased leaf area, compared to untreated plants or plants harvested from untreated seed.

In some embodiments, ent of the seed es direct application of the at least one phosphate solubilizing microorganism and the at least one LCO and/or at least one CO (collectively "actives") onto the seed, which may then be planted or stored for a period of time prior to planting. Treatment of the seed may also include indirect treatment such as by introducing the actives into the soil (known in the art as row application). The actives may be used together in a single composition, or may be ated in separate compositions for concomitant or tial treatment. In yet other embodiments, the at least one LCO and/or at least one CO may be applied to the plant that germinates from the seed, and the at least one phosphate solubilizing microorganism is applied to the seed, directly or indirectly. In some embodiments, the seed are treated with one of the actives and then stored, and the other active is used to treat the seed at the time of planting. In yet other ments, the seed is treated with the at least one phosphate solubilizing microorganism and then stored, and the plant that germinates from the seed is treated with the at least one LCO and/or at least one CO. The compositions and methods may further include use of other plant signal molecules and/or other agronomically beneficial .

The method of the present ion is applicable to legumes and non-legumes alike. In some embodiments, the leguminous seed is soybean seed.

In some other embodiments, the seed that is treated is guminous seed such as a field crop seed, 9.9., a cereal such as corn, or a vegetable crop seed such as potato.

BRIEF DESCRIPTION OF THE DRAWINGS Figs. 1 and 2 show the chemical structures of hitooligosaccharide compounds (LCO) useful in the practice of the present invention.

DETAILED PTION As used herein, hate solubilizing microorganism” is a microorganism that is able to increase the amount of phosphorous available for a plant. Phosphate solubilizing microorganisms include fungal and bacterial strains.

In embodiment, the phosphate solubilizing microorganism is a spore forming microorganism. miting examples of phosphate solubilizing microorganisms include species from a genus selected from the group consisting of Acinetobacter, Arthrobacter, botrys, Aspergillus, Azospirillum, Bacillus, Burkholderia, Candida Chryseomonas, bacter, Eupenicillium, Exiguobacterium, ella, Kluyvera, Microbacterium, Mucor, Paecilomyces, Paenibaci/lus, Penicillium, Pseudomonas, Serratia, Stenotrophomonas, Streptomyces, Streptosporangium, Swaminathania, Thiobacillus, Torulospora, Vibrio, Xanthobacter, and Xanthomonas. miting examples of phosphate solubilizing microorganisms are selected from the group consisting Acinetobacter calcoaceticus, Acinetobacter sp, Arthrobacter sp., Arthrobotrys oligospora, Aspergillus niger, Aspergillus sp., Azospirillum halopraeferans, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans,Baci/lus licheniformis, Bacillus subtilis, Burkholderia cepacia, Burkholderia vietnamiensis, Candida krissii, Chryseomonas luteola, Enterobacter aerogenes, Enterobacter asburiae, Enterobacter sp., Enterobacter taylorae, cillium parvum, Exiguobacterium sp., Klebsiella sp., Kluyvera escens, Microbacterium sp., Mucor ramosissimus, omyces hepialid, Paecilomyces marquandii, Paenibaci/lus macerans, Paenibacillus ginosus, a aglomerans, Penicil/ium expansum, Pseudomonas corrugate, Pseudomonas fluorescens, Pseudomonas lutea, Pseudomonas poae, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas trivia/is, Serratia marcescens, Stenotrophomonas maltophi/ia, Streptomyces sp., Streptosporangium sp., Swaminathania salitolerans, Thiobacillus ferrooxidans, Toru/ospora globosa, Vibrio proteolyticus, Xanthobacter agilis, and Xanthomonas campestris.

In a particular embodiment, the phosphate lizing microorganism is a strain of the fungus Penicillium. Strains of the fungus Penicillium that may be useful in the practice of the present invention include P. e (formerly known as P. bilaii), P. albidum, P. iogriseum, P. chrysogenum, P. citreonigrum, P. citrinum, P. digitatum, P. ntas, P. fuscum, P. ivorus, P. glabrum, P. griseofulvum, P. implicatum, P. janthinellum, P. lilacinum, P. minioluteum, P. montanense, P. nigricans, P. oxalicum, P. pinetorum, P. pinophilum, P. ogenum, P. rad/cans, P. radicum, P. raistrickii, P. rugulosum, P. simplicissimum, P. m, P. variabile, P. velutinum, P. viridicatum, P. m, P. orus, and P. expansum.

In one particular embodiment the Penicillium s is P. bilaiae. In another particular embodiment the P. bilaiae strains are ed from the group consisting of ATCC 20851, NRRL 50169, ATCC 22348, ATCC 18309, NRRL 50162 (Wakelin, et al., 2004. Biol Fertil Soils 40:36—43). In another particular embodiment the Penicillium species is P. gaestrivorus, e.g., NRRL 50170 (see, Wakelin, supra).

In some embodiments, more than one phosphate solubilizing microorganism is used, such as, at least two, at least three, at least four, at least five, at least 6, including any combination of the Acinetobacter, Arthrobacter, Arthrobotrys, Aspergillus, Azospirillum, Bacillus, Burkholderia, Candida Chryseomonas, Enterobacter, Eupenicillium, Exiguobacterium, Klebsiella, Kluyvera, Microbacterium, Mucor, Paecilomyces, Paenibacillus, Penicillium, Pseudomonas, Serratia, Stenotrophomonas, Streptomyces, Streptosporangium, Swaminathania, Thiobacillus, Torulospora, , Xanthobacter, and Xanthomonas, including one species selected from the following group: Acinetobacter calcoaceticus, Acinetobacter sp, Arthrobacter sp., Arthrobotrys oligospora, Aspergillus niger, Aspergillus sp., Azospiri/lum ha/opraeferans, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circu/ans,Baci/Ius licheniformis, Bacillus subti/is, Burkho/deria cepacia, Burkho/deria vietnamiensis, Candida i, Chryseomonas luteo/a, bacter aerogenes, Enterobacter asburiae, Enterobacter sp., Enterobacter taylorae, Eupenicillium parvum, Exiguobacterium sp., K/ebsie/la sp., Kluyvera Ciyocrescens, Microbacterium sp., Mucor ramosissimus, Paecilomyces hepialid, Paecilomyces marquandii, Paenibacillus macerans, Paenibacillus mucilaginosus, Pantoea ag/omerans, Penicillium um, Pseudomonas corrugate, Pseudomonas fluorescens, Pseudomonas lutea, Pseudomonas poae, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas trivia/is, Serratia marcescens, Stenotrophomonas maltophilia, omyces sp., Streptosporangium sp., Swaminathania salitolerans, Thiobacillus xidans, Torulospora globosa, Vibrio proteolyticus, Xanthobacter agiiis, and Xanthomonas campestris.

In some embodiments, two different strains of the same s may also be combined, for example, at least two different strains of Penicillium are used.

The use of a combination of at least two ent Penicillium strains has the following advantages. When applied to soil already containing insoluble (or sparingly soluble) phosphates, the use of the combined fungal strains will result in an se in the amount of phosphorus available for plant uptake compared to the use of only one Penicillium strain. This in turn may result in an increase in phosphate uptake and/or an increase in yield of plants grown in the soil compared to use of individual s alone. The combination of strains also enables insoluble rock phosphates to be used as an ive fertilizer for soils which have inadequate amounts of available phosphorus. Thus, in some embodiments, one strain of P. bilaiae and one strain of P. gaestrivorus are used. In other embodiments, the two strains are NRRL 50169 and NRRL 50162. In further embodiments, the at least two strains are NRRL 50169 and NRRL 50170. In yet further embodiments, the at least two strains are NRRL 50162 and NRRL 50170.

The phosphate solubilizing microorganisms may be ed using any suitable method known to the person skilled in the art, such as, solid state or liquid fermentation using a le carbon source. The ate solubilizing microorganism is preferably prepared in the form of a stable spore.

In an embodiment, the phosphate solubilizing microorganism is a Penicillium fungus. The Penicillium fungus according to the invention can be grown using solid state or liquid fermentation and a suitable carbon source. Penicillium es may be grown using any le method known to the person skilled in the art. For example, the fungus may be cultured on a solid growth medium such as potato dextrose agar or malt extract agar, or in flasks containing suitable liquid media such as Czapek—Dox medium or potato dextrose broth. These culture methods may be used in the preparation of an inoculum of Penicillium spp. for treating (e.g., coating) seeds and/or application to an agronomically acceptable carrier to be applied to soil. The term lum" as used in this specification is intended to mean any form of phosphate solubilizing microorganism (fungal cells, mycelium or fungal spores, ial cells or bacterial ), which is e of propagating on or in the soil when the conditions of temperature, moisture, etc., are favorable for fungal growth.

Solid state production of Penicillium spores, for example, may be achieved by inoculating a solid medium such as a peat or vermiculite-based substrate, or grains including, but not limited to, oats, wheat, barley, or rice. The sterilized medium (achieved through autoclaving or irradiation) is inoculated with a spore suspension (1x102-1x107 cfu/ml) of the appropriate Penicillium spp. and the moisture adjusted to 20 to 50%, depending on the substrate. The material is incubated for 2 to 8 weeks at room temperature. The spores may also be produced by liquid fermentation (Cunningham et al., 1990. Can J Bot. 68:2270-2274). Liquid production may be achieved by cultivating the fungus in any suitable media, such as potato dextrose broth or sucrose yeast extract media, under riate pH and temperature conditions that may be determined in accordance with standard procedures in the art.

The resulting material may be used directly, or the spores may be harvested, concentrated by centrifugation, formulated, and then dried using air drying, freeze drying, or fluid bed drying techniques (Friesen, et al., 2005, Appl iol hnol 68:397-404) to produce a wettable powder. The le powder is then suspended in water, applied to the surface of seeds, and allowed to dry prior to planting. The wettable powder may be used in conjunction with other seed treatments, such as, but not limited to, chemical seed treatments, carriers (e.g., talc, clay, , silica gel, kaolinite) or polymers (e.g., methylcellulose, polyvinylpyrrolidone). Alternatively, a spore suspension of the appropriate Penicillium spp. may be applied to a suitable soil-compatible carrier (e.g., peat-based powder or granule) to appropriate final moisture content. The al may be incubated at room temperature, typically for about 1 day to about 8 weeks, prior to use.

Aside from the ients used to cultivate the phosphate solubilizing microorganism, including, e.g., ingredients referenced above in the ation of Penicillium, the phosphate solubilizing microorganism may be formulated using other agronomically acceptable carriers. As used herein in tion with "carrier", the term "agronomically acceptable" refers to any material which can be used to r the actives to a seed, soil or plant, and preferably which carrier can be added (to the seed, soil or plant) without having an adverse effect on plant , soil structure, soil drainage or the like. Suitable rs comprise, but are not limited to, wheat chaff, bran, ground wheat straw, peat—based powders or granules, gypsum-based granules, and clays (e.g., kaolin, bentonite, montmorillonite). When spores are added to the soil a granular formulation will be preferable. Formulations as liquid, peat, or wettable powder will be le for coating of seeds. When used to coat seeds, the material can be mixed with water, applied to the seeds and allowed to dry. Example of yet other carriers include ned bran, dried, sieved and applied to seeds prior coated with an adhesive, e.g., gum arabic. In embodiments that entail formulation of the actives in a single composition, the agronomically acceptable r may be aqueous.

The amount of the at least one phosphate solubilizing microorganism is effective to enhance growth such that upon harvesting the plant exhibits at least one of increased plant yield measured in terms of bushels/acre, increased root number, increased root length, sed root mass, increased root volume and increased leaf area, compared to untreated plants or plants harvested from untreated seed (with either active). The suitable application rates vary according to the type of seed or soil, the type of crop plants, the amounts of the source of phosphorus and/or micronutrients present in the soil or added thereto, etc. A suitable rate can be found by simple trial and error experiments for each particular case. ly, for Penicillium, for example, the application rate falls into the range of 0.001-1.0 Kg fungal spores and mycelium (fresh weight) per hectare, or 102-106 colony forming units (cfu) per seed (when coated seeds are used), or on a granular carrier applying between 1x106 and 1x1011 colony forming units per hectare. The fungal cells in the form of e.g., spores and the carrier can be added to a seed row of the soil at the root level or can be used to coat seeds prior to planting, as described in more detail below.

In embodiments, for example, that entail use of at least two strains of a phosphate solubilizing microorganism, such as, two strains of Penicillium, cial fertilizers may be added to the soil instead of (or even as well as) natural rock phosphate. The source of phosphorous may contain a source of phosphorous native to the soil. In other embodiments, the source of phosphorous may be added to the soil. In one embodiment the source is rock phosphate. In another embodiment the source is a manufactured fertilizer. Commercially available manufactured phosphate fertilizers are of many types. Some common ones are those ning monoammonium phosphate (MAP), triple super phosphate (TSP), diammonium phosphate, ordinary superphosphate and ammonium polyphosphate. All of these fertilizers are ed by chemical processing of 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 d to the soil while still maintaining the same amount of phosphorus uptake from the soil.

In a further embodiment, the source or phosphorus is organic. An organic fertilizer refers to a soil amendment derived from natural sources that guarantees, at least, the minimum percentages of nitrogen, phosphate, and potash.

Examples include plant and animal by—products, rock powders, seaweed, ants, and conditioners. Specific entative examples include bone meal, meat meal, animal manure, compost, sewage sludge, or guano.

Other fertilizers, such as nitrogen sources, or other soil amendments may of course also be added to the soil at imately the same time as the phosphate solubilizing microorganism or at other times, so long as the other materials are not toxic to the fungus.

Lipo-chitooligosaccharide compounds (LCO's), also known in the art as tic Nod signals or Nod factors, consist of an oligosaccharide backbone of B-l,4-linked N-acetyl-D-glucosamine ("GIcNAc") residues with an N-linked fatty acyl chain condensed at the non-reducing end. LCO's differ in the number of GIcNAc es in the backbone, in the length and degree of tion 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: CHZORS NH-R7 in which: G is a hexosamine which can be substituted, for example, by an acetyl group on the nitrogen, a sulfate group, an acetyl group and/or an ether group on an oxygen, R1, R2, R3, R5, R6 and R7, which may be cal or different, represent H, CH3 CO--, CX Hy CO-- where x is an integer between 0 and 17, and y is an integer between 1 and 35, or any other acyl group such as for example a carbamyl, R4 represents a mono-, di- or triunsaturated aliphatic chain containing at least 12 carbon atoms, and n is an integer between 1 and 4.

LCOs may be obtained (isolated and/or purified) from ia such as Rhizobia, e.g., Rhizobium sp., hizobium sp., Sinorhizobium sp. and zobium sp. LCO structure is characteristic for each such bacterial species, and each strain may produce multiple LCO's with different structures. For example, specific LCOs from S. meliloti have also been described in US. Patent 5,549,718 as having the formula II: (CH2)5 \(CH2)5 in which R represents H or CH3 CO-- and n is equal to 2 or 3.

Even more specific LCOs e NodRM, 1, NodRM-3. When acetylated (the R=CH3 CO--), they become AcNodRM-1, and AcNodRM-3, respectively (US. Patent 5,545,718).

LCOs from Bradyrhizobium cum are described in US.

Patents 5,175,149 and 5,321,011. Broadly, they are pentasaccharide phytohormones comprising methylfucose. A number of these B. japonicum-derived LCOs are described: BjNod-V (C1821); BjNod—V (Ac, C184), BjNod-V (C164); and V (Ac, C16;0), with "V" indicating the presence of five N—acetylglucosamines; "Ac" an acetylation; the number following the "C" indicating the number of carbons in the fatty acid side chain; and the number following the ":" the number of double bonds.

LCO's used in embodiments of the invention may be obtained (i.e., isolated and/or ed) 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.

LCO's are the primary determinants of host specificity in legume symbiosis (Diaz, et al., Mol. Plant-Microbe Interactions 13:268-276 (2000)). Thus, within the legume family, specific genera and species of rhizobia develop a symbiotic nitrogen-fixing onship with a specific legume host. These plant-host/bacteria combinations are described in a, et al., Soil Biol. Biochem. 29:819-830 (1997), Examples of these bacteria/legume symbiotic partnerships include 8. meliloti/alfalfa and sweet clover; R. leguminosarum biovar viciae/peas and s; R. nosarum biovar phaseo/i/beans; Bradyrhizobium japonicum/soybeans; and R. leguminosarum biovar ii/red clover. Hungria also lists the effective oid Nod gene inducers of the rhizobial species, and the specific LCO structures that are produced by the ent rhizobial species.

However, LCO specificity is only required to establish nodulation in legumes. In the practice of the present invention, use of a given LCD is not limited to treatment of seed of its symbiotic legume partner, in order to achieve sed plant yield measured in terms of bushels/acre, increased root number, increased root length, increased root mass, increased root volume and increased leaf area, compared to plants harvested from untreated seed, or compared to plants harvested from seed treated with the signal molecule just prior to or within a week or less of planting.

Thus, by way of example, an LCO obtained from B. japonicum may be used to treat leguminous seed other than soybean and non-leguminous seed such as corn. As another example, the pea LCO obtainable from R. leguminosarum illustrated in Fig. 1 (designated LCO-V (C1821), SP104) can be used to treat leguminous seed other than pea and non-legumes too.

Also encompassed by the present invention is use of LCOs obtained (i.e., isolated and/or ed) from a hizal fungi, such as fungi of the group Glomerocycota, e.g., Glomus intraradicus. The structures of representative LCOs obtained from these fungi are described in and (the LCOs described n also referred to as "Myc s").

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

LCO's may be utilized in various forms of purity and may be used alone or in the form of a culture of LCO-producing bacteria or fungi. For e, OPTIMIZE® (commercially available from Novozymes BioAg Limited) contains a culture of B. japonicum that produces an LCD (C18:1, MeFuc), MOR116) that is illustrated in Fig. 2. s to provide ntially 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 enhanced by repeated HPLC, and the purified LCO molecules can be freeze-dried for long-term storage.

Chitooligosaccharides (00s) as bed above, may be used as starting materials for the production of synthetic LCOs. COs are known in the art as [54 linked N actyl glucosamine structures identified as chitin oligomers, also as N-acetylchitooligosaccharides. CO's have unique and ent side chain decorations which make them different from chitin molecules [(C8H13N05)n, CAS No. 1-4], and chitosan molecules [(C5H11NO4)n, CAS No. 90124].

Representative literature bing the structure and production of COs is as follows: Van der Holst, et al., t 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. r 27, "The Molecular Immunology of Complex Carbohydrates" in Advances in Experimental Medicine and Biology, Springer Science; Wan, eta/., Plant Cell 21:1053-69 (2009); PCT/F100/00803 (9/21/2000); and Demont—Caulet, et al., Plant Physiol. 120(1):83-92 . Two COs suitable for use in the present ion may be easily d from the LCOs shown in Figs. 1 and 2 (minus the fatty acid chains). The COs may be synthetic or recombinant. Methods for preparation of recombinant COs are known in the art.

See, e.g., Samain, et al. (supra); Cottaz, et al., Meth. Eng. 7(4):311-7 (2005) and Samain, etal., J. Biotechnol. 47 (1999).

The LCO and CO may be used alone, or in combination. Thus, in some embodiments, the present ion entails use of an LCO and a CO.

Seeds may be treated with the LCO and/or CO in several ways such as spraying or dripping. Spray and drip treatment may be conducted by formulating an ive amount of the LCO or CO in an agriculturally acceptable carrier, typically aqueous in nature, and spraying or dripping the composition onto seed via a continuous treating system (which is calibrated to apply treatment at a predefined rate in tion to the continuous flow of seed), such as a drum-type of treater.

These methods advantageously employ relatively small volumes of carrier so as to allow for relatively fast drying of the d seed. In this fashion, large volumes of seed can be efficiently treated. Batch systems, in which a predetermined batch size of seed and signal molecule compositions are delivered into a mixer, may also be employed. Systems and apparatus for performing these processes are commercially available from numerous suppliers, e.g., Bayer ience (Gustafson).

In another embodiment, the treatment entails coating seeds. One such s involves coating the inside wall of a round container with the composition, adding seeds, then rotating the container to cause the seeds to contact the wall and the composition, a process known in the art as "container coating".

Seeds can be coated by combinations of coating methods. Soaking typically entails use of an aqueous solution ning the plant growth enhancing agent. 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 hr, 6 hr, 12 hr, 24 hr). Some types of seeds (e.g., soybean seeds) tend to be sensitive to moisture. Thus, g such seeds for an extended period of time may not be desirable, in which case the soaking is typically carried out for about 1 minute to about 20 minutes.

In those embodiments that entail storage of seed after application of the LCO or CO, adherence of the LCO or CO to the seed over any portion of time of the storage period is not al. Without intending to be bound by any particular theory of operation, Applicants believe that even to the extent that the ng may not cause the plant signal molecule to remain in contact with the seed surface after treatment and during any part of storage, the LCO or CO may achieve its intended effect by a phenomenon known as seed memory or seed perception. See, Macchiavelli, et al., J. Exp. Bot. 55(408):1635—40 . Applicants also believe that ing treatment the LCO or CO, es toward the young developing radicle and activates symbiotic and developmental genes which results in a change in the root architecture of the plant. Notwithstanding, to the extent desirable, the itions ning the LCO or CO may further contain a sticking or coating agent. For aesthetic purposes, the compositions may further n a coating polymer and/or a colorant.

The amount of the at least one LCO and/or at least one CO is effective to enhance growth such that upon harvesting the plant exhibits at least one of sed plant yield measured in terms of bushels/acre, increased root number, increased root length, increased root mass, increased root volume and increased leaf area, compared to untreated plants or plants harvested from ted seed (with either active). The effective amount of the LCO or CO used to treat the seed, expressed in units of concentration, generally ranges from about 10'5 to about 10'14 M (molar concentration), and in some embodiments, from about 10'5 to about 10'11 M, and in some other embodiments from about 10'7 to about 10'8 M. Expressed in units of , the effective amount generally ranges from about 1 to about 400 ug/hundred weight (cwt) seed, and in some embodiments from about 2 to about 70 ug/cwt, and in some other embodiments, from about 2.5 to about 3.0 ug/cwt seed.

For purposes of treatment of seed indirectly, i.e., in-furrow treatment, the effective amount of the LCO or CO generally ranges from 1 ug/acre to about 70 ug/acre, and in some embodiments, from about 50 ug/acre to about 60 ug/acre. For purposes of application to the plants, the effective amount of the LCO or CO generally ranges from 1 pg/acre to about 30 pg/acre, and in some embodiments, from about 11 pg/acre to about 20 pg/acre.

Seed may be d with the at least one phosphate solubilizing microorganism (e.g., Penicil/ium) and the at least LCO and/or at least one CO just prior to or at the time of planting. Treatment at the time of planting may include direct application to the seed as described above, or in some other embodiments, by introducing the actives into the soil, known in the art as in-furrow treatment. In those embodiments that entail treatment of seed followed by storage, the seed may be then packaged, e.g., in 50-lb or 100—lb bags, or bulk bags or containers, in accordance with standard techniques. The seed may be stored for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 , and even longer, e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 , or even longer, under appropriate storage conditions which are known in the art. Whereas soybean seed may have to be planted the following season, corn seed can be stored for much longer periods of time including upwards of 3 years.

The present invention may also include treatment of seed or plant with a plant signal molecule other than an LCD or CO. For purposes of the present invention, the term "plant signal molecule", which may be used interchangeably with "plant growth—enhancing agent" broadly refers to any agent, both naturally occurring in plants or es, and synthetic (and which may be non-naturally occurring) that directly or indirectly activates a plant biochemical y, resulting in increased plant growth, measureable at least in terms of at least one of increased yield measured in terms of bushels/acre, increased root , increased root length, increased root mass, increased root volume and increased leaf area, compared to untreated plants or plants ted from ted seed. Representative examples of plant signal molecules that may be useful in the practice of the present invention include chitinous compounds (other than COs), flavonoids, jasmonic acid, ic acid and linolenic acid and their derivatives, and karrikins.

Chitins and ans, which are major components of the cell walls of fungi and the exoskeletons of insects and crustaceans, are also composed of GlcNAc residues. Chitinous compounds e chitin, (IUPAC: N-[5-[[3- acetylamino-4,5-dihydroxy(hydroxymethyl)oxan-2yl]methoxymethyl]—2-[[5- acetylamino-4,6-dihydroxy(hydroxy methyl)oxan—3-yl]methoxymethyl]hydroxy- 6-(hydroxymethyl)oxanys]ethanamide), and chitosan, (IUPAC: o[5- amino[5-amino-4,6-dihydroxy-2(hydroxymethyl)oxan—3-yl]oxy—4—hydroxy-2— (hydroxymethyl)oxanyl]oxy-2(hydroxymethyl)oxane-3,4-diol). These compounds may be obtained commercially, e.g., from Sigma-Aldrich, or prepared from insects, crustacean shells, or fungal cell walls. Methods for the preparation of chitin and chitosan are known in the art, and have been described, for example, in U.S.

Patent 4,536,207 (preparation from crustacean shells), Pochanavanich, et al., Lett.

Appl. Microbiol. 35:17-21 (2002) (preparation from fungal cell walls), and U.S.

Patent 5,965,545 (preparation from crab shells and hydrolysis of commercial chitosan). Deacetylated chitins and chitosans may be obtained that range from less than 35% to greater 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 of 0.5 to 2kD; “practical grade" an with a molecular weight of about 15kD; and high molecular weight chitosan of up to 70kD. Chitin and chitosan compositions formulated for seed ent are also commercially available. Commercial products include, for example, ELEXA® (Plant Defense Boosters, Inc.) and BEYONDT'V' ouse, Inc.).

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, e.g., as beneficial ing molecules, and as protection against insects, animals, fungi and bacteria. Classes of flavonoids e chalcones, anthocyanidins, coumarins, flavones, flavanols, flavonols, flavanones, and isoflavones. See, Jain, et al., J. Plant Biochem. & hnol. 11:1-10 (2002); Shaw, et al., Environmental Microbiol. 11:1867—80 (2006).

Representative oids that may be useful in the ce of the present invention include genistein, daidzein, formononetin, naringenin, etin, luteolin, and apigenin. Flavonoid compounds are commercially available, e.g., from Natland International Corp., Research Triangle Park, NC; MP Biomedicals, Irvine, CA; LC Laboratories, Woburn MA. Flavonoid compounds may be isolated from plants or seeds, e.g., as described in US. Patents 5,702,752; 5,990,291; and 6,146,668. Flavonoid compounds may also be produced by cally engineered organisms, such as yeast, as described in Ralston, et al., Plant logy 137:1375-88 (2005).

Jasmonic acid (JA, [1 R-[1d,2B(Z)]]—3-oxo (pentenyl)cyclopentaneacetic acid) and its derivatives, linoleic acid ((Z,Z)—9,12- Octadecadienoic acid) and its derivatives, and linolenic acid ((Z,Z,Z)—9,12,15- octadecatrienoic acid) and its derivatives, may be used in the practice of the present invention. Jasmonic acid and its methyl ester, methyl jasmonate (MeJA), tively known as jasmonates, are octadecanoid—based nds that occur naturally in plants. Jasmonic acid is produced by the roots of wheat seedlings, and by fungal microorganisms such as Botryodiploo‘ia theobromae and Gibbrella fujikuroi, yeast aromyces cerevisiae), and pathogenic and non-pathogenic strains of Escherichia coli. ic acid and linolenic acid are produced in the course of the biosynthesis of jasmonic acid. Jasmonates, linoleic acid and linoleic acid (and their derivatives) are reported to be inducers of nod gene expression or LCD production by rhizobacteria. See, e.g., Mabood, Fazli, Jasmonates induce the expression of nod genes in Bradyrhizobiumjaponicum, May 17, 2001; and Mabood, Fazli, "Linoleic and linolenic acid induce the expression of nod genes in Bradyrhizobium japonicum," USDA 3, May 17, 2001.

Useful derivatives of linoleic acid, linolenic acid, and jasmonic acid that may be useful in the practice of the present ion include esters, amides, glycosides and salts. Representative esters are compounds in which the carboxyl group of ic acid, linolenic acid, or jasmonic acid has been replaced with a --COR group, where R is an --OR1 group, in which R1 is: an alkyl group, such as a 01-08 ched or branched alkyl group, e.g., a methyl, ethyl or propyl group; an alkenyl group, such as a C2-C8 unbranched or branched alkenyl group; an alkynyl group, such as a 02-08 unbranched or branched 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, n the heteroatoms in the aryl group can be, for example, N, O, P, or 8. Representative amides are compounds in which the carboxyl group of linoleic acid, linolenic acid, or jasmonic acid has been ed with a --COR group, where R is an NR2R3 group, in which R2 and R3 are independently: hydrogen; an alkyl group, such as a 01-08 unbranched or branched alkyl group, e.g., a methyl, ethyl or propyl group; an alkenyl group, such as a 02-08 unbranched or branched alkenyl group; an alkynyl group, such as a 02—08 unbranched or branched 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 may be prepared by known methods, such as acid-catalyzed nucleophilic addition, wherein the carboxylic acid is reacted with an alcohol in the presence of a catalytic amount of a mineral acid. Amides may also be prepared by known methods, such as by reacting the carboxylic acid with the appropriate amine in the presence of a coupling agent such as ohexyl carbodiimide (DCC), under neutral conditions. Suitable salts of linoleic acid, linolenic acid, and jasmonic acid e e.g., base addition salts. The bases that may be used as reagents to prepare metabolically acceptable base salts of these compounds include those d from cations such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium). These salts may be readily ed by mixing together a solution of ic acid, linolenic acid, orjasmonic acid with a solution of the base. The salt may be precipitated from on and be collected by filtration or may be recovered by other means such as by evaporation of the solvent.

Karrikins are vinylogous 4H-pyrones e.g., 2H-furo[2,3-c]pyranones including derivatives and analogues thereof. Examples of these compounds are represented by the following structure: R3 Z R4 n; Z is O, S or NR5; R1, R2, R3, and R4 are each ndently H, alkyl, alkenyl, alkynyl, phenyl, benzyl, hydroxy, hydroxyalkyl, alkoxy, phenyloxy, benzyloxy, CN, COR6, COOR=, halogen, NR6R7, or N02; and R5, R6, and R7 are each independently H, alkyl or alkenyl, or a biologically acceptable salt thereof. es of biologically acceptable salts of these compounds may include acid on salts formed with biologically acceptable acids, examples of which include hydrochloride, hydrobromide, sulphate or bisulphate, phosphate or hydrogen phosphate, acetate, benzoate, ate, fumarate, maleate, lactate, citrate, tartrate, gluconate; methanesulphonate, benzenesulphonate and p-toluenesulphonic acid. Additional ically acceptable metal salts may include alkali metal salts, with bases, examples of which include the sodium and potassium salts. Examples of compounds embraced by the structure and which may be suitable for use in the present invention include the following: yl—2H-furo[2,3-c]pyranone (where R1=CH3, R2, R3, R4=H), 2H—furo[2,3—c]pyran—2—one (where R1, R2, R3, R4=H), 7- methyl-2H-furo[2,3-c]pyran—2—one (where R1, R2, R4=H, R3=CH3), 5-methyl-2H- furo[2,3-c]pyran-2—one (where R1, R2, R3=H, R4=CH3), 3,7-dimethyl-2H-furo[2,3- c]pyranone (where R1, R3=CH3, R2, R4=H), 3,5-dimethyl-2H-furo[2,3-c]pyran-2— one (where R1, R4=CH3, R2, R3=H), 3,5,7—trimethyl-2H-furo[2,3-c]pyran-2—one (where R1, R3, , R2=H), 5-methoxymethyl-3—methyl-2H-furo[2,3-c]pyran-2—one (where R1=CH3, R2, R3=H, OCH3), o—3,7-dimethyl-2H-furo[2,3-c]pyran-2—one (where R1, R3=CH3, R2=Br, R4=H), 3-methylfuro[2,3-c]pyridin-2(3H)-one (where Z=NH, R1=CH3, R2, R3, R4=H), 3,6—dimethylfuro[2,3—c]pyridin-2(6H)—one (where Z=N- -CH3, R1=CH3, R2, R3, R4=H). See, US. Patent 213. These molecules are also known as karrikins. See, Halford, supra.

The present invention may further include treatment of the seed or the plants that germinate from the seed with an agriculturally/agronomically beneficial agent. As used herein and in the art, the term "agriculturally or agronomically beneficial" refers to agents that when applied to seeds result in enhancement (which may be statistically significant) of plant characteristics such as plant stand, growth, vigor or yield in comparison to non—treated seeds. Representative examples of such agents that may be useful in the practice of the present invention include herbicides, fungicides and insecticides.

Suitable ides include bentazon, acifluorfen, chlorimuron, lactofen, clomazone, fluazifop, glufosinate, glyphosate, ydim, imazethapyr, imazamox, fomesafe, lorac, imazaquin, and clethodim. Commercial products containing each of these compounds are readily available. Herbicide concentration in the ition will generally correspond to the labeled use rate for a particular herbicide.

A "fungicide" as used herein and in the art, is an agent that kills or inhibits fungal growth. As used herein, a ide "exhibits activity against" a particular species of fungi if treatment with the fungicide s in g or growth inhibition of a fungal population (e.g., in the soil) relative to an untreated population.

Effective fungicides in accordance with the invention will suitably exhibit activity against a broad range of pathogens, including but not limited to Phytophthora, Rhizoctonia, Fusarium, Pythium, Phomopsis or Se/erotinia and sora and ations thereof.

Commercial fungicides may be suitable for use in the present invention. Suitable commercially available fungicides include PROTEGE, RIVAL or ALLEGIANCE FL or L8 (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 ina, Buenos Ares, Argentina). Active ingredients in these and other commercial fungicides include, but are not limited to, fludioxonil, mefenoxam, azoxystrobin and metalaxyl. Commercial fungicides are most suitably used in accordance with the manufacturer's instructions at the recommended concentrations.

As used herein, an insecticide "exhibits ty against" a particular species of insect if treatment with the insecticide results in killing or inhibition of an insect tion relative to an untreated population. ive insecticides in accordance with the invention will ly exhibit activity against a broad range of insects including, but not limited to, wireworms, ms, grubs, corn rm, seed corn maggots, flea beetles, chinch bugs, aphids, leaf beetles, and stink bugs.

Commercial insecticides may be suitable for use in the present invention. Suitable commercially-available insecticides e 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 suitably used in accordance with the manufacturer's instructions at the recommended concentrations.

The methods of the present invention are applicable to leguminous seed, representative examples of which include n, alfalfa, peanut, pea, lentil, bean and clover. The methods of the present invention are also applicable to non-leguminous seed, e.g., Poaceae, Cucurbitaceae, Malvaceae. ceae, Chenopodiaceae and Solonaceae. Representative examples of non-leguminous seed include field crops such as corn, rice, oat, rye, barley and wheat, cotton and canola, and vegetable crops such as potatoes, es, cucumbers, beets, lettuce and cantaloupe.

All patent and non-patent publications cited in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein incorporated by reference to the same extent as if each individual publication or patent ation were specifically and individually ted to be incorporated by reference.

Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and ations of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without ing from the spirit and scope of the present invention as defined by the appended claims.

Claims (12)

1. A method of enhancing plant growth, comprising: a) treating plant seed with an effective amount of at least one phosphate solubilizing microorganism, and b) treating the seed and/or the plant that germinates from the seed with an effective amount of at least one lipo-chitooligosaccharide (LCO) and/or at least one chitooligosaccharide (CO), wherein, upon harvesting, the plant exhibits at least one of sed plant yield measured in terms of bushels/acre, increased root number, sed root length, increased root mass, increased root volume and increased leaf area, compared to untreated plants or plants harvested from untreated seed.

2. The method of claim 1, wherein the at least one phosphate solubilizing microorganism is d to the seed at the time of ng.

3. The method of claim 1, wherein the at least one phosphate solubilizing microorganism is applied to the seed in furrow.

4. The method of claim 1, wherein the at least one LCO and/or at least one CO is applied to the seed at the time of planting.

5. The method of claim 1, wherein the at least one LCO and/or at least one CO is applied to the seed in furrow.

6. The method of claim 1, wherein the at least one LCO and/or at least one CO is applied to the plant via foliar treatment.

7. The method of claim 1, wherein the at least one phosphate solubilizing rganism and the at least one LCO and/or at least one CO are applied to the seed via a single composition.

8. The method of claim 1, wherein the at least one phosphate solubilizing microorganism and the at least one LCO and/or at least one CO are d to the seed via different compositions.

9. The method of claim 1, r comprising applying one or more chitins and/or one or more chitosans to the seed and/or to the plant that germinates from the seed.

10. The method of claim 1, further sing applying at least one flavonoid to the seed and/or to the plant that germinates from the seed.

11. The method of claim 1, further comprising applying jasmonic acid or a derivative thereof to the seed and/or to the plant that germinates from the seed.

12. The method of claim 1, further comprising ng linoleic acid or a derivative thereof to the seed and/or to the plant that germinates from the seed. 11245245