RU2588162C2 - Chitooligosaccharides and methods for use thereof to enhance soya growth - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: invention relates to agriculture. Enhancing growth of soyabean comprises treating soyabean seeds and/or soybean plant with an effective amount of at least one chitooligosaccharide represented by formula:

in which R₁ is hydrogen or methyl; R₂ is hydrogen or methyl; R₃ is hydrogen, acetyl or carbamoyl; R₄ is hydrogen, acetyl or carbamoyl; R₅ is hydrogen, acetyl or carbamoyl; R₆ is hydrogen, arabinosyl, fucosyl, acetyl, sulphate, 3-0-S-2-0-MeFuc, 2-0-MeFuc and 4-0-AcFuc; R₇ is hydrogen, or mannosyl, glycerol; R₈ is hydrogen, methyl or -CH₂OH; R₉ is hydrogen, fucosyl or arabinosyl; R₁₀ is hydrogen, acetyl or fucosyl; and n is 0, 1, 2 or 3.

EFFECT: invention enables to increase yield of soybean.

63 cl, 8 dwg, 1 tbl, 4 ex

Description

BACKGROUND

A symbiosis has been documented between Gram-negative soil bacteria, Rhizobiaceae and Bradyrhizobiaceae, and legumes such as soy. The biochemical basis of these relationships involves the exchange of molecular signals, the compounds that transmit signals from plants to bacteria include flavones, isoflavones and flavanones, and the compounds that transmit signals from bacteria to plants include the final products of expression of the bradyrisobial and rhizobial nod genes known as lipochito oligosaccharides (LHO). The symbiosis between these bacteria and legumes allows legumes to bind the atmospheric nitrogen necessary for plant growth, and thereby eliminate the need for nitrogen fertilizers. Since nitrogen fertilizers can significantly increase the cost of crops and are accompanied by a number of polluting effects, research is ongoing in agriculture on the use of this biological relationship and the development of new tools and methods for improving plant yields without increasing the use of nitrogen-based fertilizers.

In U.S. Patent 6979664 describes a method for improving seed germination or the emergence of crop sprouts, comprising the steps of preparing a composition that comprises an effective amount of at least one lipochito oligosaccharide and an agriculturally suitable carrier, and applying the composition in close proximity to the seeds or sprouts in an amount effective to improve seed germination or the appearance of sprouts compared to untreated seeds or sprouts.

Further development of this approach is presented in WO 2005/062899, relating to combinations of at least one plant inducer, namely LHO, in combination with a fungicide, insecticide or a combination thereof to improve plant characteristics such as plant density, growth, power and / or plant productivity. It is indicated that the compositions and methods are applicable to legumes and non-leguminous plants and can be used to treat seeds (immediately before sowing), sprouts, roots or plants.

Similarly, WO 2008/085958 describes compositions for enhancing plant growth and increasing yields of leguminous and non-leguminous plants that contain LHO in combination with another active agent such as chitin or chitosan, a flavonoid or herbicide, and which can be applied to seeds and / or plants simultaneously or sequentially. As with WO 2005/062899, WO 2008/085958 describes seed treatment immediately before sowing.

Recently, Halford, "Smoke Signals" at Chem. Eng. News (April 12, 2010) on pages 37-38 reported that carricins or butenolides contained in smoke, after a forest fire, act as growth stimulants and activators of seed germination and can activate seeds such as corn, tomato, lettuce and onions that were in storage. These molecules are the subject of U.S. patent. 7576213.

However, there is still a need for systems to improve or enhance plant growth.

SUMMARY OF THE INVENTION

The first object of the present invention is a method of enhancing the growth of soybean plants, comprising a) processing (for example, applying) seeds of soybean or soybean plant that sprouts from the seeds, an effective amount of at least one chitooligosaccharide (XO), where after harvesting the soybean plant is characterized by at least at least one of the following: increased plant productivity, measured in units of bushel / acre, increased number of roots, increased root length, increased root mass, increased root volume and increased area Strongly leaves as compared to untreated plants soybeans or soybean plants grown from untreated soybean seed.

In some embodiments, at least two XOs are used. In some embodiments, soybean seed treatment involves directly applying at least one XO to soybean seeds, which can then be sown or stored for some time before sowing. Soybean seed treatment may include indirect treatment, for example, by introducing at least one chemical agent into the soil (which is known in the art as applying to furrows). In other embodiments, at least one XO can be applied to plants that germinate from seeds, for example, by foliar spraying. The methods may additionally include the use of other agronomically useful agents, such as micronutrients, fatty acids and their derivatives, plant signaling molecules ((except XO), such as lipochitooligosaccharides, chitin compounds (except XO), flavonoids, jasmonic acid and its derivatives, linoleic acid and its derivatives, linolenic acid and its derivatives and carricins and their derivatives, herbicides, fungicides and insecticides and phosphate solubilizing microorganisms.

As the working examples show, in which the data of experiments conducted in greenhouses and in the field are summarized, the results obtained by the methods proposed in the present invention show that applying at least one CW to soybean seeds or soybean plants that germinate from seeds results to enhanced plant growth. These results seem unexpected, especially given the point of view according to which CWs participate in systemic acquired resistance (STR), but do not necessarily participate in direct enhancement of plant growth. The results described in the present invention show that in some cases, the methods proposed in the present invention lead to essentially the same effect or, in some other cases, lead to a more significant increase in plant growth than that provided by LHO. In this regard, the results obtained through experiments in greenhouses are especially important, since they are carried out under conditions where there are practically no diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

In FIG. 1a and 2a show the chemical structures of chitooligosaccharides (XO), applicable in the practical implementation of the present invention.

In FIG. 1b and 2b show the chemical structures of lipo-chitooligosaccharides (LCOs), which correspond to the CIs shown in FIG. 1a and 2a, and which are also applicable in the practical implementation of the present invention.

In FIG. 3a and 4a show the chemical structures of other chemical agents useful in the practice of the present invention.

In FIG. 3b and 4b show the chemical structures of Myc factors that correspond to the XO shown in FIG. 3a and 3b, and which are also applicable in the practical implementation of the present invention.

In FIG. 5 is a bar chart that illustrates the effect of the XO shown in FIG. 2a, compared with the LHO shown in FIG. 2b, with a mixture of chemical agents produced by chitinase, isoflavonoid and control during soybean seed treatment, expressed using leaf surface area.

In FIG. 6 is a bar chart that illustrates the effect of the XO shown in FIG. 2a, the LHO shown in FIG. 1b, an isoflavonoid and mixtures of the chitin compounds not obtained in the present invention (obtained from chitosan by an enzymatic process) during the processing of soybean seeds, expressed using the average weight of soybean plants in a dry state.

In FIG. 7 is a bar chart that illustrates the effect of XO shown in FIG. 2a, individually or in combination with one of two different fatty acids, compared to the LHO shown in FIG. 1b, for soybean seeds, expressed using the average root length.

In FIG. 8 is a bar chart that illustrates the effect of the XO shown in FIG. 2a, compared with the LHO shown in FIG. 1b, and a mixture of chemical agents produced by chitinase in the processing of soybean plants, expressed using the average dry biomass of the plant.

DETAILED DESCRIPTION OF THE INVENTION

Chitooligosaccharides

XOs are known in the art as β-1-4-linked N-acetylglucosamine structures identified as chitin oligomers, as well as N-acetylchitooligosaccharides. CWs have special and different side chain structures that make them different from chitin molecules [(C ₈ H ₁₃ NO ₅ ) _n , CAS No. 1398-61-4] and chitosan molecules [(C ₅ H ₁₁ NO ₄ ) _n , CAS No. 9012-76-4]. See, for example, Hamel, et al., Planta 232: 787-806 (2010) (for example, Fig. 1, which shows the structures of chitin, chitosan, Nod factors (LHO) and the corresponding CW (which does not contain 18C, 16C or 20C acyl groups)). The XOs of the present invention are also relatively soluble in water compared to chitin and chitosan, and in some of the embodiments described below in the present invention are pentameric. Typical literature that describes the structure and preparation of XO, which may be suitable for use in the present invention, is as follows: Muller, et al., Plant Physiol. 124: 733-9 (2000) (for example, FIG. 1 in this publication); Van der Holst, et al., Current Opinion in Structural Biology, 11: 608-616 (2001) (e.g., Fig. 1 in this publication); Robina, et al., Tetrahedron 58: 521-530 (2002); D′Haeze, et al., Glycobiol. 12 (6): 79R-105R (2002); 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).

CWs in structure differ from LHOs mainly in that they do not have a side group of a fatty acid. The synthetic derivatives formed from mycorrhiza XO and their naturally occurring synthetic derivatives that can be used in the practical implementation of the present invention can be represented by the following formula:

,

,

wherein R ₁ and R ₂ are each independently hydrogen or methyl; R ₃ means hydrogen, acetyl or carbamoyl; R ₄ means hydrogen, acetyl or carbamoyl; R ₅ means hydrogen, acetyl or carbamoyl; R ₆ means hydrogen, arabinosyl, fucosyl, acetyl, sulfate, 3-0-S-2-0-MeFuc, 2-0-MeFuc and 4-0-AcFuc; R ₇ means hydrogen, mannosyl or glycerin; R ₈ is hydrogen, methyl or —CH ₂ OH; R ₉ means hydrogen, arabinosyl or fucosyl; R ₁₀ means hydrogen, acetyl or fucosyl; and n is 0, 1, 2, or 3. The structures of the corresponding rhizobial LHO are described in D′Haeze, et al., supra.

Two XOs suitable for use in the present invention are shown in FIG. 1a and 2a. They correspond to LHO produced by Bradyrhizobium japonicum and Rhizobium leguminosarum biovar viciae, respectively, which interact symbiotic with soy and peas, respectively, but they do not have side chains of fatty acids. The corresponding LHOs produced by these mycorrhiza (and which are also applicable in the practice of the present invention) are shown in FIG. 1b and 2b.

Structures of other CWs that may be suitable for use in the practice of the present invention can be easily formed from CLCs obtained (i.e., isolated and / or purified) from mycorrhizal fungi, such as Glomerocycota fungi, for example, Glomus intraradices. See, for example, WO 2010/049751 and Maillet, et al., Nature 469: 58-63 (2011) (the LHOs described therein are also called “Myc factors”). Typical CWs formed from mycorrhizal fungi are described by the following structure:

,

,

in which n = 1 or 2; R ₁ is hydrogen or methyl; and R ₂ is hydrogen or SO ₃ H. Two other XOs suitable for use in the present invention, one of which is sulfated and the other is not sulfated, are shown in FIG. 3a and 4a, respectively. They correspond to two different LHOs produced by mycorrhizal fungi Glomas intraradices, which are shown in FIG. 3b and 4b (and which are also applicable in the practical implementation of the present invention).

XO can be synthetic or recombinant. Methods for producing synthetic XO are described, for example, in the publication Robina, see above. Methods for producing recombinant XO, for example, using E. coli as a host, are known in the art. See, for example, Dumon, et al., ChemBioChem 7: 359-65 (2006), Samain, et al., Carbohydrate Res. 302: 35-42 (1997); Cottaz, et al., Met. Eng. 7 (4): 311-7 (2005) and Samain, et al., J. Biotechnol. 72: 33-47 (1999) (for example, FIG. 1 in this publication, which shows XO structures that can be formed recombinantly in E. coli containing various combinations of nodBCHL genes). For the objectives of the present invention, at least one CW is structurally different from chitins, chitosans and other chitooligosaccharides obtained enzymatically using chitin as a starting material.

For the objectives of the present invention, in embodiments in which at least one XO is recombinant, at least one recombinant XO has a purity of at least 60%, for example, a purity of at least 60%, a purity of at least 65% purity of not less than 70%, purity of not less than 75%, purity of not less than 80%, purity of not less than 85%, purity of not less than 90%, purity of not less than 91%, purity , constituting not less than 92%, purity, was at least 93%, purity at least 94%, purity at least 95%, purity at least 96%, purity at least 97%, purity at least 98%, purity at least less than 99%, up to a purity of 100%.

Soybean seeds can be treated with at least one XO according to various methods, such as spraying or spraying. Spraying and spraying can be carried out by preparing an effective amount of at least one CW in an agriculturally acceptable carrier, usually aqueous in nature, and spraying or spraying the composition onto the seeds using a continuous treatment system (which is calibrated to apply the treatment agent at a predetermined speed in consistent with a continuous flow of seeds), such as a drum type treater. These techniques preferably use relatively small volumes of carrier to allow relatively quick drying of the treated seeds. In this way, large volumes of seeds can be effectively treated. Batch systems can also be used in which predetermined quantities of seeds and signaling molecule compositions are fed to the mixer. Systems and equipment for carrying out such processing are sold by numerous suppliers, for example, Bayer CropScience (Gustafson).

In another embodiment, the treatment comprises applying to the soybean seeds a coating of at least one XO. One such technique involves applying the composition to the inner wall of a round container, adding seeds and then rotating the container so that the seeds come into contact with the wall and composition, this technique is known in the art as “container coating”. Apply seed coatings using a combination of coating techniques. Soaking usually involves the use of an aqueous solution containing an agent for enhancing plant growth. For example, seeds can be soaked for about 1 minute to about 24 hours (for example, for at least 1 minute, 5 minutes, 10 minutes, 20 minutes, 40 minutes, 80 minutes, 3 hours, 6 hours, 12 hours, 24 h). Some types of seeds (such as soybean seeds) are sensitive to moisture. Soaking these seeds for a long period of time may then be undesirable, in which case the soaking is usually carried out for from about 1 minute to about 20 minutes.

In those embodiments that include storing soybean seeds after application of at least one XO, adhesion of XO to the seeds during any part of the storage time is not critical. If not limited to any particular theory, the applicants believe that, although the treatment may not ensure that the signal molecule of the plant comes into contact with the surface of the seed after treatment and during any part of the storage time, XO may have the intended effect due to a phenomenon known like seed memory, or seed perception. See Macchiavelli, et al., J. Exp. Bot. 55 (408): 1635-40 (2004). Applicants also believe that after treatment, XO diffuses toward the young developing root and activates genes associated with symbiosis and development, which leads to a change in the structure of plant roots. However, compositions containing XO may additionally contain the desired amount of tackifying agent or coating agent. For aesthetic reasons, the compositions may further comprise a coating polymer and / or dye.

The amount of at least one CW is effective for enhancing growth, so that after harvesting the soybean plant is characterized by at least one of the following: increased plant yield, measured in units of bushel / acre, increased number of roots, increased root length, increased root mass, increased volume roots and increased foliage compared to untreated soybean plants or soybean plants grown from untreated soybean seeds (any active substance). An effective amount of at least one CW used to treat soybean seeds, expressed in units of concentration, is usually in the range of 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. The effective amount expressed in units of weight is usually in the range of from about 1 to about 400 μg / centner (c) of seed, and in some embodiments exercise from about 2 of about 70 g / q, and in certain other embodiments from about 2.5 to about 3.0 g / n seeds.

For indirect processing of soybean seeds, i.e. furrow treatment, an effective amount of at least one XO is typically in the range of from about 1 μg / acre to about 70 μg / acre, and in some embodiments, from about 50 μg / acre to about 60 μg / acre. For application to plants, an effective amount of CI is typically in the range of from about 1 μg / acre to about 30 μg / acre, and in some embodiments, from about 11 μg / acre to about 20 μg / acre.

Soybean seeds can be treated with at least one XO immediately before sowing or during sowing. Processing during sowing may include direct application to the seeds, as described above, or in some other embodiments, by applying the active substances to the soil, known in the art, as applying to the furrows. In those embodiments that include treating the seeds after storage, the seeds can then be packaged in, for example, 50 lb or 100 lb bags, or large bags, or containers in accordance with standard techniques. Seeds can be stored for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months and even longer, for example, for 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or even longer under suitable storage conditions that are known in the art technicians.

Other agronomically useful agents

The present invention may further include treating soybean seeds or soybean plants that germinate from the seeds with at least one agricultural / agronomically useful agent. As used in the present invention and in the art, the term “agricultural or agronomically beneficial” means agents which, when applied to soybean seeds or soybean plants, result in an improvement (which may be statistically significant) of the characteristics of the soybean plant, such as plant density, growth (for example, as defined in connection with CW) or power compared to untreated soybean seeds or soybean plants. These agents can be prepared together with at least one XO or applied to seeds or plants using a separate composition. Typical examples of such agents that may be useful in the practice of the present invention include micronutrients (e.g. vitamins and micronutrients), fatty acids and their derivatives, plant signaling molecules (except XO), herbicides, fungicides and insecticides, phosphate solubilizing microorganisms, diazotrophs (rhizobial inoculum) and / or mycorrhizal fungi.

Micronutrients

Typical vitamins that may be useful in the practice of the present invention include calcium pantothenate, folic acid, biotin, and vitamin C. Typical microelements that may be useful in the practice of the present invention include boron, chlorine, manganese, iron, zinc, copper, molybdenum, nickel, selenium and sodium.

The amount of at least one micronutrient used for seed treatment, expressed in units of concentration, is usually in the range of 10 ppm to 100 ppm, and in some embodiments, from about 2 ppm to about 100 ppm The effective amount expressed in units of weight in one embodiment is usually in the range of about 180 μg to about 9 mg / centner (s) of seed, and in some embodiments, when foliar application is from about 4 μg to about 200 μg / plant. In other words, in seed treatment, an effective amount of at least one micronutrient is typically in the range of 30 μg / acre to about 1.5 mg / acre, and in some embodiments, when foliar application is from about 120 mg / acre to about 6 g / acre.

Fatty acid

Typical fatty acids that may be useful in the practice of the present invention include fatty acids, which are substituents in natural LHOs, such as stearic and palmitic acids. Other fatty acids that may be used include the saturated C ₁₂ -C ₁₈ fatty acids, which (except for stearic and palmitic acids) include lauric acid, myristic acid, and unsaturated C ₁₂ -C ₁₈ fatty acids such as miristolenovuyu acid palmitolenovuyu acid , sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linolenic acid and linoelaidic acid. Linoleic acid and linolenic acid are formed during the biosynthesis of jasmonic acid (which, as described below, is also an agronomically useful agent for the tasks of the present invention). Linoleic acid and linoleic acid (and their derivatives) are reported to be inducers of nod gene expression or LHO production by rhizobacteria. See, for example, Mabood, Fazli, "Linoleic and linolenic acid induce the expression of nod genes in Bradyrhizobium japonicum," USDA 3, May 17, 2001.

Useful derivatives of fatty acids that may be useful in the practice of the present invention include esters, amides, glycosides and salts. Typical esters are compounds in which the carboxy group of a fatty acid, for example linoleic acid and linolenic acid, is replaced by a --COR group, in which R is a --OR ¹ group, in which R ¹ is: an alkyl group, such as straight or branched C ₁ -C ₈ alkyl group, for example, methyl, ethyl or propyl group; an alkenyl group, such as a straight or branched C ₂ -C ₈ alkenyl group; an alkynyl group such as a straight or branched C ₂ -C ₈ alkynyl group; an aryl group containing, for example, from 6 to 10 carbon atoms; or a heteroaryl group containing, for example, from 4 to 9 carbon atoms, where the heteroatoms in the heteroaryl group can be, for example, N, O, P or S. Typical amides are compounds in which the carboxy group of a fatty acid, for example, linoleic acid and linolenic an acid replaced by a --COR group in which R is an NR ² R ³ group in which R ² and R ^{3 are} independently hydrogen; an alkyl group such as a straight or branched C ₁ -C ₈ alkyl group, for example a methyl, ethyl or propyl group; an alkenyl group, such as a straight or branched C ₂ -C ₈ alkenyl group; an alkynyl group such as a straight or branched C ₂ -C ₈ alkynyl group; an aryl group containing, for example, from 6 to 10 carbon atoms; or a heteroaryl group containing, for example, from 4 to 9 carbon atoms, where the heteroatoms in the heteroaryl group can be, for example, N, O, P or S. Esters can be obtained by known methods, such as acid-catalyzed nucleophilic addition, in which carboxylic acid interacts with alcohol in the presence of a catalytic amount of an inorganic acid. Amides can also be obtained by known methods, for example, by reacting a carboxylic acid with the corresponding amine in the presence of a coupling reagent, such as dicyclohexylcarbodiimide (DCC), in a neutral medium. Suitable salts of fatty acids, for example linoleic acid and linolenic acid, include, for example, base addition salts. Bases that can be used as reagents to produce the metabolically acceptable salts of these compounds with bases include those formed from cations, such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium). These salts can be easily prepared by mixing a fatty acid solution with a base solution. Salt can be precipitated from the solution and collected by filtration, or can be recovered by other means, such as evaporation of the solvent.

The amounts of fatty acid or a derivative thereof used to treat soybean seeds or soybean plants typically range from about 10% to about 30%, and in some embodiments, about 25%, based on the amount of at least one XO.

Plant signaling molecules

The present invention may also include treating soybean or soybean seeds with a non-XO plant signal molecule. For the purposes of the present invention, the term “plant signal molecule”, which can be used interchangeably with the term “plant growth enhancer”, in the broad sense means any agent naturally found in plants or microbes, and synthetic (and which may not be present in nature), which directly or indirectly activates the biochemical pathway of the plant, which leads to enhanced growth of soybean plants, which can be measured using at least one of the following: increased plant yield Measured in units bu / acre, an increased amount of roots, increased root length, enhanced root mass, enhanced root volume and increased leaf area compared with untreated plants soybean or soybean plants, grown from untreated seeds of soybean. Typical examples of plant signaling molecules that may be useful in the practice of the present invention include lipo-chitooligosaccharides, chitin compounds (other than XO), flavonoids, jasmonic acid, linoleic acid and linolenic acid and their derivatives (see above) and carricins and their derivatives.

Lipo-chitooligosaccharides (LHOs), also known in the art as symbiotic Nod signals or Nod factors, consist of the oligosaccharide backbone of β-1,4-linked N-acetyl-D-glucosamine ("GIcNAc") residues attached to N atom with a fatty acyl chain condensed at the non-reducing end. LHOs differ in the number of GIcNAc residues in the main chain, in the length and degree of saturation of the fatty acyl chain, and the degree of substitution with reducing and non-reducing sugar residues. See, for example, Denarie, et al., Ann. Rev. Biochem. 65: 503-35 (1996), Hamel, et al., Supra., Prome, et al., Pure  Appl. Chem. 70 (1): 55-60 (1998). An example of LHO is given below in the form of formula I

,

,

wherein:

G means hexosamine, which may be substituted, for example, with an acetyl group at the nitrogen atom, a sulfate group, an acetyl group and / or an ether group at the oxygen atom,

R ₁ , R ₂ , R ₃ , R ₅ , R ₆ and R ₇ , which may be the same or different, mean H, CH ₃ CO--, C _x H _y CO--, where x is an integer equal to 0 to 17, and y is an integer of 1 and 35, or any other acyl group, for example, carbamoyl,

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

LHO can be obtained (isolated and / or purified) from bacteria such as Rhizobia, for example, Rhizobium sp., Bradyrhizobium sp., Sinorhizobium sp. and Azorhizobium sp. The structure of LHO is characteristic for each such type of bacteria, and each strain can produce many LHOs with different structures. For example, specific LHOs from S. meliloti have also been described in U.S. Patent. 5549718, as having the formula II:

,

,

in which R is H or CH ₃ CO-- and n is 2 or 3.

Even more specific LHOs include NodRM, NodRM-1, NodRM-3. In the case of acetylation (R = CH ₃ CO--), they are converted to AcNodRM-1 and AcNodRM-3, respectively (patent US 5545718).

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

LHOs used in embodiments of the present invention can be obtained (i.e., isolated and / or purified) from bacterial strains that produce LHO, such as strains of Azorhizobium, Bradyrhizobium (including B. japonicum), Mesorhizobium, Rhizobium (including R. leguminosarum), Sinorhizobium (including S. meliloti) and bacterial strains that have been given genetic engineering ability to produce LHO.

LHOs are the primary determinants of host specificity in legume symbiosis (Diaz, et al., Mol. Plant-Microbe Interactions 13: 268-276 (2000)). Thus, in the legume family, specific genera and species of mycorrhiza form a symbiotic nitrogen-binding relationship with a specific legume host. These plant-host / bacterial combinations are described in Hungria, et al., Soil Biol. Biochem. 29: 819-830 (1997). Examples of this symbiotic partnership of bacteria / legumes include S. meliloti / alfalfa and white melilot; R. leguminosarum biovar viciae / peas and lentils; R. leguminosarum biovar phaseoli / beans; Bradyrhizobium japonicum / soybeans; and R. leguminosarum biovar trifolii / red clover. The Hungria publication also lists the effective inducers of the flavonoid gene Nod of rhizobial species and the specific structures of LHOs that are produced by various rhizobial species. However, the specificity of LHO is necessary only for the formation of nodule formation in leguminous plants. In the practical implementation of the present invention, the use of this LHO is not limited to treating the seeds of its symbiotic bean partner to provide increased plant yields, measured in units of bushel / acre, increased number of roots, increased root length, increased root mass, increased root volume and increased foliage area by compared to plants grown from untreated seeds, or compared to plants grown from seeds treated directly with signaling molecules but before sowing, or for a week or less time after sowing.

Thus, as further examples, LHOs and their non-naturally occurring synthetic derivatives that can be used in the practice of the present invention are described by the following formula:

,

,

in which R ₁ means C14: 0, 3OH-C14: 0, iso-C15: 0, C16: 0, 3-OH-C16: 0, iso-C15: 0, C16: 1, C16: 2, C16: 3 , iso-C17: 0, iso-C17: 1, C18: 0, 3OH-C18: 0, C18: 0/3-OH, C18: 1, OH-C18: 1, C18: 2, C18: 3, C18 : 4, C19: 1 carbamoyl, C20: 0, C20: 1, 3-OH-C20: 1, C20: 1/3-OH, C20: 2, C20: 3, C22: 1 and C18-26 (ω- 1) -OH (which, according to the publication of D'Haeze, et al., See above, include C18, C20, C22, C24 and C26 hydroxylated compounds and C16: 1Δ9, C16: 2 (Δ2.9) and C16: 3 (Δ2,4,9)); R ₂ means hydrogen or methyl; R ₃ means hydrogen, acetyl or carbamoyl; R ₄ means hydrogen, acetyl or carbamoyl; R ₅ means hydrogen, acetyl or carbamoyl; R ₆ means hydrogen, arabinosyl, fucosyl, acetyl, sulfate, 3-0-S-2-0-MeFuc, 2-0-MeFuc and 4-0-AcFuc; R ₇ means hydrogen, mannosyl or glycerin; R ₈ is hydrogen, methyl or —CH ₂ OH; R ₉ means hydrogen, arabinosyl or fucosyl; R ₁₀ means hydrogen, acetyl or fucosyl; and n is 0, 1, 2, or 3. The structures of natural rhizobial LHOs described by this structure are described in D'Haeze, et al., supra.

As other further examples, LHO obtained from B. japonicum shown in FIG. 1b can be used to treat non-soy bean seeds and non-leguminous seeds such as corn. As another example, LHO, which can be obtained from R. leguminosarum shown in FIG. 2b (designated LHO-V (C18: 1), SP104) can be used to treat the seeds of leguminous plants other than peas and also non-leguminous plants.

The scope of the present invention also encompasses the use of LHOs obtained (i.e., isolated and / or purified) from mycorrhizal fungi, such as Glomerocycota fungi, for example Glomus intraradices. The structures of typical LHO obtained from these fungi are described in WO 2010/049751 and WO 2010/049751 (the LHO described therein is also referred to as “Myc factors”). Typical chemical agents formed from mycorrhizal fungi, and their derivatives not found in nature, are described by the following structure:

,

,

in which n = 1 or 2; R ₁ means C16, C16: 0, C16: 1, C16: 2, C18: 0, C18: 1Δ9Z or C18: 1Δ11Z; and R ₂ is hydrogen or SO ₃ H. In some embodiments, the LHOs produced by the mycorrhizal fungi shown in FIG. 3b and 4b.

The scope of the present invention also includes the use of synthetic LHO compounds, such as those described in WO 2005/063784, and recombinant LHO obtained by genetic engineering. The basic structure of natural LWOs may contain modifications or substituents found in natural LWOs, such as those described in Spaink, Crit. Rev. Plant Sci. 54: 257-288 (2000) and D′Haeze, et al., Glycobiology 12: 79R-105R (2002). Precursor oligosaccharide molecules (XO, which, as described below, are also applicable as plant signaling molecules in the present invention) used to obtain LXO, can also synthesize genetically engineered microorganisms, for example, as described in Samain publications, et al., Carbohydrate Res. 302: 35-42 (1997); Cottaz, et al., Met. Eng. 7 (4): 311-7 (2005) and Samain, et al., J. Biotechnol. 72: 33-47 (1999) (for example, FIG. 1 in this publication, which shows LHO structures that can be formed recombinantly in E. coli containing various combinations of nodBCHL genes).

You can use LHO of different purity and they can be used in pure form or in the form of a culture of LHO producing bacteria or fungi. For example, OPTIMIZE® (sold by Novozymes BioAg Limited) contains a B. japonicum culture that produces LHO (LHO-V (C18: 1, MeFuc), MOR116), which is shown in FIG. 1b. Techniques for preparing substantially pure LHOs include simply removing microbial cells from a mixture of LHO and microbes, or continuing to isolate and purify LHO molecules by separating the LHO phase of the solvent, followed by purification by HPLC (high performance liquid chromatography), as described, for example, in U.S. Patent. 5549718. Purification can be improved by repeating HPLC and for long-term storage, purified LHO molecules can be freeze dried. Chitooligosaccharides (XO) described above can be used as starting materials for the production of synthetic LHO. For the purposes of the present invention, recombinant LHOs have a purity of at least 60%, for example, purity of at least 60%, purity of at least 65%, purity of at least 70%, purity of at least 75%, purity of at least 80%, purity of at least 85%, purity of at least 90%, purity of at least 91%, purity of at least 92%, purity of at least 93%, purity of at least 94%, with a purity of at least 95%, with a purity of boiling at least 96%, a purity of not less than 97%, a purity of not less than 98%, a purity of not less than 99%, up to a purity of 100%.

Chitins and chitosans, which are the main components of the cell walls of fungi and exoskeletons of insects and crustaceans, also consist of GIcNAc residues. Chitin compounds include chitin (IUPAC: N- [5 - [[3-acetylamino-4,5-dihydroxy-6- (hydroxymethyl) oxan-2-yl] methoxymethyl] -2 - [[5-acetylamino-4,6- dihydroxy-2- (hydroxymethyl) oxan-3-yl] methoxymethyl] -4-hydroxy-6- (hydroxymethyl) oxan-3-yl] ethanamide) and chitosan (IUPAC: 5-amino-6- [5-amino-6 - [5-amino-4,6-dihydroxy-2 (hydroxymethyl) oxan-3-yl] oxy-4-hydroxy-2- (hydroxymethyl) oxan-3-yl] oxy-2 (hydroxymethyl) oxan-3,4 diol). These compounds can be obtained, for example, from Sigma-Aldrich, or obtained from insects, crustacean shells or fungal cell walls. Techniques for producing chitin and chitosan are known in the art and are described, for example, in U.S. Patent. 4,536,207 (obtaining from crustacean shells), Pochanavanich, et al., Lett. Appl. Microbiol. 35: 17-21 (2002) (preparation from fungal cell walls), and in U.S. 5965545 (production of crab from shells and hydrolysis of commercially available chitosan). See also Jung, et al., Carbohydrate Polymers 67: 256-59 (2007); Khan, et al., Photosynthetica 40 (4): 621-4 (2002). You can get deacetylated chitins and chitosans, the degree of deacetylation of which is from less than 35% to more than 90%, and which are characterized by a wide range of molecular masses, for example, low molecular weight chitosan oligomers having molecular masses of less than 15 kDa, and chitin oligomers having molecular masses equal to from 0.5 to 2 kDa; a “practical brand” of chitosan having a molecular weight of about 150 kDa; and high molecular weight chitosan having a molecular weight of up to 700 kDa. Chitin and chitosan formulations prepared for seed treatment are also commercially available. Commercially available products include, for example, ELEXA® (Plant Defense Boosters, Inc.) and BEYOND ™ (Agrihouse, Inc.).

Flavonoids are phenolic compounds, the general structure of which contains two aromatic rings connected by a bridge of three carbon atoms. Flavonoids are produced by plants and perform many functions, for example, they are excellent signaling molecules and protection against insects, animals, fungi and bacteria. Classes of flavonoids include chalcones, anthocyanidins, coumarins, flavones, flavanols, flavonols, flavanones and isoflavones. See publications Jain, et al., J. Plant Biochem. & Biotechnol. 11: 1-10 (2002); Shaw, et al. Environmental Microbiol. 11: 1867-80 (2006).

Typical flavonoids that may be useful in the practice of the present invention include genistein, daidzein, formononetin, naringenin, hesperitin, luteolin and apigenin. Flavonoid compounds are commercially available from, for example, 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 U.S. patents. 5,702,752; 5,990,291; and 6146668. Flavonoid compounds can also produce genetically engineered microorganisms, such as yeast, as described in Ralston, et al., Plant Physiology 137: 1375-88 (2005).

Jasmonic acid (JA, [1R- [1α, 2β (Z)]] - 3-oxo-2- (pentenyl) cyclopentane acetic acid) and its derivatives (which include linoleic acid and linolenic acid (which are described above in connection with fatty acids and their derivatives) can be used in the practice of the present invention Jasmonic acid and its methyl ester, methyl jasmonate (MeJA), collectively known as jasmonates, are octadecanoid-based compounds that are naturally found in plants. The roots of wheat germ and produce jasmonic acid gr 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 obtained during the biosynthesis of jasmonic acid. Linoleic acid and linoleic acid are reported to (and their derivatives) are inducers of nod gene expression or LHO production by rhizobacteria See, for example, Mabood, Fazli, Jasmonates induce the expression of nod genes in Bradyrhizobium japonicum, May 17, 2001.

Useful jasmonic acid derivatives that may be useful in the practice of the present invention include esters, amides, glycosides, and salts. Typical esters are compounds in which the carboxy group of jasmonic acid is replaced by a —COR group, in which R is a —OR ¹ group, in which R ¹ is an alkyl group, such as a straight or branched C ₁ -C ₈ alkyl group, for example methyl, ethyl or propyl group; an alkenyl group, such as a straight or branched C ₂ -C ₈ alkenyl group; an alkynyl group such as a straight or branched C ₂ -C ₈ alkynyl group; an aryl group containing, for example, from 6 to 10 carbon atoms; or a heteroaryl group containing, for example, from 4 to 9 carbon atoms, where the heteroatoms in the heteroaryl group can be, for example, N, O, P or S. Typical amides are compounds in which the carboxy group of jasmonic acid is replaced by the group --COR, in where R means a group NR ² R ³ in which R ² and R ³ independently mean: hydrogen; an alkyl group such as a straight or branched C ₁ -C ₈ alkyl group, for example a methyl, ethyl or propyl group; an alkenyl group, such as a straight or branched C ₂ -C ₈ alkenyl group; an alkynyl group such as a straight or branched C ₂ -C ₈ alkynyl group; an aryl group containing, for example, from 6 to 10 carbon atoms; or a heteroaryl group containing, for example, from 4 to 9 carbon atoms, where the heteroatoms in the heteroaryl group can be, for example, N, O, P or S. Esters can be obtained by known methods, such as acid-catalyzed nucleophilic addition, in which carboxylic acid interacts with alcohol in the presence of a catalytic amount of an inorganic acid. Amides can also be obtained by known methods, for example, by reacting a carboxylic acid with the corresponding amine in the presence of a coupling reagent, such as dicyclohexylcarbodiimide (DCC), in a neutral medium. Suitable jasmonic acid salts include, for example, base addition salts. Bases that can be used as reagents to produce the metabolically acceptable salts of these compounds with bases include those formed from cations, such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium). These salts can be easily obtained by mixing a solution of linoleic acid, linolenic acid or jasmonic acid with a solution of the base. Salt can be precipitated from the solution and collected by filtration, or can be recovered by other means, such as evaporation of the solvent.

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

,

,

in which: Z is O, S or NR ₅ ; R ₁ , R ₂ , R ₃ and R ₄ all independently mean H, alkyl, alkenyl, alkynyl, phenyl, benzyl, hydroxy, hydroxyalkyl, alkoxy, phenyloxy, benzyloxy, CN, COR ₆ , COOR =, halogen, NR ₆ R ₇ or NO ₂ ; and R _5, R ₆ and R ₇ are each independently mean H, alkyl or alkenyl, or a biologically acceptable salt. Examples of biologically acceptable salts of these compounds may include acid addition salts formed with biologically acceptable acids, examples of which include hydrochloride, hydrobromide, sulfate or bisulfate, phosphate or hydrogen phosphate, acetate, benzoate, succinate, fumarate, maleate, lactate, citrate, tartrate, gluconate; methanesulfonate, benzenesulfonate and p-toluenesulfonate. Additional biologically acceptable metal salts may include alkali metal salts with bases, examples of which include sodium and potassium salts. Examples of compounds described by this structure and which may be suitable for use in the present invention include the following: 3-methyl-2H-furo [2,3-c] pyran-2-one (where R ₁ = CH ₃ , R ₂ , R ₃ , R ₄ = H), 2H-furo [2,3-c] pyran-2-one (where R ₁ , R ₂ , R ₃ , R4 = H), 7-methyl-2H-furo [2, 3-c] pyran-2-one (where R ₁ , R ₂ , R ₄ = H, R ₃ = CH ₃ ), 5-methyl-2H-furo [2,3-c] pyran-2-one (where R ₁ , R ₂ , R ₃ = H, R ₄ = CH ₃ ), 3,7-dimethyl-2H-furo [2,3-c] pyran-2-one (where R ₁ , R ₃ = CH ₃ , R ₂ , R ₄ = H), 3,5-dimethyl-2H-furo [2,3-c] pyran-2-one (where R ₁ , R ₄ = CH ₃ , R ₂ , R ₃ = H), 3,5,7-trimethyl-2H-furo [2,3-c] pyran-2-one (where R ₁ , R ₃ , R ₄ = CH ₃ , R ₂ = H), 5-methoxymethyl-3-methyl -2H-furo [2,3-c] pyran-2-one (where R ₁ = CH ₃ , R ₂ , R ₃ = H, R ₄ = CH ₂ OCH ₃ ), 4-bromo-3,7-dimethyl-2H-furo [2,3-c] pyran-2-one (where R ₁ , R ₃ = CH ₃ , R ₂ = Br, R ₄ = H), 3-methylfuro [2,3-c] pyridin-2 (3H) -one (where Z = NH, R ₁ = CH ₃ , R ₂ , R ₃ , R ₄ = H ), 3,6-dimethyl-furo [2,3-c] pyridin-2 (6H) -one (where Z = N - CH _3, R ₁ = CH _3, R _2, R _3, R ₄ = H). See US Pat. No. 7,576,213. These molecules are also known as carricins. See the Halford publication above.

The amount of at least one signaling plant molecule used to treat soybean seeds, expressed in units of concentration, is usually in the range of 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. The effective amount expressed in units of weight is usually in the range of from about 1 to about 400 μg / centner (c) of seed and some embodiments m 2 to 70 g / q, and in certain other embodiments from about 2.5 to about 3.0 g / n seeds.

For indirect processing of soybean seeds, i.e. furrow treatment, an effective amount of at least one signal molecule of a plant is usually in the range of 1 μg / acre to about 70 μg / acre, and in some embodiments, from about 50 μg / acre to about 60 μg / acre. For application to soybean plants, an effective amount of at least one signal molecule of the plant is usually in the range of 1 μg / acre to about 30 μg / acre and in some embodiments, from about 11 μg / acre to about 20 μg / acre.

Herbicides, fungicides and insecticides

Suitable herbicides include bentazone, acifluorfene, chlorimuron, lactophen, clomazone, fluazifop, glufosinate, glyphosate, sethoxydim, imazetapyr, imazamox, fomesaf, flumiclorac, imazahine and globodim. Commercially available products containing each of these compounds are readily available. The concentration of the herbicide in the composition usually corresponds to the rate of application of the specific herbicide indicated on the label.

"Fungicide" when used in the present invention and in the art, means an agent that destroys fungi or inhibits the growth of fungi. When used in the present invention, the fungicide "has activity with respect to" a particular type of fungus, if the fungicide treatment destroys or suppresses the growth of the fungal population (for example, in soil) compared to the untreated population. Effective fungicides in accordance with the present invention will have appropriate activity against a wide range of pathogens, including, but not limited to Phytophthora, Rhizoctonia, Fusarium, Pythium, Phomopsis or Selerotinia and Phakopsora, and combinations thereof.

Commercially available fungicides may be suitable for use in the present invention. Suitable commercially available fungicides include PROTÉGÉ, RIVAL or ALLEGIANCE FL or LS (Gustafson, Plano, TX), WARDEN RTA (Agrilance, St. Paul, MN), APRON XL, APRON MAXX RTA or RFC, MAXIM 4FS or XL (Syngenta, Wilmington, DE), KAPTAN (Arvesta, Guelph, Ontario) and PROTREAT (Nitragin Argentina, Buenos Ares, Argentina). Active ingredients in these and other commercially available fungicides include, but are not limited to, fludioxonil, mefenoxam, azoxystrobin, and metalaxyl. Commercially available fungicides are most preferably used in accordance with the manufacturer's instructions at recommended concentrations.

When used in the present invention, the insecticide "has activity against" a particular type of insect, if the treatment with the insecticide destroys or inhibits the growth of the insect population compared to the untreated population. Effective insecticides in accordance with the present invention will have appropriate activity against a wide range of insects, including, but not limited to wireworms, scoops, larvae, corn beetle, sprout fly larvae, earthen fleas, earthen bugs, aphids, leaf beetles and shield bugs .

Commercially available insecticides may be suitable for use in the present invention. Suitable commercially available insecticides include CRUISER (Syngenta, Wilmington, DE), GAUCHO and PONCHO (Gustafson, Plano, TX). The active ingredients in these and other commercially available insecticides include thiamethoxam, clothianidin, and imidacloprid. Commercially available insecticides are most preferably used in accordance with the manufacturer's instructions at recommended concentrations.

Solubilizing phosphate microorganisms, diazotrophs (rhizobial inoculum) and / or mycorrhizal fungi

The present invention may further include treating the seeds with phosphate solubilizing microorganisms. As used in the present invention, “phosphate solubilizing microorganism” means a microorganism that is capable of increasing the amount of phosphorus available to the plant. Solubilizing phosphate microorganisms include strains of fungi and bacteria. In an embodiment, the phosphate solubilizing microorganism is a spore-forming microorganism.

Non-limiting examples of phosphate-solubilizing microorganisms include species of the genus selected from the group consisting of Acinetobacter, Arthrobacter, Arthrobotrys, Aspergillus, Azospirillum, Bacillus, Burkholderia, Candida Chryseomonas, Enterobacterium, Pauperiobiumerium, Kupella Microbe, Kigella Microbius , Pseudomonas, Serratia, Stenotrophomonas, Streptomyces, Streptosporangium, Swaminathania, Thiobacillus, Torulospora, Vibrio, Xanthobacter and Xanthomonas.

Non-limiting examples of phosphate-solubilizing microorganisms are selected from the group consisting of Acinetobacter calcoaceticus, Acinetobacter sp, Arthrobacter sp., Arthrobotrys oligospora, Aspergillus niger, Aspergillus sp., Azospirillacillus bilillius bilillus francis bacillus amylis bilillifens , Burkholderia vietnamiensis, Candida krissii, Chryseomonas luteola, Enterobacter aerogenes, Enterobacter asburiae, Enterobacter sp., Enterobacter taylorae, Eupenicillium parvum, Exiguobacterium sp., Klebsiella sp., Klucimececium ricemicecium, Icocomeccemsracemiumcericeramis riumcicemsmaceramisriceramisceramisrymeciumceramis rimecismeria Paenibacillus macerans, Paenibacillus mucilaginosus, Pantoea aglomerans, Penicillium expansum, Pseudomonas corrugate, Pseudomonas fluorescens, Pseudomonas lutea, Pseudomonas poae, Pseudomonas putida, Pseudom onas stutzeri, Pseudomonas trivialis, Serratia marcescens, Stenotrophomonas maltophilia, Streptomyces sp., Streptosporangium sp., Swaminathania salitolerans, Thiobacillus ferrooxidans, Torulospora globosa, Vibrio proteolyticus, Xanthanthantobrisanthantobrisanthus

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

In one preferred embodiment, the Penicillium species is P. bilaiae. In another preferred embodiment, P. bilaiae strains are selected from the group consisting of ATCC 20851, NRRL 50169, ATCC 22348, ATCC 18309, NRRL 50162 (Wakelin, et al., 2004. Biol Fertil Soils 40: 36-43). In another preferred embodiment, the Penicillium species is P. gaestrivorus, for example, NRRL 50170 (see Wakelin above).

In some embodiments, more than one phosphate solubilizing microorganism is used, for example at least 2, at least 3, at least 4, at least 5, and at least 6, including any combination of Acinetobacter, Arthrobacter, Arthrobotrys, Aspergillus, Azospirillum , Bacillus, Burkholderia, Candida Chryseomonas, Enterobacter, Eupenicillium, Exiguobacterium, Klebsiella, Kluyvera, Microbacterium, Mucor, Paecilomyces, Paenibacillus, Penicillium, Pseudomonas, Serratia, Stenotrophomonas, Streptomyces, Streptosporangium, Swaminathania, Thiobacillus, Torulospora, Vibrio, Xanthobacte and Xanthomonas, including one species selected from the following group: Acinetobacter calcoaceticus, Acinetobacter sp, Arthrobacter sp., Arthrobotrys oligospora, Aspe rgillus niger, Aspergillus sp., Azospirillum halopraeferans, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans, Bacillus licheniformis, Bacillus subtilis, Burkholderia cepaceroberobeola kerida bida Eupenicillium parvum, Exiguobacterium sp., Klebsiella sp., Kluyvera cryocrescens, Microbacterium sp., Mucor ramosissimus, Paecilomyces hepialid, Paecilomyces marquandii, Paenibacillus macerans, Paenibacillus mucilaginosus, Pantoea aglomerans, Penicillium expansum, Pseudomonas corrugate, Pseudomonas fluorescens, Pseudomonas lutea, Pseudomonas poae , Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas trivialis, Serratia marcescens, Stenotrophomonas maltophilia, Streptomyces sp., Streptosporangium sp., Swaminathania salitolerans, Thiobacillus ferrooxidans, Torulososporhobrobolosa globola

In some embodiments, two different strains of the same species can also be combined, for example, at least two different Penicillium strains are used. The use of a combination of at least two different Penicillium strains has the following advantages. When applied to soil already containing insoluble (or moderately soluble) phosphates, the use of combined fungal strains will lead to an increase in the amount of phosphorus available for assimilation by plants, compared to the case of using only one Penicillium strain. In turn, this can lead to an increase in the absorption of phosphate and / or an increase in the yield of plants growing on this soil, in comparison with the cases when only certain strains are used. The combination of strains also allows the use of insoluble phosphorites as an effective fertilizer for soils that contain insufficient amounts of available phosphorus. Thus, in some embodiments, one strain of P. bilaiae and one strain of P. gaestrivorus are used. In other embodiments, these two strains are NRRL 50169 and NRRL 50162. In other embodiments, these at least two strains are NRRL 50169 and NRRL 50170. In other embodiments, these at least two strains are NRRL 50162 and NRRL 50170.

Phosphate-soluble microorganisms can be prepared by any suitable technique known to one skilled in the art, such as solid phase or liquid fermentation using a suitable carbon source. The solubilizing phosphate microorganism is preferably obtained as a stable spore.

In one embodiment, the phosphate solubilizing microorganism is Penicillium fungi. Penicillium mushrooms of the present invention can be grown using solid phase or liquid fermentation and a suitable carbon source. Penicillium isolates can be grown by any suitable technique known to a person skilled in the art. For example, mushrooms can be grown in a solid growth medium, such as potato dextrose agar or malt extract agar, or in mattresses containing suitable liquid media, such as Capek-Dox medium or potato-dextrose broth. These growth techniques can be used to prepare the Penicillium spp inoculum. for treating (eg, coating) seeds and / or applying to an agronomically acceptable carrier for application to the soil. The term "inoculum" as used herein means any form of a phosphate solubilizing microorganism, fungal cells, mycelium or spores, bacterial cells or bacterial spores that are capable of multiplying on or in the soil when temperature, humidity, and the like. favorable for mushroom growth.

The solid phase preparation of Penicillium spores can be carried out by plating in a solid medium, such as a substrate based on peat or vermiculite, or grain, including, but not limited to, oats, wheat, barley or rice. The sterilized medium (sterilization is performed by autoclaving or by irradiation) were seeded spore suspension (1 × ¹⁰ February -1 × July ¹⁰ CFU / ml) of a suitable Penicillium spp. and depending on the substrate set the humidity equal to from 20 to 50%. The material is incubated for 2 to 8 weeks at room temperature. Spores can also be obtained by liquid fermentation (Cunningham et al., 1990. Can J Bot. 68: 2270-2274). Liquid production can be carried out by growing mushrooms in any suitable medium, such as potato dextrose broth or medium with sucrose and yeast extract, at suitable pH and temperature values that can be determined by standard procedures used in the art.

The resulting material can be used without treatment or spores can be collected, concentrated by centrifugation, the composition is prepared and then dried by air drying, freeze drying or fluidized bed drying (Friesen, et al., 2005, Appl. Microbiol. Biotechnol. 68: 397 -404) and get a wettable powder. Then the wettable powder is suspended in water, applied to the surface of the seeds and allowed to dry before sowing. Wettable powder may be used in conjunction with other seed treatment agents, such as, but not limited to, seed treatment chemicals, carriers (e.g., talc, clay, kaolin, silica gel, kaolinite) or polymers (e.g. methyl cellulose, polyvinylpyrrolidone) . Alternatively, a spore suspension of a suitable Penicillium spp. can be applied to a suitable soil compatible carrier (e.g. powder or peat granules) to provide a suitable final moisture content. Prior to use, the material can be incubated at room temperature, usually for about 1 day to about 8 weeks.

In addition to the ingredients used to grow the phosphate solubilizing microorganism, including, for example, the ingredients mentioned above for growing Penicillium, other agronomically acceptable carriers can be used in the preparation of the phosphate solubilizing microorganism. As used herein in connection with a “carrier,” the term “agronomically acceptable” means any material that can be used to deliver active substances to a soybean, soil, or soybean plant, and it is preferred that the carrier can be added (to seeds, soil or plant) without adversely affecting plant growth, soil structure, soil drainage, etc. Suitable carriers include, but are not limited to, wheat chaff, bran, chopped wheat straw, peat-based powders or granules, gypsum and clay granules (e.g. kaolin, bentonite, montmorillonite). When spores are added to the soil, a granular formulation is preferred. Liquid formulations using peat or wettable powder are suitable for application to soybean seeds. When used for application on soybean seeds, the material can be mixed with water, applied to the seeds and allowed to dry. An example of yet another carrier includes wet peat, dried, sieved and applied to the seeds before applying glue, such as gum arabic. In those embodiments that include preparing the active substances as a single composition, an agronomically acceptable carrier may be aqueous.

The amount of at least one phosphate solubilizing microorganism varies depending on the type of seed or soil, the type of crop, the amount of phosphorus sources and / or micronutrients contained in or added to the soil, etc. In each case, the appropriate amount can be determined using simple successive approximation techniques. Typically, for example, for Penicillium, the applied amount is in the range of 0.001-1.0 kg of spores of fungi and mycelium (raw weight) per hectare, or 10 ² -10 ⁶ colony forming units (CFU) per one seed (when using seeds coated), or on a granular support, from 1 × 10 ⁶ to 1 × 10 ¹¹ colony forming units are applied per hectare. Fungal cells, for example, in the form of spores and carriers, can be introduced into the seed bed in the soil at the root level or can be used for application to the seeds before planting.

In embodiments where, for example, at least two strains of a phosphate solubilizing microorganism are used, for example, two Penicillium strains, commercially available fertilizers can be introduced into the soil instead of (or even together) with natural phosphorite. The source of phosphorus may contain a source of phosphorus in the soil. In other embodiments, a source of phosphorus can be added to the soil. In one embodiment, the source is phosphorite. In another embodiment, the source is finished fertilizer. There are many types of ready-made phosphate fertilizers on sale. Some common are those containing monoammonium phosphate (MAP), triple superphosphate (TSP), diammonium phosphate, ordinary superphosphate, and ammonium polyphosphate. All these fertilizers are obtained by chemical treatment of insoluble natural phosphorites at large-scale fertilizer manufacturing plants and the product is expensive. Using the present invention, it is possible to reduce the amount of these fertilizers applied to the soil while maintaining the same amount of phosphorus absorbed from the soil.

In another embodiment, the source or phosphorus is organic. Organic fertilizer means a soil supplement derived from natural sources that guarantees at least a minimum content of nitrogen, phosphate and potassium. Examples include plant and animal by-products, powder rocks, algae, bacterial fertilizers, and conditioners. Specific representative examples include bone meal, meat meal, manure, compost, sewage sludge or guano.

Of course, other fertilizers such as nitrogen sources or other soil improvers can also be added to the soil at about the same time as the phosphate solubilizing microorganism, or at other times if these other materials are non-toxic to fungi.

Diazotrophs are bacteria and archibacteria that bind atmospheric nitrogen in a more convenient form, such as ammonia. Examples of diazotrophs include bacteria of the genus Rhizobium spp. (e.g. R. cellulosilyticum, R. daejeonense, R. etli, R. galegae, R. gallicum, R. giardinii, R. hainanense, R. huautlense, R. indigoferae, R. leguminosarum, R. loessense, R. lupini , R. lusitanum, R. meliloti, R. mongolense, R. miluonense, R. sullae, R. tropici, R. undicola and / or R. yanglingense), Bradyrhizobium spp. (e.g. B. bete, B. canariense, B. elkanii, B. iriomotense, B. japonicum, B. jicamae, B. liaoningense, B. pachyrhizi and / or B. yuanmingense), Azorhizobium spp. (e.g. A. caulinodans and / or A. doebereinerae), Sinorhizobium spp. (e.g. S. abri, S. adhaerens, S. americanum, S. aboris, S. fredii, S. indiaense, S. kostiense, S. kummerowiae, S. medicae, S. meliloti, S. mexicanus, S. morelense , S. saheli, S. terangae and / or S. xinjiangense), Mesorhizobium spp., (M. albiziae, M. amorphae, M. chacoense, M. ciceri, M. huakuii, M. loti, M. mediterraneum, M . pluifarium, M. septentrionale, M. temperatureum and / or M. tianshanense) and combinations thereof. In a preferred embodiment, the diazotroph is selected from the group consisting of B. japonicum, R leguminosarum, R meliloti, S. meliloti, and combinations thereof. In another embodiment, the diazotroph is B. japonicum. In another embodiment, the diazotroph is R leguminosarum. In another embodiment, the diazotroph is R meliloti. In another embodiment, the diazotroph is S. meliloti.

Mycorrhizal fungi form a symbiotic association with the roots of a vascular plant and provide, for example, the ability to absorb water and mineral nutrients due to the relatively large surface area of the mycelium. Mycorrhizal fungi include endomycorrhizal fungi (also called vesicular arbuscular mycorrhiza, VAM, arbuscular mycorrhiza, or AM), ectomycorrhizal fungi, or a combination thereof. In one embodiment, the mycorrhizal fungi are endomycorrhiza type Glomeromycota and the genus Glomus and Gigaspora. In yet another embodiment, the endomicorizises are strains 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.

Examples of mycorrhizal fungi include endomycorrhiza type Basidiomycota, Ascomycota and Zygomycota. Other examples include 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.

Mycorrhizal fungi include ericoid mycorrhiza, arbutoid mycorrhiza, or monotropoid mycorrhiza. Arbuscular and ectomycorrhiza form ericoid mycorrhiza with many plants belonging to the order Ericales, and some Ericales form arbutoid and monotropoid mycorrhiza. In one embodiment, the mycorrhiza may be ericoid mycorrhiza, preferably of the type Ascomycota, such as Hymenoscyphous ericae or Oidiodendron sp. In another embodiment, the mycorrhiza may also be an arbutoid mycorrhiza, preferably of the type Basidiomycota. In yet another embodiment, the mycorrhiza may be monotropoid mycorrhiza, preferably of the type Basidiomycota. In yet another embodiment, the mycorrhiza may be orchid mycorrhiza, preferably of the genus Rhizoctonia.

The present invention will be described using the following non-limiting examples. Unless otherwise indicated, water was used as control (indicated as “control” or “CHK”).

Examples

Greenhouse experiments

Example 1: Processing soya with various active substances

Soybean seeds (Jung seeds, cultivar 8168NRR) were treated with various active molecules. The seeds were treated with liquid at a rate of 3 fluid ounces / 100 pounds of seeds. The seeds were allowed to dry for 2 hours and in the greenhouse were sown in plastic pots containing a 1: 1 mixture of sand: perlite. Sprouts were grown for 4 weeks, and liquid fertilizers were added from time to time, and then the plants were harvested. The central leaflet was separated from the second shamrock (from bottom to top) and its surface area was measured using a WinRhizo scanner. The rest of the plant was used to determine the dry weight of the plant (MS).

The results obtained in the experiment showed that not pea LHO proposed in the present invention, pea XO and China XO proposed in the present invention led to a significant increase in leaf surface area. However, from these three active substances, XO peas led to the largest leaf surface area (significantly larger than in the case of control (water)) and relatively larger than Chinese XO (Fig. 5). In another experiment, CW led to the largest masses of plants in a dry state when assessed by the biomass of sprouts or roots and the total biomass of plants. Thus, it was obvious that the increase in biomass when using XO was more significant than when using LHO soy or any other treatment, including water as a control and isoflavonoids as a separate signal molecule of the plant (Fig. 6).

Example 2: Soybean Seed Processing

Soybean seeds (Pioneer 9oM80) were placed in Petri dishes on moistened germination filter paper, soaked in 5 ml of a treatment solution containing water or SHO of soybeans, XO peas and XO with the addition of fatty acids. Sprout roots were separated after 48 hours and their length was measured.

LHO led to a better increase in seed root growth compared to the control and XO, but XO with the addition of palmitic acid or stearic acid led to a significant increase in the root length. XO itself was more effective than LXO for soy, but the addition of fatty acid, palmitic or stearic acid to XO could further enhance the growth of rootlets of sprouts (Fig. 7).

Example 3: Non-Root Processing of Soybean with Various Active Substances

Soybean seeds (Jung seeds, cultivar 8168NRR) were treated with various active molecules at the V4 growth stage. Plants were grown from seeds in a greenhouse in plastic pots containing a 1: 1 mixture of sand: perlite. Sprouts were grown for 4 weeks, and liquid fertilizers were added from time to time, and then the plants were harvested.

The foliar application of LHO of soybean, XO pea or China-XO did not lead to a significant effect on the increase in the biomass of plants in the dry state (Fig. 8). When using LHO, XO and China-XO, the biomass was relatively larger than for control, and the active substances were equally effective.

18-20: Field Research

Example 4: Soy

To evaluate the embodiments of the present invention with respect to grain yield when foliarly applied to soybeans, 19 field studies were conducted. Field studies were carried out in 8 states on different soils and under different environmental conditions.

In studies, the treatment was carried out with water (control), pure XO (chitooligosaccharide) - XO-V (shown in Fig. 2a) and pure LXO (lipochitooligosaccharide) - SP104 (presented in Fig. 2b). Processing with CW and Lho carried out at a molar concentration of 8 × 10 ^-8, which corresponded to the introduction of 12 g / acre. Used a variety of commercially available soybean varieties. The agents were added to the glyphosate herbicide and sprayed onto the leaves of the plants at the vegetative stage of V4-V5. At 4 ounces / acre, the processing aids combined with the herbicide and water were added at a rate of 5 to 10 gallons per acre. Soybeans were grown to maturity and the grain mass was collected and determined.

The results are shown in table 1.

Table 1 YIELD (bushel / acre) The control LHO (SP104) XO (XO-V) The average value (N = 19) 56.5 58.3 58.2 Rise (Bushel / Acre) 1.8 1.7 Increase (% compared to control) 3% 3% Percentage of studies that increased yield (%) 68,4 68,4

As a comparison of control with XO shows, with non-root treatment with XO, the yield increased by 1.7 bushels / acre, which corresponded to a 3% increase compared to the control, and yield increased in 68.4% of studies.

Compared to the increase in non-root treatment with LHC, in the case of XO, the average increase was 0.1 bushel / acre, which is less, but it represents the same percentage increase in productivity compared to the control and the same percentage of studies expressed as a percentage at which productivity increased. Therefore, with non-root treatment, both XO and LHO led to approximately the same increase in yield.

All patent and non-patent publications cited in the present description, are characteristic of the level of training of specialists in the field of technology to which the present invention relates. All of these publications are incorporated by reference to the same extent as if for each individual publication or patent application it was specifically and individually indicated to be included as a reference.

Although the present invention has been described using specific embodiments, it should be understood that these embodiments only illustrate the principles and applications of the present invention. Therefore, it must be understood that numerous changes can be made to these illustrative embodiments, and that other combinations can be formed without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims (63)

1. A method of enhancing the growth of a soybean plant, comprising treating soybean seeds and / or a soybean plant that germinates from soybean seeds with an effective amount of at least one chitooligosaccharide (XO) represented by the formula:

wherein
R ₁ means hydrogen or methyl;
R ₂ means hydrogen or methyl;
R ₃ means hydrogen, acetyl or carbamoyl;
R ₄ means hydrogen, acetyl or carbamoyl;
R ₅ means hydrogen, acetyl or carbamoyl;
R ₆ means hydrogen, arabinosyl, fucosyl, acetyl, sulfate, 3-0-S-2-0-MeFuc, 2-0-MeFuc and 4-0-AcFuc;
R ₇ means hydrogen, mannosyl or glycerin;
R ₈ is hydrogen, methyl or —CH ₂ OH;
R ₉ means hydrogen, arabinosyl or fucosyl;
R ₁₀ means hydrogen, acetyl or fucosyl; and
n is 0, 1, 2 or 3; and
where, after harvesting, the soybean plant is characterized by at least one of the following: increased plant productivity, measured in units of bushel / acre, increased number of roots, increased root length, increased root mass, increased root volume and increased foliage area, compared to untreated soy plants and / or soy plants grown from untreated soybean seeds. 2. The method according to p. 1, in which at least one HO includes HO, represented by the formula:

3. The method according to p. 1, in which at least one HO includes HO, represented by the structure:

4. The method according to p. 1, in which at least one HO includes HO, represented by the formula:

in which n = 1 or 2; R ₁ means hydrogen or methyl; and R ₂ means hydrogen or SO ₃ H. 5. The method according to p. 1, in which at least one HO includes HO, represented by the structure:

6. The method according to p. 1, in which at least one HO includes HO, represented by the structure:

7. The method according to p. 1, in which at least one CW includes synthetic CW. 8. The method according to p. 1, in which at least one HO includes recombinant HO. 9. The method of claim 8, wherein the recombinant XO is at least 60% pure. 10. The method according to p. 8, in which the recombinant XO has at least 70% purity. 11. The method according to p. 8, in which the recombinant XO has at least 80% purity. 12. The method according to p. 8, in which the recombinant XO has at least 90% purity. 13. The method according to p. 1, in which at least one CW is applied to soybean seeds before sowing and / or around the time of sowing. 14. The method according to p. 1, in which at least one CW is applied to the seeds at least one month before sowing. 15. The method according to p. 1, in which at least one CW is applied to the seeds at least two months before sowing. 16. The method according to p. 1, in which at least one CW is applied to the seeds at least three months before sowing. 17. The method according to p. 1, in which at least one CW is applied to the seeds at least six months before sowing. 18. The method according to p. 1, in which at least one CW is applied to the seeds at least one year before sowing. 19. The method according to p. 1, in which at least one CW is applied to soybean seeds in furrows. 20. The method according to p. 1, in which at least one CW is applied to the soybean plant using foliar treatment. 21. The method according to p. 1, in which the effective amount of at least one CW is from about 10 ^-5 to about 10 ^-14 M. 22. The method according to p. 1, in which the effective amount of at least one XO is from about 10 ^-5 to about 10 ^-11 M. 23. The method according to p. 1, in which an effective amount of at least one XO is from about 10 ^-7 to about 10 ^-8 M. 24. The method of claim 1, wherein the effective amount of at least one CI is from about 1 μg / acre to about 70 μg / acre. 25. The method of claim 1, wherein the effective amount of at least one CI is from about 1 μg / acre to about 30 μg / acre. 26. The method of claim 1, wherein the effective amount of at least one CI is from about 11 μg / acre to about 20 μg / acre. 27. The method of claim 1, wherein the effective amount of at least one CI is from about 50 μg / acre to about 60 μg / acre. 28. The method according to claim 1, further comprising applying to the soybean seeds and / or the plant, which sprouts from soybean seeds, at least one micronutrient. 29. The method of claim 28, wherein the at least one micronutrient comprises vitamins and / or micronutrients. 30. The method according to p. 1, further comprising applying to soybean seeds and / or a plant that germinates from soybean seeds, a fatty acid or its derivative. 31. The method according to p. 1, further comprising applying to soybean seeds and / or a plant that germinates from soybean seeds, at least one signal molecule of the plant. 32. The method according to p. 31, in which at least one signal molecule of the plant includes at least one lipochito-oligosaccharide (LHO). 33. The method according to p. 32, in which at least one LHO includes LHO, represented by the structure:

34. The method according to p. 32, in which at least one LHO includes LHO, represented by the structure:

35. The method according to p. 32, in which at least one LHO includes LHO, represented by the structure:

36. The method according to p. 32, in which at least one LHO includes LHO, represented by the structure:

37. The method according to p. 32, in which at least one LHO includes at least one LHO obtained from a strain of Rhizobium, Sinorhizobium, Azorhizobium, Mesorhizobium or Bradyrhizobium. 38. The method according to p. 32, in which at least one LHO includes at least one LHO obtained from a strain of Bradyrhizobium japonicum. 39. The method according to p. 32, in which at least one LHO includes at least one LHO obtained from a strain of Sinorhizobium meliloti. 40. The method according to p. 32, in which at least one LHO includes at least one LHO obtained from a strain of Rhizobium leguminosarum. 41. The method according to p. 32, in which at least one LHO includes at least one LHO obtained from mycorrhizal fungi. 42. The method according to p. 32, in which at least one LHO includes at least one LHO obtained from a Glomerocycota strain. 43. The method according to p. 32, in which at least one LHO includes at least one LHO obtained from a Glomus intraradices strain. 44. The method of claim 31, wherein the at least one plant signal molecule comprises a plant signal molecule selected from the group consisting of chitin compounds, flavonoids, jasmonic acid and its derivatives, linoleic acid and its derivatives, linolenic acid and its derivatives and carrickins and their derivatives. 45. The method of claim 31, wherein the at least one signaling molecule of the plant comprises chitin and / or chitosan. 46. The method according to claim 1, further comprising applying to soybean seeds and / or a plant that germinates from soybean seeds, one or more herbicides, insecticides and / or fungicides. 47. The method according to p. 1, further comprising applying to soybean seeds and / or a plant that sprouts from soybean seeds, at least one solubilizing phosphate microorganism. 48. The method of claim 47, wherein the at least one phosphate solubilizing microorganism comprises at least one Penicillium strain of fungi. 49. The method of claim 47, wherein the at least one phosphate solubilizing microorganism comprises at least one strain of P. bilaiae. 50. The method according to p. 49, in which at least one strain of P. bilaiae includes NRRL 50162, NRRL 50169, ATCC 20851, ATCC 22348 and / or ATCC 18309. 51. The method of claim 47, wherein the at least one phosphate solubilizing microorganism comprises at least one strain of P. gaestrivorus. 52. The method of claim 51, wherein the at least one P. gaestrivorus strain comprises NRRL 50170. 53. The method according to any one of paragraphs. 47-52, in which at least one solubilizing phosphate microorganism includes at least one strain of Streptomyces. 54. The method according to any one of paragraphs. 1-46, further comprising applying to the soybean seeds and / or a plant that sprouts from soybean seeds, at least one diazotroph. 55. The method of claim 54, wherein said at least one diazotroph includes one or more strains of Rhizobium, Sinorhizobium, Azorrhizobium, Mesorhizobium and / or Bradyrhizobium. 56. The method according to any one of paragraphs. 54-55, wherein said at least one diazotroph includes at least one strain of Bradyrhizobium japonicum. 57. The method according to any one of paragraphs. 54-55, wherein said at least one diazotroph includes at least one strain of Sinorhizobium meliloti. 58. The method according to any one of paragraphs. 54-55, wherein said at least one diazotroph includes at least one strain of Rhizobium leguminosarum. 59. The method according to any one of paragraphs. 54-55, wherein said at least one diazotroph includes at least one strain of Mesorhizobium ciceri. 60. The method according to any one of paragraphs. 1-46, further comprising applying to the soybean seeds and / or a plant that sprouts from soybean seeds, at least one mycorrhizal fungus. 61. The method of claim 60, wherein said at least one mycorrhizal fungus comprises at least one Glomerocycota strain. 62. Seed processed in accordance with the method according to any one of paragraphs. 1-61. 63. A plant sprouted from a seed according to paragraph 62.

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