Seed treatment methods

[0001] The symbiosis between Gram-negative soil bacteria, Rhizobiaceae and Bradyrhizobiaceae, and legumes, such as soy, is well documented. The biochemical basis for such relationships includes the exchange of molecular signaling in which plant-to-bacterial signaling compounds include flavones, isoflavones and flavanones, and bacterial-to-plant signal compounds include end products of the expression of bradyrhysobial and rhizobial nod genes, known as lipo-chitooligosaccharides ( LCO). The symbiosis between these bacteria and legumes allows the legume to bind nitrogen from the atmosphere and thus grow in soil that contains low levels of available nitrogen, which eliminates the need for nitrogen fertilizers. Due to the,

[0002] WO2005 / 087005 discloses the use of one or more signal isoflavonoid compounds that can be used with an agriculturally acceptable support for application before sowing / planting.

[0003] WO20099 / 049747 discloses a biological composition for enhancing crop growth and crop yield comprising a lipo-chitooligosaccharide and a flavonoid compound comprising flavones, flavanols, flavonols, flavanones or isoflavones.

[0004] US 6979664 discloses a method of increasing seed germination or seedling emergence in crop cultivation, comprising the steps of providing a composition that comprises an effective amount of at least one lipo-chitooligosaccharide and suitable for agricultural use, and applying the composition in the immediate vicinity of seeds or cuttings in an amount effective to increase seed germination or seedling emergence compared to untreated seeds or seedlings.

[0005] A further development of this concept disclosed in WO 2005/062899 relates to combinations of at least one plant growth stimulator, more specifically LCO, in combination with a fungicide, an insecticide or a combination thereof, to improve the plant characteristics such as habit, growth, viability and / or yield of plants. It is disclosed that the compositions and methods are useful for both legumes and non-legumes and can be used to treat seeds (just before sowing), seedlings, roots or plants.

Similarly, WO 2008/085958 discloses compositions for enhancing plant growth and yield crops, both legumes and non-legumes, which contain LCO in combination with another active agent, such as chitin or chitosan, a flavonoid compound or a herbicide, and which can be applied to seeds and / or plants simultaneously or successively. As with '899, publication' 958 discloses the treatment of seeds just before sowing.

[0007] A number of other publications describe the benefits associated with LCO in seed treatment methods, such as, Kidaj et al., "Microbiol Res 25426 (2011)," Nod factors stimulate seed germination and promoting growth and development of pea and vetch under competitive conditions ". , and May et al., "Pretreatment of Clover Seeds with Nod Factors Improves Growth and Nodulation of Trifolium pratense," J. Chem Ecol (2009) 35: 479-487.

[0008] Recently in Halford, "Smoke Signals," in Chem. Eng. News (April 12, 2010) on pages 37-38 has been reported that karrikiny or butenolides, which are contained in smoke, act as growth promoters and stimulate seed germination after forest fires and can stimulate stored plant seeds such as corn, tomatoes, lettuce and onion. These molecules are subject to US Patent No. 7,766,213.

BRIEF SUMMARY OF THE INVENTION [0009] The present invention provides methods for enhancing plant growth and plant production in which a beneficial effect of a plant signal molecule (plant growth promoting agent) can be achieved without the need to apply a plant signal molecule (plant growth enhancer) to seeds simultaneously with seeding. The present invention is based, in part, on the discovery that seed dressing with a plant signal molecule, which is LCO, followed by prolonged storage before sowing, results in enhanced plant growth, including increased plant yield and / or leaf area, and / or number, length and the weight of the roots, compared to the plants harvested from both uncultivated seeds. The present invention also provides methods for enhancing plant growth and plant production,

[0010] A first aspect of the present invention relates to a method of enhancing plant growth, comprising treating a seed at least one month (thirty days) before sowing with an effective amount of a plant signal molecule, which plant signal molecule is lipo-chitooligosaccharide. In embodiments, the seeds may be treated in accordance with the method of the present invention 2 months before sowing, for at least 3 months before sowing, for at least 5 months prior to sowing, for at least 5 months prior to sowing, for at least 6 months before sowing, for at least 9 months before sowing, for at least 1 year before sowing, for at least 2 years before sowing, and in certain embodiments at least 3 years before sowing.

[0011] The treatment is applied to obtain a plant (crop) that exhibits at least one parameter from the increased yield measured in bushels / acres, increased root numbers, increased root length, increased root mass, increased root volume and increased leaf area, compared to plants harvested from cultivation of unrepaired seeds. In specific embodiments, the treatment can be used to obtain a plant (crop) that exhibits at least one parameter from the increased yield measured in bushels / acres, increased root numbers, increased root length, increased root mass, increased root volume and increased leaf area compared to with a plant (cultivation) harvested from growing from seed treated with a signal molecule just before,

[0012] In the present invention, the plant signal molecule is lipochitooligosaccharide (LCO). In certain embodiments, the LCO is recombinant. In other embodiments, the LCO is synthetic. In other embodiments, the LCO is obtained from a microorganism, e.g. from Rhizobia species selected from Rhizobium sp., Bradyrhizobium sp., E.g. Bradyrhizobium japonicum, Sinorhizobium sp. And Azorhizobium sp. Or from an arbuscular mycorrhizal fungus.

[0013] In other embodiments, the dressing further comprises contacting the seed with at least one other agronomically advantageous agent, e.g. a diazotroph (a Rhizobium inoculant), mycorrhizal fungi, a phosphate solubilizing agent, a herbicide, an insecticide or fungicide. In certain embodiments, dressing involves spraying a composition comprising a vegetable signal molecule into seeds, and in certain other embodiments, the dressing includes instilling the seed composition.

[0014] The method of the present invention is useful in the case of leguminous plants as well as non-leguminous plants. In some embodiments, the legume seed is soybean. In certain other embodiments, the treated seed is a non-buddy seed, such as a seed of a field crop plant, e.g. corn, or a vegetable seed crop.

The seeds can be treated according to the present invention at any time from one month (thirty days) to 1 year, 2 years, and in some embodiments even up to 3 years before sowing, depending on the properties of the particular seed (viability after storage) or industry standards. For example, soybeans are generally sown in the next season, while maize seeds can be stored for much longer periods, including those up to 3 years before sowing.

[0016] As demonstrated in the embodiments, which include comparative experiments carried out both in the greenhouse and in field conditions, the benefits of using signal molecules / plant growth agents can be achieved even when the signal molecules are applied to seeds well before the sowing time and after the sowing time. long-term storage period.

[0017] As further demonstrated in embodiments, which include comparative experiments performed both in a greenhouse and in field conditions, embodiments of the present invention, which included treating soybeans with LCO from Bradyrhizobium japonicum, exhibited increased plant yield, leaf area and increased root length and root volume compared to both un-treated seeds and LCO-treated seeds just before or during the week before sowing.

BRIEF DESCRIPTION OF THE DRAWINGS [0018]

Figures 1 and 2 show chemical structures of lipochitooligosaccharide (LCO) compounds useful in practicing the present invention. Fig. 3 is a bar graph which represents the average surface area of ​​the first trifoliate leaves of 19 day soy plants germinated from seeds treated according to an embodiment of the present invention (i.e., 55 days before sowing) compared to controls (i.e. unprepared seeds and seed treated with a molecule) signal 7 days before sowing).

DETAILED DESCRIPTION [0019] For the purposes of the present invention, the term "vegetable signal molecule" which can be used interchangeably with "plant growth agent" generally refers to any agent both naturally occurring in plants or a microorganism as well as synthetic (and which can not present in nature), which directly or indirectly activates the biochemical pathway of the plant, resulting in enhanced plant growth, measurable at least with respect to at least one of the parameters including increased bushel / acre yield, increased number of roots, increased root length, increased weight roots, increased root volume and increased leaf area. Plant signaling molecules,

[0020] The plant signal molecule may be a secreted and / or purified component. The term "isolated" means that the signal molecule is removed from its natural state and separated from other molecules naturally associated with it. The term "purified" means that the concentration of the signal molecule is increased (as a result of the purification process) with respect to other components, e.g. undesirable components or with a lower value.

[0021] LCOs, also known as symbiotic Nod signals or Nod factors, consist of an oligosaccharide backbone of e-1,4-linked acetyl-D-glucosamine residues ("GlcNAc") with an N-attached fatty acid chain fused at the end. non-reducing. The LCOs differ in the number of GIcNAc residues in the backbone, the length and degree of saturation of the acyl fatty chain, and substitutions of the reducing and nonreducing sugar residues. The LCO example is shown below as formula I

<img file="PL2747565T3_D0001.tif" />

G is hexosamine, which may be substituted, for example, by an acetyl group on a nitrogen atom, a sulphate moiety, an acetyl group and / or an ether moiety at an oxygen atom,

R1, R2, R3, R5, R6 and R7, which may be the same or different, are H, CH3CO-, CxHyCO-, where x is an integer between 0 and 17, and y is an integer between 1 and 35, or any another acyl group, such as, for example, carbamyl,

R4 is a mono-, di- or tri-unsaturated and a polyunsaturated aliphatic chain containing at least 12 carbon atoms, and n is an integer between 1 and 4.

[0022] LCOs can be obtained (i.e. secreted and / or purified) from bacteria such as Rhizobia, e.g. Rhizobium sp., Bradyrhizobium sp., Sinorhizobium sp. And Azorhizobium sp. The structure of LCO is characteristic of each of these bacterial species and each strain can produce many LCOs with different construction sites. For example, specific

LCO S. meliloti is described in U.S. Patent No. 5549718 as compounds of formula II:

<img file="PL2747565T3_D0002.tif" />

wherein R is H or CH 3 CO-, and n is 2 or 3.

[0023] In particular, LCO includes NodRM, NodRM-1, NodRM-3. After acetylation (R = CH3CO-), they become AcNodRM-1 and AcNodRM-3 respectively (US patent

5545718).

[0024] LCOs from Bradyrhizobium japonicum are described in patents US 5175149 and 5321011.

Generally, these are pentasaccharide phytohormones containing methylfucose. A number of such LCOs derived from B. japonicum have been described: BjNod-V (C18: 1); BjNod-V (Ac, C18: 1), BjNod-V (C16: 1) and BjNod-V (AC, C16: 0), where "V" indicates the presence of five Nacetylglucosamines; "Ac" acetylation; the number after "C" indicates the number of carbon atoms in the fatty acid side chain; and the number after ":" means the number of double bonds.

[0025] LCOs used in embodiments of the invention can be recovered from bacterial strains that produce LCOs, such as Azorhizobium, Bradyrhizobium (including B. japonicum), Mesorhizobium, Rhizobium (including R. leguminosarum), Sinorhizobium (including S. meliloti) and bacterial strains genetically modified to produce LCO.

[0026] LCO are essential determinants of host specificity in legume symbiosis (Diaz et al., Mol. Plant-Microbe Interactions 13: 268-276 (2000)). Thus, in legume families, certain types and species of rhizobia develop a symbiotic nitrogen-binding relationship with a particular legume plant as a host. Such plant host / bacteria combinations are described in Hungria et al., Soil Biol. Biochem. 29: 819830 (1997). Examples of such symbiotic bacterial / legume partnerships include S. meliloti / alfalfa and melilot; R. leguminosarum biovar viciae / peas and lentils; R. leguminosarum biovar phaseoli / beans; Bradyrhizobium japonicum / soybeans; and R. leguminosarum biovar trifolii / grass clover. Hungria also mentions the effective flavonoid inducers of the Nod gene of rhizobes and specific LCO structures produced by various rhizobia. However, the specificity of LCO is the only one necessary for the occurrence of nodulation in legumes. In the practice of the present invention, the use of a given LCO is not limited to the seed treatment of leguminous plants being their symbiotic partner for achieving an increased crop yield measured in bushels / acres, increased roots, increased root length, increased root mass, increased root volume and increased leaf area, compared to plants harvested from un-treated seeds or in comparison to plants harvested from seed pickled with a signal molecule just before or a week or less before sowing. Thus, for example, the LCO obtained from B. japonicum can be used for seed treatment of legumes other than soybeans and seeds of non-leguminous plants, such as maize. As another example, the pea LCO obtained from R. leguminosarum, illustrated in Fig. 1 (designated as LCO-V (C18: 1), SP104) can be used for seed treatment of legumes other than peas and also non-leguminous plants.

[0027] The present invention also encompasses the use of LCOs obtained (i.e. isolated and / or purified) from arbuscular mycorrhizal fungi, such as fungi of the Glomerocycota group, e.g. Glomus intraradicus. The structure of representative LCO obtained from these fungi is described in WO 2010/049751 and WO 2010/049751 (the LCOs described therein are also referred to as "Myc factors").

[0028] Also encompassed by the present invention is the use of synthetic LCO compounds, such as those described in WO2005 / 063784, and recombinant LCOs obtained by the use of genetic engineering techniques. The structure of the basic, naturally occurring LCO, may contain modifications or substitutions found in LCO found in nature, such as described in Spaink, Crit. Rev. Plant Sci. 54: 257-288 (2000) and D'Haeze et al., Glycobiology 12: 79R-105R (2002). The precursor oligosaccharide molecules (CO, which, as described below, are also useful as plant signaling molecules in the present invention) for constructing LCO, can also be synthesized by organisms modified by genetic engineering techniques, e.g. as in Samain et al., Carb. Res. 302: 35-42 (1997).

[0029] LCOs may be used in various purity forms and may be used alone or in the form of LCO-producing bacteria or fungi. For example,

OPTIMIZE® (available at handle from Novozymes BioAg Limited) contains the B. japanese6 nicum culture which produces LCO (LCO-V (C18: 1, MeFuc), MOR116) illustrated in Figure 2. Methods of delivering substantially pure LCOs simply include microbial cells from LCO mixture and microorganism, or further treatments to purify LCO molecules by separating the LCO solvent phase, followed by HPLC chromatography as described, for example, in US Patent No. 5549718. Purification can be increased by repeating HPLC and purified LCO molecules can be freeze-dried for long-term storage.

[0030] The seeds can be treated with the plant signal molecule in several ways, but preferably by spraying or dripping. Spraying or spotting treatment can be carried out by formulating an effective amount of a plant signal molecule in an agriculturally acceptable carrier, usually of an aqueous character, and spraying or dripping a seed composition using a continuous treatment system (which is calibrated to apply the mortar at a predetermined rate) proportionally to a continuous seed flow), such as a drum-type pickling device. In such methods, relatively small volumes of the carrier are suitably used to allow relatively fast drying of the treated seeds. In this way, you can efficiently treat large volumes of seeds. Batch systems can also be used, in which the seed feed of a predetermined size and signal molecule compositions are delivered to the mixer. Systems and apparatus for conducting such processes are commercially available from numerous suppliers, e.g. Bayer CropScience (Gustafson).

[0031] In another embodiment, the dressing includes seed coating. One such method involves coating the inner wall of a circular container with the composition, adding seeds, rotating the container to bring the seed into contact with the wall and the composition, as is known in the art as "coating in a container". The seeds can be coated by combining the coating methods. The dipping comprises the use of an aqueous solution containing a plant growth enhancer. For example, the seeds may be soaked for a time from about 1 minute to about 24 hours (e.g., for at least 1 min, 5 min, 10 min, 20 min, 40 min, 80 min, 3 h, 6 h, 12 h, 24 h ). Certain types of seeds (e.g. soybeans) are generally sensitive to moisture.

[0032] Without intending to be bound by any theory of action, the Applicants believe that even if the dressing may not cause the plant signal molecule to remain in contact with the surface of the seed after treatment and during any part of storage, the signal molecule can achieve the intended effects due to the phenomenon known as seed memory. or seed perception. See Macchiavelli and Brelles-Marino, J. Exp. Bot. 55 (408): 2635-40 (2004). ThrowsMoreover, they believe that after treatment with the LCO signal molecule, it diffuses into the young developing germinal root and activates the symbiotic and developmental genes, which results in a change in the root architecture of the plant. Nevertheless, compositions containing a plant signal molecule may further comprise an adhesion or coating agent to aid adherence of the signaling molecule to the seed. For aesthetic purposes, the compositions may further comprise a coating polymer and / or a coloring agent.

[0033] The effective amount of the plant signaling molecule used for seed dressing, expressed in concentration units, is typically from about 10^-5 to around 10^-14 M, and in some embodiments from about 10^-5 to around 10^-11 M, and in some other embodiments from about 10 to about 10 M. The effective amount in units of pulp is usually from about 1 to about 400 μg / quintal (q) of seed, in some embodiments from about 2 to about 70 pg / q, and in certain other embodiments, from about 2.5 to about 3.0 μg / q of seed. However, an effective amount of the plant signal molecule can be obtained in a suitable dose response test, preferably in a greenhouse and / or field study.

[0034] The dressing may also comprise contacting the seed, before, simultaneously with or after contact with the plant signal molecule, with the agronomically / agronomically beneficial agent. As used herein and in the art, the term "agriculturally or agronomically advantageous" refers to agents which, when applied to seeds, cause an improvement (which can be statistically significant) of the plant's characteristics, such as habit, growth, viability or crop yield, compared to with unrepaired plants. Representative examples of such agents that may be useful in practicing the present invention include, but are not limited to, diazotrophs, mycorrhizal fungi, herbicides, fungicides, insecticides, and phosphate solubilizing agents.

[0035] Suitable herbicides include bentazone, acifluorfen, chlorimuron, lactofen, clomazone, fluazifop, glufosinate, glyphosate, setoxydim, imazethapyr, imazamox, fomesaf, flumichlorak, imazachin and clethodim. Commercial products containing each of these compounds are readily available. The concentration of the herbicide in the composition will usually correspond to the dose of application indicated on the label for a particular herbicide.

[0036] A "fungicide" as used herein and in the field is an agent that kills fungi or inhibits their growth. As used herein, a fungicide "exhibits activity against" specific fungal species if treatment with a fungicide results in killing or inhibiting the growth of fungal populations (e.g., in soil) relative to the untreated population. The effective fungicides of the present invention will conveniently exhibit activity against a wide range of pathogens, including, but not limited to, Phytophthora, Rhizoctonia, Fusarium, Pythium, Phomopsis or Selerotinia and Phakopsora, and combinations thereof.

[0037] Commercial fungicides may be suitable for use in the present invention. Useful fungicides available in handle include PROTEGE, RIVAL or ALLEGIANCE FL or LS (Gustafson, Piano, TX), WARDEN RTA (Agrilance, St. Paul, MN), APRON XL, APRON MAXX RTA or RFC, MAXIM 4FS or XL (Syngenta, Wilmington, DE), CAPTAN (Arvesta, Guelph, Ontario) and PROTREAT (Nitragin Argentina, Buenos Aires, Argentina). Active ingredients in these and other commercial fungicides include, but are not limited to, fludioxonil, mefenoxam, azoxystrobin and metalaxyl. Commercial fungicides are most conveniently used according to the manufacturer's instructions at the recommended concentrations.

As used herein the insecticide "exhibits activity against" specific insect species if the insecticide treatment kills or inhibits the growth of the insect population relative to the untreated population. Effective insecticides according to the present invention will conveniently exhibit activity against a broad range of insects including, but not limited to, larvae, dark larvae, beetle larvae, corn stalk, soil trash, earthworms, bugs, aphids, leaf beetles and shields.

[0039] Commercial insecticides may be suitable for use in the present invention. Useful insecticides commercially available include CRUISER (Syngenta, Wilmington, DE), GAUCHO and PONCHO (Gustafson, Piano, TX). Active ingredients in these and other commercial insecticides include thiamethoxam, clothianidin and imidacloprid. Commercial insecticides are most conveniently used according to the manufacturer's instructions at the recommended concentrations.

[0040] As used herein, phosphate solubilizing agents include, but are not limited to, microorganisms solubilizing phosphates. As used herein, a "microorganism solubilizing phosphate" is a microorganism that is capable of increasing the amount of phosphorus available to a plant. The phosphate solubilising microorganisms include fungal and bacterial strains. In one embodiment, the phosphate solubilizing microorganism is a spore-forming microorganism.

[0041] Non-limiting examples of microorganisms solubilizing phosphates include species selected from the group consisting of Acinetobacter, Arthrobacter, Arthro- botrys, Aspergillus, Azospirillum, Bacillus, Burkholderia, Candida Chryseomonas, Enterobacter, Eupenicillium, Exiguobacterium, Klebsiella, Kluyvera, Microbacterium, Mucor, Paecilomyces, Paenibacillus, Penicillium, Pseudomonas, Serratia, Stenotrophomonas, Streptomyces, Streptosporangium, Swaminathania, Thiobacillus, Torulospora, vibrio, Xanthobacter and Xanthomonas.

Pseudomonas fluorescens, Pseudomonas lutea, Pseudomonas poae, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas trivialis, Serratia marcescens, Stenotrophomonas maltophilia, Streptomyces sp., Streptosporangium sp., Swaminathania salitolerans, Thiobacillus ferrooxidans, Torulospora globosa, vibrio proteolyticus, Xanthobacter agilis and Xanthomonas campestris. [0043] Preferably the microorganism solubilizing phosphate is a strain of Penicillium fungus. Strains of the Penicillium fungi that may be useful in practicing 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.

[0044] More preferably the microorganism solubilizing the phosphates of the Penicillium species is P. bilaiae, P. gaestrivorus and / or a combination thereof. Most preferably, the P. bilaiae strains are selected from the group consisting of ATCC 20851, NRRL 50169, ATCC 22348, ATCC 18309,

NRRL 50162 (Wakelin et al., 2004. Biol Fertil Soils 40: 36-43), and the P. gaestrivorus strain is NRRL 50170 (see, Wakelin, supra).

Thiobacillus ferrooxidans, Torulospora globosa, vibrio proteolyticus, Xanthobacter agilis and Xanthomonas campestris. [0046] Diazotrophs are bacteria and archaea that bind atmospheric nitrogen gas into a more useful form, such as ammonia. Examples of diazotrophs include bacteria from the species 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. temperatum, and / or M. tianshanense) and combinations thereof. In a particular 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 disinfectant is R leguminosarum. In another embodiment, the diazotroph is R meliloti. In another embodiment, the diazotroph is S. meliloti. S. kummerowiae, S. medicae, S. meliloti, S. mexicanus, S. morelense, S. saheli, S. terangae, and / or S. xinjiangense), Mesorhizobium spp., (M. albiziae, M. amorphae, M. chacoense, M. ciceri, M. huakuii, M. loti, M. mediterraneum, M. pluifarium, M. septentrionale, M. temperatum, and / or M. tianshanense) and combinations thereof. In a particular embodiment, the 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 disinfectant is R leguminosarum. In another embodiment, the diazotroph is R meliloti. In another embodiment, the diazotroph is S. meliloti. S. kummerowiae, S. medicae, S. meliloti, S. mexicanus, S. morelense, S. saheli, S. terangae, and / or S. xinjiangense), Mesorhizobium spp., (M. albiziae, M. amorphae, M. chacoense, M. ciceri, M. huakuii, M. loti, M. mediterraneum, M. pluifarium, M. septentrionale, M. temperatum, and / or M. tianshanense) and combinations thereof. In a particular embodiment, the 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 disinfectant is R leguminosarum. In another embodiment, the diazotroph is R meliloti. In another embodiment, the diazotroph is S. meliloti. chacoense, M. ciceri, M. huakuii, M. loti, M. mediterraneum, M. pluifarium, M. septentrionale, M. temperatum, and / or M. tianshanense) and combinations thereof. In a particular embodiment, the 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 disinfectant is R leguminosarum. In another embodiment, the diazotroph is R meliloti. In another embodiment, the diazotroph is S. meliloti. chacoense, M. ciceri, M. huakuii, M. loti, M. mediterraneum, M. pluifarium, M. septentrionale, M. temperatum, and / or M. tianshanense) and combinations thereof. In a particular embodiment, the 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 disinfectant is R leguminosarum. In another embodiment, the diazotroph is R meliloti. In another embodiment, the diazotroph is S. meliloti. In another embodiment, the diazotroph is B. japonicum. In another embodiment, the disinfectant is R leguminosarum. In another embodiment, the diazotroph is R meliloti. In another embodiment, the diazotroph is S. meliloti. In another embodiment, the diazotroph is B. japonicum. In another embodiment, the disinfectant is R leguminosarum. In another embodiment, the diazotroph is R meliloti. In another embodiment, the diazotroph is S. meliloti.

[0047] Mycorrhizal fungi form symbiotic connections with the roots of the vascular plant and provide e.g. absorption capacity for water and mineral nutrients due to the relatively large surface area of ​​the mycelium. Mycorrhizal fungi include endomycorrhizal fungi (also called vesicular arbuscular mycorrhizae, VAM, arbuscular mycorrhiza or AM), ectomycorrhizal fungi or a combination thereof. In one embodiment, mycorrhizal fungi are Glomeromycota endomicorros and Glomus and Gigaspora species. In a further embodiment, endomycorrosis is a strain of Glomus aggregatum, Glomus brasilianum, Glomus clarum, Glomus deserticola, Glomus etunicatum, Glomus fasciculatum, Glomus intraradices, Glomus monosporum or Glomus mosseae, Gigaspora margarita or a combination thereof.

[0048] Examples of mycorrhizal fungi include Basidiomycota, Ascomycota and Zygomycota ectomycorrhizases. Other examples include Laccaria bicolor, Laccaria laccata, Pisolithus tinctorius, Rhizopogon amylopogon, Rhizopogon fulvigleb, Rhizopogon luteolus, Rhizopogon villosuli, Scleroderma cepa, Scleroderma citrinum or a combination thereof.

[0049] Mycorrhizal fungi include erythroid mycorrisms, arbutoid mycorrhisms or monotropoid mycorrhizals. Arbuscular mycorrhisms and ectomycorrhizia form elliptic mycorrhizas with many plants belonging to the order Ericales, while some Ericales form arbutoid and monotropoid mycorrhisms. In one embodiment, the mycorrhisms may be erosionic mycorrheisms, preferably of the Ascomycota type, such as Hymenoscyphous ericae or Oidiodendron sp. In another embodiment the mycorrhisms may be arbutoid mycorrheisms, preferably of the Basidiomycota type. In yet another embodiment, mycorrhisms may be monotripoid mycorrhizes, preferably of the Basidiomycota type. In yet another embodiment, the mycorrhiza may be orchidic mites, preferably from the Rhizoctonia species.

[0050] The methods of the present invention are useful in the case of legume seeds, representative examples of which include soy, alfalfa, peanut, peas, lentils, beans and clover. The methods of the present invention are also useful in the case of non-legume plants, e.g. Poaceae, Cucurbitaceae, Malvaceae, Asteraceae, Chenopodiaceae and Solonaceae. Representative examples of non-legume seeds include field crops such as corn, cereals, such as rice, barley and wheat, cotton and canola, and vegetable crops such as potatoes, tomatoes, cucumbers, beets, beans and cantaloupe.

[0051] After treatment and for storage purposes, the seeds are then packaged e.g. into 50-pound or 100-pound bags or in collection bags or containers, in accordance with standard techniques. The seeds are stored for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or 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 months or even longer under appropriate storage conditions known in the art. As used herein, the term "month" will mean 30 days. In the sense used in the description year will mean 365 days. While soybeans should be sowed in the next season, corn seeds can be stored much longer, up to 3 years inclusive.

[0052] The plant signal molecule may be applied in any suitable way, such as in the form of a seed treatment composition, which comprises at least one vegetable signal molecule and an agriculturally acceptable carrier.

[0053] Any suitable agriculturally acceptable carrier may be used, for example a solid carrier, a semi-solid carrier, an aqueous liquid carrier, a non-aqueous liquid carrier, a suspension, emulsion or emulsifiable concentrate. The agriculturally acceptable carriers may include, for example, adjuvants, inert ingredients, dispersing agents, surfactants, tackifiers, binders, stabilizers and / or polymers.

The seed dressing composition may further comprise one or more agronomically / agronomically preferred agents (i.e., in addition to the signal molecule), such as one or more diazotrophs, mycorrhizal fungi, herbicides, fungicides, insecticides and / or phosphate solubilizing agents.

[0055] The present invention will now be described by means of the following non-limiting examples.

Summary of Embodiments [0056] Examples 1 and 2 describe comparative field experiments using soybeans that show that the claimed invention provides an increased yield of plants. The seeds were seasoned in accordance with the present invention for 5 months prior to commercial sowing of Optimize®, which is a combination of Bradyrhizobium japonicum and LCO-V (C18: 1, MeFuc) inoculant (illustrated in Fig. 2), and pure LCO alone, and at 4, 5 months before sowing non-commercial (i.e. less pure) Optimize® species, and LCO itself, and, for comparison, the same plant signaling molecules during sowing. Unprepared seeds were used as a different control. The results, which were expressed as differences in grain yield, measured in bushel / acre units, show

[0057] Examples 3 and 4 describe comparative experiments conducted in greenhouses that demonstrate that the claimed invention provides improvement in other plant growth characteristics. Example 3 describes the experience that involves treating soybeans with pure LCO-V (C18: 1, MeFuc) for one month and one year before sowing. Soybean plants (including roots) were harvested ten days after sowing. The results, which are presented as differences in root length and volume, show that the methods of the present invention provide dramatic increases in these parameters. Finally, Example 4 describes experiments conducted with Optimize® soybean seeds 55 days before sowing, and, in comparison, with soybean seeds treated 7 days before sowing and un-treated seeds. Results,

EXAMPLE 1 [0058] A field trial was carried out to evaluate the embodiments of the present invention in terms of grain yield when applied to soybeans. The field test site was located near Whitewater, WI, and characterized by soil in the form of Milford clay. Soil testing, carried out six months before sowing, showed a soil pH of 6.8, an organic content of 5.3% and a phosphorus and potassium content of 39 ppm and 139 ppm, respectively.

[0059] In the trials, Optimize®, non-commercial Optimize® (NI-50S-1), pure LCO-V (C18: 1, MeFuc) (NI-50GREN-1) and non-commercial LCO-V species were used as plant signaling molecules ( C18: 1, MeFuc) (NI-50S-2CF). The test used Stine S2118 soybeans. The plant signaling molecules were sprayed onto the seeds after dilution / without dilution to a concentration of 4.8 fl oz / quintal.

[0060] The study was carried out in a random block arrangement on a plot of 10 feet by 50 feet (0.011 acres), with a spacing of 7.5 inches. Four replications were made. The seeds were seeded with plant signal particles 4.5 or 5 months before sowing and just prior to sowing and planted to a depth of 1 inch with a seeding density of 225,000 seeds per acre using a John Deere 750 NT corn seed drill. Pesticides, Extreme® and AMPS® were both used 11 days before sowing (pre-emergence) at 3.0 pints and 2.5 pounds respectively. Assure II®, Roundup WeatherMax® and AMPS® were applied 46 days after seeding (post-emergence), at 6.0 ounce, 21 ounce and 2.5 lb respectively. Plants were harvested 4 months and 20 days after sowing.

[0061] Control seeds were treated with an amount (by weight) of water corresponding to the amount (by weight) of the experimental composition of the signal molecule (signal molecule + vehicle). Prior to sowing, the control seeds were stored under the same conditions as the experimental seeds and sown at the same time as the experimental seeds into the same soil.

[0062] The results of the study are shown below in Table 1.

Table 1

<td></td><td>TREATED GROUP</td><td>GRAIN YIELD at 13%</td>

<td>1</td><td>Control - unvaccinated</td><td>62.5</td>

<td>2</td><td>Optimize - for sowing</td><td>64.2</td>

<td>3</td><td>Optimize - 5 months</td><td>65.7</td>

<td>4</td><td>NI-50GREN-1 - for sowing</td><td>62.2</td>

<td>5</td><td>NI-50GREN-1 - 5 months</td><td>70.5</td>

<td>6</td><td>NI-50S-1 - 4.5 months</td><td>67.2</td>

<td>7</td><td>NI-50S-2CF - 4.5 months</td><td>69.6</td>

[0063] As reflected by the comparison between the comparative Group 2 (not according to the invention) and Group 3 according to the invention, soybean seed treatment with Optimize® species for 5 months prior to sowing increased the soybean yield by 1.5 bushel soy. As reflected by the comparison between Group 4 and Group 5 according to the invention, dressing soybean seeds for 5 months before sowing with pure LCO-V alone (C18: 1, MeFuc) resulted in an increase in soybean harvest by 8.3 bushel / acre. As reflected by the comparison between Group 2 and Group 6 of the invention, dressing soybeans 4.5 months before sowing non-commercial Optimize® species resulted in an increase in soybean crop by 3.0 bushel / acre. Finally, as shown by the comparison between Group 4 and Group 7 according to the invention, the treatment of soybeans with the non-commercial LCO-V species (C18: 1, MeFuc) per 4, 5 months before sowing, it increased the soybean yield by 7.4 bushels / acres. Grain yield measurements were made at the grain moisture level of 13%.

EXAMPLE 2 [0064] A soybean trial was conducted to evaluate the effect of an embodiment of the present invention on grain yield when applied to soybeans. The field test site was located near Whitewater, WI, and characterized by soil in the form of Milford clay. Soil testing, carried out six months before sowing, showed a soil pH of 6.6, an organic content of 4.8% and a phosphorus and potassium content of 41 ppm and 131 ppm, respectively.

[0065] The plant signaling molecules used in the assay were the same as in Example 1. Stine S2118 was used as soybeans in the study. The plant signaling molecules were sprayed onto the seeds after dilution / without dilution to a concentration of 4.8 fl oz / quintal.

[0066] The study was conducted in a random block arrangement on a plot of 10 feet by 50 feet (0.011 acres), with a spacing of 7.5 inches. Four replications were made. The seeds were seeded with plant signal particles 4.5 or 5 months before sowing and just prior to sowing and planted to a depth of 1 inch with a seeding density of 225,000 seeds per acre using a John Deere 750 NT corn seed drill. Pesticides, Extreme® and AMPS®, both were applied 10 days prior to sowing (pre-emergence) in an amount of 3.0 pints and 2.5 pounds respectively. Assure II®, Roundup WeatherMax® and AMPS® were applied 45 days after seeding (post-emergence), at 6.0 ounce, 21 ounce and 2.5 lb respectively. Plants were harvested 4 months and 21 days after sowing.

[0067] Control seeds were treated with the amount (by weight) of water corresponding to the amount (by weight) of the experimental composition of the signal molecule (signal molecule + vehicle). Prior to sowing, the control seeds were stored under the same conditions as the experimental seeds and sown at the same time as the experimental seeds into the same soil.

[0068] The test results are shown below in Table 2.

Table 2

<td></td><td>TREATED GROUP</td><td>GRAIN YIELD at 13%</td>

<td>1</td><td>Control - unvaccinated</td><td>62.4</td>

<td>2</td><td>Optimize - for sowing</td><td>64.1</td>

<td>3</td><td>Optimize - 5 months</td><td>68.6</td>

<td>4</td><td>NI-50GREN-1 - for sowing</td><td>65.8</td>

<td>5</td><td>NI-50GREN-1 - 5 months</td><td>64.0</td>

<td>6</td><td>NI-50S-1 - 4.5 months</td><td>69.4</td>

<td>7</td><td>NI-50S-2CF - 4.5 months</td><td>66.6</td>

[0069] As reflected by the comparison between the comparative Group 2 (not according to the invention) and Group 3 according to the invention, soybean seed treatment with Optimize® species for 5 months prior to sowing has resulted in a soybean yield increase of 4.5 soybeans. As reflected by the comparison between Group 2 and Group 6 of the invention, dressing soybeans 4.5 months before sowing the non-commercial Optimize® species resulted in a soybean crop increase of 5.3 bushel / acre. As shown by the comparison between Group 4 and Group 7 according to the invention, treating soybeans with the same non-dairy LCO-V species (C18: 1, MeFuc) for 4.5 months before sowing increased the soybean crop by 0.8 bushel / acre. The only negative response reflected by the comparison between Group 4 not according to the invention and Group 5 according to the invention showed that soybean seed treatment for 5 months before sowing pure LCO alone resulted in a 1.8 bushel / acre drop, which is the result attributed to the unexplained variability associated with field trials. Grain yield measurements were made at the grain moisture level of 13%.

Experiences in a greenhouse

EXAMPLE 3 [0070] Soybean seeds were applied 10^-7M pure LCO-V (C18: 1, MeFuc) and stored at 15 ° C. The treated seeds and uncultivated (control) seeds were sown at 1 and 12 months after setting in greenhouse pots containing sand and perlite (1: 1 mixture). The seedlings were cultivated for 10 days after sowing the seeds, after which the seedlings were harvested, their roots were cleaned and measured with a Winrhizo® scanner. Control seeds were treated with the amount (by weight) of water corresponding to the amount (by weight) of the experimental composition of the signal molecule (signal molecule + vehicle). Prior to sowing, the control seeds were stored under the same conditions as the experimental seeds and sown at the same time as the experimental seeds into the same soil. The results are shown in Table 3.

TABLE 3

<td>Treatment</td><td colspan="2">One month after treatment</td><td colspan="2">1 year after treatment</td>

<td></td><td>The length of the root ^(Cm)</td><td>Root volume ^(Cm3)</td><td>The length of the root ^(Cm)</td><td>Root volume ^(Cm3)</td>

<td>Checklist</td><td>128</td><td>0.455</td><td>115.5</td><td>0.403</td>

<td>LCO</td><td>135 *</td><td>0.468</td><td>159.3 *</td><td>0.540 *</td>

<td>% increase</td><td>5.46</td><td>2.86</td><td>37.92</td><td>34</td>

[0071] The results obtained with both embodiments of the invention (LCO treated seeds for 1 month and 12 months before sowing), and especially the results obtained after 1 year of treatment, are spectacular, given that it is known from the knowledge that soybeans are susceptible to spoiling over time.

EXAMPLE 4 [0072] Soybean seeds treated with Optimize® were kept at 15 ° C in a refrigerator. The seeds were sown 7 (7 dpz) and 55 (55dpz) days after treatment in rooting trays containing the peat: perlite mixture. The area of ​​their leaves was measured (cm²) for the first trifoliate leaves after 19 days. As illustrated in Fig. 3 and shown in Table 4, the leaves that grew out of seeds treated in accordance with the present invention showed a 50% larger increase in leaf area area compared to non-inventive embodiments (42% vs. 28%).

TABLE 4

<td></td><td>Average</td><td>Deviation standard</td><td>Reply</td><td>Reply, %</td>

<td>Average, not repaired (NRC)</td><td>146.25</td><td>18.7539</td><td></td><td></td>

<td>Average 7dp</td><td>187.05</td><td>29.8215</td><td>40.81</td><td>28%</td>

<td>Average 55dpz</td><td>207.18</td><td>20.5278</td><td>60.93</td><td>42%</td>

[0073] As the bacterial titer (Bradyrhizobium japonicum) is known to diminish over time, the increase in average surface area exhibited by plants that have sprouted from seeds aged 55 days prior to sowing can be attributed to the rhizobial

LCO.