NZ710171B2 - Seed treatment methods and compositions - Google Patents

Abstract

Disclosed herein is a method of enhancing plant growth, comprising treating a seed at least one month prior to planting with a chitooligosaccharide (CO), such as those shown in the figures, wherein, upon harvesting, the plant exhibits at least one of increased plant yield measured in terms of bushels/acre, increased root number, increased root length, increased root mass, increased root volume and increased leaf area, as compared to a plant harvested from a control seed, optionally a control seed treated with the CO just prior to or within a week or less of planting. s/acre, increased root number, increased root length, increased root mass, increased root volume and increased leaf area, as compared to a plant harvested from a control seed, optionally a control seed treated with the CO just prior to or within a week or less of planting.

Description

SEED TREATMENT METHODS AND ITIONS OUND OF THE INVENTION The symbiosis between the gram-negative soil bacteria, Rhizobiaceae and Bradyrhizobiaceae, and legumes such as soybean, is well documented. The Zbiochemica' basis for these relationships includes an exchange of molecular signaling, n the plant-to-bacteria signal compounds include es, isoflavones and flavanones, and the bacteria-to-plant signal compounds, which include the end ts of the sion of the hizobial and Rhizobial nod genes, known as lipo—chitooligosaccharides (LCOs). The symbiosis between these bacteria and th legumes onablos tho legume to fix atmospheric nitrogen and thereby grow in soil that has low assimilable nitrogen , thus obviating a need for nitrogen fertilizers. Since nitrogen fertilizers can significant'y increase the cost of crops and are associated with a number 0: polluting effects, the agricultural industry continues ius e "orts to exploit this biological relationship and develop new agents and methods for improving plant yield without sing the use of nitrogen-based fertilizers.

U.S. Patent 6,979,664 teaches a method for enhancing seed germination or seedling emergence of a plant crop, comprising the steps of providing a composition that comprises an e "ective amount of at least one lipo-chitooligosaccharide and an agriculturally le carrier and applying the composition in the immediate ty A: o_ a seed or seedling in an effective amount for enhancing seed germination of seedling emergence in comparison to an untreated seed or seedling..

Further development on this concept is taught in WO 2005/062899, directed to combinations of at least one plant inducer, namely an LCD, in combination with a fungicide, insecticide, or combination thereof, to enhance a plant characteristic such as plant stand, growth, vigor and/or yield. The compositions and methods are taught to be applicable to both legumes and non—legumes, and may be used to treat a seed (just prior to planting), ng, root or plant.

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

A number of other publications describe the benefit of LCOs in seed treatment processes, such as, Kidaj et al., ”Nod factors stimulate seed germination and promote growth and nodulation of pea and vetch under competitive conditions,” iol Res 25426 (2011) and Maj et al., “Pretreatment of Clover Seeds with Nod Factors Improves Growth and Nodulation of Trifolium pratense,” J. Chem Ecol (2009) 35:479-487.

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

BRIEF SUMMARY OF THE INVENTION ] In a first aspect, the present invention provides a method of enhancing plant growth, comprising ng a seed at least one month prior to planting with a chitooligosaccharide (CO) wherein, upon harvesting, the plant exhibits at least one of sed plant yield measured in terms of bushels/acre, increased root number, increased root length, increased root mass, increased root volume and increased leaf area, as compared to a plant harvested from a control seed. 1106451_1 [0006B] In a second aspect, the present invention provides a seed treated according to the method the first aspect. [0006C] In a third aspect, the present invention provides a plant germinated from the seed of the second aspect.

The present invention provides methods of enhancing plant growth and crop production in which the beneficial effect of a plant signal molecule (plant growth-enhancing agent) may be obtained without the need to apply the plant signal molecule (plant enhancing agent) to the seed contemporaneously with planting. The present ion is based, in part, on the discovery that treatment of seeds with a plant signal molecule such as an LCO, followed by prolonged storage prior to planting, results in enhanced plant growth, ing greater plant yield and/or leaf surface area and/or root , length and mass, compared to plants harvested 1106451_1 from both untreated seeds. The present invention also provides methods of enhancing plant growth and crop production in which additional improvements may be obtained over plants crops produced from seeds treated just prior to or within a week or less of planting.

A first aspect of the t invention is directed to a method of ing plant , comprising treating seed at least one month (thirty days) prior to planting with an effective amount of a plant signal molecule.

In embodiments, the seed may be treated in accordance with the present method at 2 months prior to planting, at least 3 months prior to ng, at least 4 months prior to planting, at least 5 months prior to planting, at least 6 months prior to planting, at least 9 months prior to planting, at least 1 year prior to planting, at least 2 years prior to planting and in some embodiments, at least 3 years prior to planting.

The treatment is used to produce a plant (crop) that exhibits at least one of increased yicld measur d in terms or bushels/acre, increased root number, increased root length, increased root mass, increased root volume and increased lea: area, compared to plants harvested from untreated seed. In particular embodiments, the treatment may be used to produce a plant (crop) that exhibits at least one of increased. yield measured in terms of bushels/acre, increased root number, increased root length, increased. root mass, increased root volume and increased. leaf area compared. to a plant (crop_ harvested from seed treated. with the signal molecule just prior to or within a week or less of planting.

In certain embodiments of the present ion, the plant signal molecule is a lipo-chitooligosaccharide (LCO).

In some embodiments, the LCO is inant. In other embodiments, the LCO is synthetic. In other embodiments, the LCO is obtained from. a microorganism, e.g¢, a species of Rhizobium selected from. ium sp., Bradyrhizobium Sp., e.g., Bradyrhizobium japonicum, Sinorhizobium Sp. and Azorhizobium sp, or from an arbuscular mycorrhizal fungus.

In other embodiments, the plant signal le is a chitinous compound. such. as a chito-oligomer (CO). In some embodiments, the CO is recombinant. In other embodiments, the CO is synthetic. In other embodiments, the CO is ed from a microorganism as per LCO's.

In other embodiments, the plant signal le is a flavonoid. In other embodiments, the plant signal molecule is jasmonic acid, linoleic acid, linolenic acid or a derivative thereof. In other embodiments, the plant signal le is a Combinations of two or more different plant signal molecules (or types thereof) may be used to treat the seed.

In other embodiments, the treating further comprises contacting the seed. with at least one other agronomically beneficial agent, e.g., diazotroph (Rhizobial inoculant), mycorrhizal fungi, a phosphate solubilizing agent, herbicide, insecticide or a fungicide. In some embodiments, the treating entails spraying' a composition comprising the plant signal molecule onto the seed, and. in some other‘ embodiments, the treating entails dripping the composition onto the seeds.

The method of the t invention is applicable to legumes and non-legumes alike. In some embodiments, the leguminous seed is soybean seed. In some other embodiments, the seed. that is treated. is non-leguminous seed. such. as a field crop seed, e.g., corn, or a vegetable crop seed.

The seed may be treated in ance with the present method anywhere from one month (thirty days) up to 1 year, 2 years and in some embodiments, even 3 years prior to planting, depending on particular seed properties (viability after storage) or industry standards. For example, soybean seeds are generally planted the following season, whereas corn seed can be stored for much longer periods of time including s of 3 years prior to ng.

The present invention also relates to seeds treated with a plant signal molecule/plant growth—enhancing agent, such as an LCO or CO, which have been stored for at least thirty-days 1m) to 1 year, 2 years and 111 some embodiments, even 3 years prior to planting.

Yet another aspect of the present invention is directed. to a d, seed. which. was treated. with. a plant signal Hm'ecule/plant growth—enhancing agent, such as an LCD or CO, which have been stored for at least thirty-days up to 1 year, 2 years and in some embodiments, even 3 years prior to A related aspect of the present invention is directed to a package comprising the treated seeds according to the present invention for purposes of planting subsequent to the treatment.

As trated in the working examples, which include comparative ments conducted under both greenhouse and field conditions, the benefits of signal molecules/plant growth—enhancing agents may be obtained even though the signal les are applied to a seed significant'y prior to the time of ng and after prolonged storage period.

As further demonstrated in the working es, which include comparative ments conducted under both greenhouse and. field. conditions, embodiments o: the present invention that entailed treatment of soybean seed with an LCD from Rradyrhizobium japonicum exhibited increased plant yield, "ea" surface area, and increased root length and root volume compared to both untreated seed and seed treated with the LCD just prior to or within a week of planting.

BRIEF DESCRIPTION OF THE DRAWINGS Figs. 1 and 2 show the chemical ures of lipo-chitooligosaccharide compounds (LCO) useful in the practice of the present invention.

Fig. 3 is a bar graph that shows mean surface area of first—trifoliate leaves on 19—day old soybean plants germinated from seed treated in accordance with an embodiment of the present invention (e.g;, 55 days pre-planting) as compared to controls (i.e., untreated seed and seed treated with the signal molecule 7 days prior to planting).

DETAILED DESCRIPTION For purposes of the present invention, the term "plant signal molecule", which. may' be used interchangeably with "plant growth—enhancing agent" broadly .C reiers to any agent, both naturally' ing' in plants or microbes, and synthetic (and which may be non-naturally occurring) that directly or indirectly activates a plant biochemical pathway, resulting in increased plant growth, eable at least in terms of at least one of increased yield measured in terms of bushels/acre, sed root number, increased root length, sed root mass, increased root volume and increased leaf area. Representative es of plant signal molecules that may be useful in the practice of the t invention include lipo-chitooligosaccharide compounds (LCO's), chito- oligosaccharides (COs), chitinous nds, flavonoids, jasmonic acid, ic acid and linolenic acid and their derivatives, and karrikins.

The plant signal molecule may be isolated and/or purified component. The term. “isolated” means the signal le is removed from its natural state and separated from other molecules naturally ated with it. The term “purified" means that the concentration of the signal molecule is ircreased. (by a purification. process) relative to Other components, e.g., unwanted or inferior components.

LCO's, also known in the art as symbiotic Nod signals or Nod factors, consist of an oligosaccharide backbone of B—l,4—linked N—acetyl—D-glucosamine ("GIcNAc") residues with an N—linked fatty acyl chain condensed at the non- reducing end. LCO's differ in the number of GIcNAc residues in the backbone, in the length and degree of saturation of the fatty' acyl chain, and. in the substitutions of reducing' and nonreducing sugar residues. An example of an LCO is presented below as formula I CH2OR3 0R4 G NH-R7 in which: G is a mine which can be substituted, for e, by an acetyl group on the nitrogen, a sulfate group, an acetyl group and/or an ether group on an oxygen, R1, t9, Ra R5, kg and iRm which. may' be identical or di""erent, represent H, CH3 CO--, CX Hy CO-- where >< is an integer between 0 and 17, and. y is an integer between 1 and 35, or any other acyl group such as for example a carbamyl, R4 represents a mono-, di—, or triunsaturated and tetraunsaturated aliphatic chain containing at least 12 carbon atoms, and n is an integer between 1 and 4.

LCOs may be obtained. (e.g., isolated and/or purified) from bacteria such as Rhizobia, e.g., Rhizobium sp., Bradyrhizobium sp., Sinorhizobium sp. and Azorhizobium sp. LCO structure is characteristic for each such bacterial species, and each strain may produce multiple LCO's with different structures. For e, specific LCOs from S. ti have also been described. in U.S. Patent 5,549,718 as having' the formula II: Hgkl (CH2)5 \\‘@Wk in which R represents H or CH3CO—— and n is equal to 2 or 3.

Even more specific LCOs include NodRM, NodRM—l, B. When acetylated (the O--), they become AcNodRM- 1, and AcNodRM-B, respectively (U.S. Patent 5,545,718).

LCOs from Bradyrhizobium cum are described in U.S. Patents 5,175,149 and 5,321,011. Broadly, they are pentasaccharide phytohormones comprising methylfucose. A number of these B. japonicum-derived LCOs are described: BjNod—V (c1821); BjNod-V (AC, CW1), BjNod-V (C16zi); and BjNod—V (AC, Clfio), with "V" indicating the presence of five N-acetylglucosamines; "Ac" an acetylation; the number following the "C" indicating the number of carbons in the fatty acid side chain; and the number following the ":" the number of double bonds.

LCO's used. in embodiments of the invention. mayr be red from bacterial strains that produce LCO's, such as strains of Azorhizobium, Bradyrhizobium ding B. japonicum), Mesorhizobium, Rhizobium (including R. leguminosarum), izobium (including S. meliloti), and bacterial strains genetically engineered to e LCO's.

LCO's are the primary determinants of host specificity in legume symbiosis (Diaz, et al., Mol. Plant- Microbe Interactions 13:268—276 (2000)). Thus, within the legume family, specific genera and s of rhizobia develop a symbiotic nitrogen—fixing relationship with a specilic legume host. These plant-host/bacteria combinations are described in Hungria, et al., Soil Biol. Biochem. 29:819-830 , Examples of these bacteria/legume symbiotic partnerships include S. meliloti/alfalfa and sweet clover; R. leguminosarum biovar Viciae/peas and lentils; R. leguminosarum biovar phaseoli/beans; Bradyrhizobium japonicum/soybeans; and R. leguminosarum .biovar‘ ii/red. clover. Hungria also lists the effective oid Nod gene inducers of the rhizobial species, and the specific LCO structures that are produced by the different rhizobial species. However, LCO specificity is only required to ish nodulation in legumes. In the ce of the present invention, use or a given LCD is not limited to treatment of seed 0: its symbiotic legume partner, in order to achieve increased. plant yield measured in terms .C ol bushels/acre, increased root number, increased root length, increased. root mass, increased root volume and increased leaf area, compared to plants harvested from untreated seed, or compared to plants harvested from seed treated with the sigral leecule just prior to or withir a week or less of planting. Thus, by way of e, an LCD ed from B. japonicwn may be used to treat leguminous seed other than soybean and non-leguminous seed such as corn.

As another example, the pea LCO obtainable from R. leguminosarum illustrated in Fig. 1 (designated LCO—V (Cl8zl), SP104) can be used to treat leguminous seed other than pea and non—legumes too.

Also encompassed by the present invention is use of LCOs obtained (e.g., isolated and/or purified) from arbuscular mycorrhizal fungi, such as fungi of the group Glomerocycota, e.g., Glomus intraradicus. The structures 0: representative LCOs obtained from these fungi are described in and WO 2010/049751 (the LCOs described therein also referred to as "Myc factors").

Further encompassed by the present invention is use of synthetic LCO compounds, such as those bed in WO2005/063784, and recombinant LCO's produced through genetic engineering. The basic, naturally occurring LCO ure may contain modifications or substitutions found in naturally occurring LCO's, such as those described in Spaink, Crit. Rev.

Plant Sci. 54:257-288 (2000) and e, et al., Glycobiology 12:79R—105R (2002). Precursor oligosaccharide molecules (COs, which as described. below, are also useful as plant signal molecules in the present invention) for the construction of LCOs may also be synthesized by genetically engineered organisms, e.g., as in Samain, et al., Carb. Res. 302:35-42 LCO's may be utilized in various forms 0: purity and may be used alone or in the form of a culture 0: LCO-producing bacteria or fungi. For example, OPTIM Zfi® (commercially avai'able from Novozymes BioAg Limited) cortains a culture Oi B. japonicum that produces an LCO (Cl8:l, MeFuc), MOR”6) that is illustrated in Fig. 2. Methods to provide substantially pure LCO's include simply ng the microbial cells from a mixture of LCOs and the microbe, or continuing to e and purify the LCO molecules through LCO solvent phase separation ed by HPLC chromatography as described, for example, in U.S. Patent 5,549,718. cation can be enhanced by repeated HPLC, and the purifed LCO les can be freeze-dried for long-term storage.

Chitins and chitosans, which are major components of the cell walls of fungi and the exoskeletons of insects and crustaceans, are also composed of GIcNAc residues. Chitinous nds include chitin, (IUPAC: N—[5-[[3-acetylamino-4,5- dihydroxy—6—(hydroxymethyl)oxan—2yl]methoxymethyl]—2—[[5— acetylamino-4,6-dihydroxy—2—(hydroxy methyl)oxan—3— y:]methoxymethyl]—4—hydroxy—6—(hydroxymethyl)oxan ys]ethanamide), and an, (IUPAC: 5-amino[5-amino[5- amino—4,6—dihydroxy—2(hydroxymethyl)oxan-3—yl]oxy—4—hydroxy—2— (hydroxymethyl)oxan—3—yl]oxy—2(hydroxymethyl)oxane-3,4-diol).

These compounds may be obtained commercially, e.g., from Sigma—Aldrich, or prepared from insects, crustacean shells, or fungal cell walls. Methods for the preparation of chitin and chitosan are known in the art, and have been described, for example, in U.S. Patent 4,536,207 (preparation from crustacean shells), Pochanavanich, et al., Lett. Appl. Microbiol. 35:17— 21 (2002) (preparation from fungal cell walls), and U.S.

Patent 5,965,545 ration from crab shells and hydrolysis of commercial chitosan). Deacetylated chitins and chitosans may be obtained that range from less than 35% to greater than 90% deacetylation, and. cover‘ a broad. spectruH1 of molecular weights, e.g., low molecular weight chitosan oligomers 0: less than 15k) and chitin oligomers of 0.5 to 2kD; "practical grade" chitosan with a leecular weight of about 150k); and high. molecular weight chitosan of up to 700k). Chitin and chitosan compositions formulated for seed treatment are also commercially available. Commercial products include, for example, fitfiXA® (Plant e rs, Inc.) and %fiYONDm (Agrihouse, Inc.).

Yet other chitinous compounds that are suitab'e for use in the present invention include COs (e.g., ed and/or purified). COs are known in the art as 0—1—4 linked N actyl amine structures identified as chitin oligomers, also as ylchitooligosaccharides. CO's have unique and different side chain decorations which make them different from chitin molecules [(Cfih3N05)n, CAS No. 13984], and an molecules [(C5HHNO4)n, CAS No. 9012—76—4].

Representation literature describing the structure and production of COs is as follows: Van der Holst, et al., Current n in Structural Biology, 11:608—616 (2001); Qobina, et al., Tetrahedron 58:521—530 (2002); Hanel, et al., ?lanta 232:787—806 (2010); Rouge, et al. Chapter 27, "The olecu'ar "mmunology of Complex Carbohydrates" in es in Experimental ne 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).

Two COs suitable for use in the present invention may be easily d from the LCOs shown in Figs. 1 and 2 (minus the fatty acid chains), which are the CO sors to the LCOs shown in Figs. 1 and 2. Methods for preparation of recombinant COs are known in the art. See, e.g., Samain, et a1. (supra.); Cottaz, et al., Meth. Eng. 11-7 (2005) and Samain, et al., J. hnol. 72:33-47 (1999).

Flavonoids are phenolic compounds having the general structure of two aromatic rings connected by a three-carbon bridge. Flavonoids are produced by plants and have many functions, e.gu, as beneficial signaling molecules, and as protection against insects, animals, fungi and bacteria.

Classes of flavonoids include chalcones, anthocyanidins, coumarins, flavones, flavanols, flavonols, flavanones, and isoflavones. See, Jain, et al., J. Plant 3iochem. & 3io:echnol. 11:1—10 (2002); Shaw, et al., Environmental icrobiol. 11:1867-80 (2006).

Representative flavonoids that may be useful in the practice 0: the present invention include genistein, daidzein, formononetin, enin, hesperetin, luteolin, and apigenin.

Flavonoid compounds are commercially ble, e.g., from Natland. ational Corp., Research. Triangle Park, NC; MP Riomedicals, Trvine, CA; LC tories, Woburn MA. Flavonoid compounds may be isolated from plants or seeds, e.g., as described in U.S. Patents 5,702,752; 5,990,291; and 6,146,668.

Flavonoid compounds may also be produced by genetically engineered organisms, such as yeast, as described in Ralston, et al., Plant Physiology 137:1375—88 (2005).

In other embodiments, the seed are treated with jasmonic acid (JA, [1R—[1d,26(Z)]]—3—oxo—2— (pentenyl)cyclopentaneacetic acid) and its derivatives, ic acid ((Z,Z)—9,12—Octadecadienoic acid) and its derivatives, and linolenic acid ((Z,Z,Z)—9,l2,15— octadecatrienoic acid) and its derivatives. Jasmonic acid and its methyl ester, methyl jasmonate (MeJA), collectively known as jasmonates, are octadecanoid—based compounds that occur naturally in plants. Jasmonic acid is ed by the roots of wheat seedlings, and by fungal microorganisms such as Botryodiplodia theobromae and Gibbrella fujikuroi, yeast (Saccharomyces cerevisiae), and pathogenic and non-pathogenic strains of Escherichia coli. Linoleic acid and linolenic acid are ed in the course of the biosyntthesis of ic acid. Jasmonates, linoleic acid and linoleic acid (and their derivatives) are reported to be inducers of nod gene expression or LCO production. by' rhizobacteriad See, e.g., Mabood, Fazli, Jasmonates induce the expression of nod genes in Bradyrhizobium japonicum, May 17, 2001; and Mabood, Fazli, "Linoleic and linolenic acid induce the expression or nod genes in Bradyrhizobium japonicum," USDA 3, May 17, 2001.

Useful derivatives of linoleic acid, linolenic acid, and jasmonic acid that may be useful in the ce 0: ,he present invention include esters, amides, glycosides and salts. Representative esters are compounds in which the carboxyl group of linoleic acid, linolenic acid, or jasmonic acid has been replaced with a ——COR group, where R is an ——OR1 group, in which R1 is: an alkyl group, such as a C1-C8 unbranched or ed alkyl group, e.g., a , ethyl or propyl group; an alkenyl group, such as a C2-C8 unbranched or branched alkenyl group; an alkynyl group, such as a C2-C8 unbranched or branched alkynyl group; an aryl group having, for example, 6 to 10 carbon atoms; or a heteroaryl group having, for example, 4 to 9 carbon atoms, wherein the heteroatoms in the heteroaryl group can be, for example, N, O, P, or S. Representative amides are compounds in which the carboxyl group of ic acid, nic acid, or jasmonic acid has been ed with a ——COR group, where R is an NRfig group, in which R2 and R3 are independently: en; an alkyl group, such. as a C1-C8 'unbranched. or branched. alkyl group, e.g., a methyl, ethyl or propyl group; an alkenyl group, such as a Cb-Cg unbranched or branched alkenyl group; an alkynyl group, such as a C2-C8 unbranched or branched alkynyl group; an aryl group having, for example, 6 to 10 carbon atoms; or a heteroaryl group having, for example, 4 to 9 carbon atoms, wherein the heteroatoms in the heteroaryl group can be, for example, N, O, P, or S. Esters may' be prepared. by known methods, such as acid-catalyzed nucleophilic addition, wherein the ylic acid is reacted with an alcohol in the presence of a catalytic amount of a mineral acid. Amides may also be prepared by known methods, such as by reacting the ylic acid with the appropriate amine in the presence of a coupling agent such as dicyclohexyl iimide (DCC), under neutral conditions. Suitable salts of linoleic acid, linolenic acid, and jasmonic acid include e.g., base addition salts. The bases that may be used as reagents to prepare metabolically acceptable base salts of these compounds include those derived from cations such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium). These salts Hay 1x3 readily prepared Tux mixing together a solution of ic acid, linoleric acid, or jasmonic acid with a solution of the base. The salt may be itated from solution and be collected by filtration or may be recovered by other means such as by evaporation of the solvent.

In other embodiments, the seed are treated with a vinylogous 4H—pyrone e.g., o[2,3-c]pyranones including derivatives and ues f, es of which are represented by the following structure: n; Z is O, S or NR5; R2, R2, R3, and R4 are each independently H, alkyl, alkenyl, alkynyl, phenyl, benzyl, y, hydroxyalkyl, alkoxy, phenyloxy, benzyloxy, CN, CO?“ COOR=, halogen, NR&%, or Iflb; and IQ” R& and. R7 are each independently H, alkyl or l, or a biologica"y acceptable salt thereof. Examples of biologically acceptable salts of these compounds may include acid addition salts formed with biologically acceptable acids, examples of which include hydrochloride, hydrobromide, sulphate or bisulphate, phosphate or hydrogen phosphate, acetate, benzoate, succinate, fumarate, maleate, lactate, citrate, tartrate, gluconate; methanesulphonate, benzenesulphonate and p-toluenesulphonic acid. Additional biologically acceptable metal salts may include alkali metal salts, with. bases, es of which irclude the sodium and potassium salts. Examples of compounds embraced by the structure and which may be suitable for use in :te present invention include the following: 3—methyl—2 — iLro[2,3-c]pyran—2—one (where , R2, R3, R4=E), 2 — iLro[2,3—c]pyran—2—one (where R2, R2, R3, R4=H), 7—He:hyl—2 — itro[2,3—c]pyran—2—one (where R2, R2, R4=H, R3=CH3), 5—methyl— 2E—furo[2,3—c]pyran—2—one (where R1, IQ, R3=H, R4=Ci3), 3,7— dimethyl—ZH—furo[2,3—c]pyran—2—one (where Rh Ih=Ci3,'%b IQ=H), methyl-2H—furo[2,3—c]pyran—2-one (where Rh R4=CP3, 'R” R3=H), 3,5,7—trimethyl—2H—furo[2,3-c]pyran—2—one (where {1, {& R4=Cfly R2=H), 5—methoxymethyl—3-methyl—2H—furo[2,3—c]pyran—2— one (where Ih=CH3, R2, R3=H, Rr4HbOCH3), 4—bromo—3,7—dimethyl— 2H—furo[2,3—c]pyran—2—one (where ZRh R3=CH& R2=Br, R4=H), 3— methylfuro[2,3—c]pyridin-2(3H)-one (where Z=NH, , R2, R& R4=H), 3,6—dimethylfuro[2,3-c]pyridin-2(6H)-one (where Z=N—— CH3, R2=CH3, R2, R3, R4=H). See, U.S. Patent 7,576,213. These molecules are also known as karrikins. See, Halford, supra.

Seeds may be treated with the plant signal molecule in several ways but preferably Via spraying or dripping.

Spray and drip treatment may be conducted by ating an e "ective amount of the plant signal le in an agriculturally' acceptable carrier, typically' aqueous in nature, and spraying or ng the composition onto seed via a uous ng system. (which. is calibrated. to apply ent at a predefined rate in proportion to the uous flow 0" seed), such as a drum-type of treater. These methods advantageously employ relatively small vo_umes of carrier so as to allow for relatively fast drying of the treated seed.

In this fashion, large s of seed. can be efficiently treated. Batch systems, in which a predetermined batch size of seed and signal molecule compositions are delivered into a mixer, may also be employed. Systems and apparati for performing these processes are commercially available from numerous suppliers, e.g., Bayer CropScience (Gustafson).

In another embodiment, the treatment entails coating seeds. One such process involves g the inside wall of a round container with the composition, adding seeds, then rotating the container to cause the seeds to contact the wall and the composition, a process known in the art as "container coating". Seeds can be coated by combinations 0; coating methods. Soaking lly entails use of an aqueous solution containing the plant growth enhancing agent. For example, seeds can be soaked for about 1 minute to about 24 hours (e.g., for at least 1 min, 5 min, 10 min, 20 min, 40 min, 80 min, 3 hr, 6 hr, 12 hr, 24 hr). Some types of seeds (e.g” soybean seeds) tend to be sensitive to moisture. Thus, soaking such seeds for an extended period of time may not be desirable, in which case the soaking is typically d out for about 1 minute to about 20 minutes.

Without intending to be bound by any particular theory of operation, Applicants believe that even to the extent that the treating may not cause the plant signal molecule to remain in contact with the seed surface after treatment and during any part of storage, the signal molecule may achieve intended effects by a phenomenon known as seed memory' or seed. perceptiond See, Macchiavelli and. Brelles— Marino, J. Exp. Bot. 55(408):2635—40 (2004) . Applicants also believe that following' ent the signal molecule, e.g., the LCD, es toward. the young developing radicle and aCtivates syHbiotic and developmental genes which results in a change in the root architecture of the plant.

NOtwithstanding, the compositions ning the plant signal molecule may further contain a sticking or coating agent to assist in adherence of the signal molecule to the seed. For aesthetic purposes, the compositions may further contain a coating polymer and/or a colorant.

The effective amount of the plant signal molecule used to treat the seed, expressed in units of concentration, generally ranges from about 10—5 to about 10'14 M, and in some embodiments, from about 10$ to about 10—“ M, and in some other embodiments from about 10—7 to about 10—8 M. Expressed in units of , the effective amount generally ranges from about 1 to about 400 ug/hundred weight (cwt) seed, and in some embodiments from about 2 to about 70 ug/cwt, and in some other embodiments, from about 2.5 to about 3.0 ug/cwt seed. The e e amount of the plant signal molecule, however, may be obtained by a suitable dosage response assay, preferably, in a greenhouse and/or field study.

The treatment may also e contaCting the seed, prior, simultaneously with or sequentially to the contacting with the plant signal molecule, with an agriculturally/agronomically beneficial agent. As used herein and in the art, the term. "agriculturally or argonomically beneficial" refers to agents that when applied to seeds result in enhancement (which. may’ be statistica ly' significant) of plant teristics such as plant stand, , vigor or yield in comparison to eated seeds. Representative examples of such agents that may be useful in the practice of the present invention includes, but is not limited to, diazotrophs, mycorrhizal fungi, herbicides, fungicides, insecticides, and phosphate solubilizing agents.

Suitable herbicides include bentazon, acifluorfen, chlorimuron, lactofen, clomazone, fluazifop, glufosinate, glyphosate, sethoxydim, imazethapyr, imazamox, fomesafe, flumiclorac, imazaquin, and. clethodim. Commercial ts containing each of these compounds are readily available.

Herbicide concentration in the composition will generally correspond to thc labclcd use rate for a particuiar herbicide.

A cide" as used herein and in the art, is an agent that kills or inhibits fungal growth. As used herein, a fungicide "exhibits activity against" a particular s of fungi if treatment with the fungicide results in killing or growth inhibition of a fungal population (e.g., in the soil) relative to an ted population. ive fungicides in accordance with the invention will suitably exhibit activity against a broad range of pathogens, including but not limited to Phytophthora, Rhizoctonia, Evsarium, Pythium, Phomopsis or Selerotinia and Phakopsora and combinations thereof. cial fungicides may be suitable for use in the present invention. le commercially available fungicides include PROTEGE, RIVAL or ALLEGIANCE FL or LS fson, '?lano, TX), WARDEN RTA (Agrilance, St. Paul, M1\), APROI\ XL, A?RON MAXX RTA or RFC, MAXIM 4FS or XL (Syngenta, Wilmington, DE), CAPTAN (Arvesta, Guelph, Ontario) and PROTRL-lJAT gin ina, 3uenos Ares, Argentina). Active ingredients in these and other commercial fungicides include, but are not limited to, fludioxonil, mefenoxam, azoxystrobin and metalaxyl. Commercial fungicides are most suitably' used in accordance with the manufacturer's instructions at the recommended concentrations.

As used. herein, an insecticide "exhibits activity against" a ular species of insect if treatment with the insecticide results in killing or inhibition of an insect population relative to an untreated population. Effective insecticides in accordance with. the invention will suitably exhibit activity t a broad range of s including, but not limited to, wireworms, cutworms, grubs, corn rootworm, seed corn maggots, flea. beetles, chinch. bugs, aphids, leaf beetles, and stink bugs.

Commercial insecticides may be suitable for use in the present invention. Suitable commercially-available insecticides include CRUISER (Syngenta, Wilmington, DE), GAUCHO and PONCHO (Gustafson, Plano, TX). Active ingredients in these and other commercial insecticides include thoxam, anidin, and imidacloprid. Commercial insecticides are most suitably' used. in accordance with the manufacturer's instructions at the recommended concentrations.

As used herein, phosphate lizing agents include, but are not limited to, phosphate solubilizing microorganisms. As used herein, “phosphate solubilizing microorganism” is a microorganism that is able to increase the amount of phosphorous available for a plant. Phosphate solubilizing microorganisms include fungal and bacterial strains. In embodiment, the phosphate solubilizing microorganism is a spore forming microorganism.

Non-limiting es of phosphate lizing rganisms include species from a genus selected from the group consisting o: obacter, Arthrobacter, Arthrobotrys, illus, 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.

Non—limiting es of phosphate solubilizing microorganisms are selected from the group consisting Acinetobacter calcoaceticus, obacter sp, Arthrobacter sp., Arthrobotrys oligospora, Aspergillus .niger, Aspergillus sp., Azospirillum halopraeferans, us iquefaciens, Bacillus atrophaeus, Bacillus circulans,Bacillus licheniformis, Bacillus subtilis, Burkholderia cepacia, Burkholderia vietnamiensis, Candida krissii, Chryseomonas luteola, Enterobacter nes, Enterobacter asburiae, Enterobacter sp., Enterobacter taylorae, Eupenicillium parvum, Exiguobacterium sp., Klebsiella sp., Kluyvera cryocrescens, Microbacterium sp., Mucor ramosissimus, Paecilomyces hepialid, Paecilomyces ndii, Paenibacillus macerans, Paenibacillus mucilaginosus, Pantoea rans, Penicillium expansum, Pseudomonas corrugate, Pseudomonas fluorescens, Pseudomonas lutea, Pseudomonas poae, Pseudomonas putida, Pseudomonas ri, Pseudomonas trivialis, Serratia marcescens, Stenotrophomonas maltophilia, Streptomyces sp., Streptosporangium sp., athania salitolerans, Thiobacillus ferrooxidans, Torulospora globosa, Vibrio proteolyticus, Xanthobacter agilis, and Xanthomonas campestris.

Preferably, the phosphate solubilizing microorganism is a strain of the fungus Penicillium. Strains of the fungus Penicillium that may be useful in the practice of the present ion include P. bilaiae rly known as P. bilaii), P. albidum, P. iogriseum, P. chrysogenum, P. citreonigrum, P. citrinum, P. digitatum, P. frequentas, P. fuscum, P. gaestrivorus, .P. glabrum, £2 griseofulvum, .P. implicatum, £2 janthinellum, P. lilacinum, P. minioluteum, P. montanense, P. nigricans, P. um, P. pinetorum, P. pinophilum, P. purpurogenum, P. radicans, P. radicum, P. raistrickii, P. rugulosum, P. simplicissimum, P. solitum, P. variabile, P. velutinum, .P. Viridicatunu .P. glaucum, .P. orus, and 1% expansum.

More preferably, the phosphate solubilizing microorganism Penicillium species is P. bilaiae, P. gaestrivorus, and/or a ation thereof. Most preferably, the P. bilaiae strains are ed from the group consisting of ATCC 20851, NRRL 50169, ATCC 22348, ATCC 18309, NRRL 50162 (Wakelin, et al., 2004. Biol Fertil Soils 40:36-43) and the P. gaestrivorus strain is NRRL 50170 (see, Wakelin, supra.). ing to the invention, it is envisioned that more than one phosphate solubilizing microorganism. may be used, such as, at least two, at least three, at least :our, at least five, at least six, including' any' combination. of the Acinetobacter, Arthrobacter, Arthrobotrys, Aspergillus, Azospirillum, Bacillus, Burkholderia, Candida Chryseomonas, Enterobacter, Eupenicillium, Exiguobacterium, Klebsiella, Kluyvera, Microbacterium, MUcor, Paecilomyces, Paenibacillus, Penicillium, Pseudomonas, Serratia, Stenotrophomonas, Streptomyces, Streptosporangium, Swaminathania, Thiobacillus, Torulospora, Vibrio, Xanthobacter, and xanthomonas, including one species selected from the following group: Acinetobacter calcoaceticus, Acinetobacter sp, Arthrobacter sp., Arthrobotrys oligospora, Aspergillus .nigery Aspergillus sp., Azospirillum halopraeferans, us amyloliquefaciens, us atrophaeus, Bacillus circulans,Bacillus licheniformis, Bacillus subtilis, Burkholderia cepacia, lderia vietnamiensis, Candida i, Chryseomonas luteola, Enterobacter aerogenes, bacter asburiae, Enterobacter sp., Enterobacter taylorae, Eupenicillium parvum, Exiguobacterium sp., Klebsiella sp., Kluyvera cryocrescens, Microbacterium sp., Mucor ramosissimus, Paecilomyces hepialid, Paecilomyces marguandii, acillus macerans, Paenibacillus mucilaginosus, Pantoea aglomerans, Penicillium expansum, Pseudomonas corrugate, Pseudomonas scens, monas lutea, Pseudomonas poae, Pseudomonas putida, monas stutzeri, Pseudomonas trivialis, Serratia marcescens, Stenotrophomonas maltophilia, Streptomyces sp., Streptosporangium sp., Swaminathania salitolerans, Thiobacillus ferrooxidans, Torulospora globosa, Vibrio proteolyticus, bacter , and Xanthomonas campestris.

Diazotrophs are bacteria and archaea that fix atmospheric nitrogen gas into a more usable form such as ammonia. Examples of diazotrophs e bacteria from the genera Rhizobium spp. (e.g., R. osilyticum, R. daejeonense, R. etli, R. galegae, R. gallicum, R. giardinii, R. hainanense, R. ense, R. ferae, nosarum, R. loessense, R. lupini, R. lusitanum, meliloti, R. mongolense, R. miluonense, R. sullae, tropici, R. undicola, and/or R. yanglingense), hizobi Spp- (e.g., B. bete, B. canariense, 3. e kanii, iriomotense, B. japonicum, B. jicamae, B. liaoningense, 3. 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), izobium spp., (M. albiziae, M. ae, M. chacoense, M. ciceri, M. huakuii, M. loti, M. mediterraneum, M. pluifarium, M. septentrionale, M. temperatum, and/or M. tianshanense), and combinations thereo;. 211 a particular embodiment, the diazotroph is selected from the group consisting of B. japonicum, R leguminosarum R meliloti, S. meliloti, and combinations f. 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. tic associations with the roots of a vascular plant, and provide, e.g., absorptive capacity for water and mineral nutrients due to the comparatively large surface area of myceliunu Mycorrhizal fungi e endomycorrhizal fungi (also called vesicular arbuscular mycorrhizae, VAMs, arbuscular mycorrhizae, or AMs), an ectomycorrhizal fungi, or a combination thereof. In one embodiment, the mycorrhizal fungi is an corrhizae of the phylum Glomeromycota and genera Glomus and Gigaspora. In still a further ment, the endomycorrhizae is a strain of Glomus aggregatum, Glomus brasilianum, Glomus clarum, Glomus deserticola, Glomus etunicatum, Glomus fasciculatum, Glomus intraradices, Glomus monosporum, or Glomus e, Gigaspora margarita, or a combination thereof.

Examples of mycorrhizal fungi include ectomycorrhizae of the phylum Basidiomycota, Ascomycota, and Zygomycota. Other examples include a strain 0: Laccaria bicolor, Laccaria laccata, thus tinctorius, Rhizopogon amylopogon, Rhizopogon fulvigleba, ogon luteolus, Rhizopogon villosuli, Scleroderma cepa, Scleroderma citrinum, or a combination thereof.

The mycorrhizal fungi include ecroid mycorrhizae, arbutoid mycorrhizae, or opoid mycorrhizae. ular and ectomycorrhizae form ericoid mycorrhiza with many plants belonging to the order Ericales, while some Ericales form arbutoid and monotropoid mycorrhizae. In one embodiment, the mycorrhiza. may' be an ericoid. mycorrhiza, preferably' of the phylum Ascomycota, such as Hymenoscyphous ericae or Oidiodendron sp. In another embodiment, the mycorrhiza also may be an arbutoid mycorrhiza, ably of the phylum 3asidiomyco:a. In yet another embodiment, the mycorrhiza may be a Hmnotripoid hiza, ably of the phylum 3asidiomyco:a. In still yet another embodiment, the mycorrhiza may be an orchid. mycorrhiza, preferably 0" she genus Rhizoctonia.

The methods of the present invention are applicable to leguminous seed, representative examples of which e soybean, alfalfa, peanut, pea, , bean and clover. The methods of the present invention are also applicable to non- leguminous seed, e.g., Poaceae, Cucurbitaceae, Malvaceae.

Asteraceae, Chenopodiaceae and ceae. Representative examples of non—leguminous seed include field crops such as corn, cereals such as rice, barley and wheat, cotton and , and vegetable crops such as potatoes, tomatoes, cucumbers, beets, lettuce and cantaloupe.

Following treatment, and for purpose of storage, the seed is then packaged, e.g., in 50—lb or lOO-lb bags, or bulk bags or containers, in accordance with. standard. ques.

The seed is stored for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, l0, ll, or 12 months, and even longer, e.g., l3, I4, 15, l6, l7, l8, 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 which are known in the art. As used herein, the term “month” shall mean 30 days. As used , a year shall mean 365 days. Whereas soybean seed. may' have to be planted the following season, corn seed can be stored for much longer periods of time including upwards of 3 years.

The plant signal molecule may be applied in any suitable manner, such as, in the fOIHI of a seed treatment composition which comprises at least one plant signal molecule and an agriculturally acceptable carrier.

Any' suitable lturally' able carrier‘ may be used, for example, a solid carrier, semi—solid carrier, an aqueous-based liquid carrier, a non—aqueous based liquid carrier, a suspension, an emulsion or an emulsiliable concentrate. Agriculturally—acceptable carriers may include, e.g., nts, inert components, dispersants, surlacuants, tackifiers, s, stabilizing agents, and/or polymers.

The seed treatment composition may furuher include one or more agriculturally/agronomically beneficial agents (that is in on to the signal molecule), such as, one or more diazotrophs, mycorrhizal fungi, herbicides, ides, insecticides, and/or phosphate lizing agents.

The present invention will now be described by way 0: the following non—limiting examples. They are presented solely for purposes of illustration, and are not intended to limit the invention in any way.

Summary of Wbrking Examples Examples 1 and 2 describe comparative field experiments using soybean seed that trate that the claimed invention achieves increased plant yield. Seed were treated in accordance with the present invention at 5 Hwnths prior to planting with the commercial product Optimize® which is a ation of Bradyrhizobium japonicum inoculant and LCO-V (Cl8zl, MeFuc)(illustrated in Fig. 2), and with the pure LCO alone, and at 4.5 months prior to planting with non— commercial (i.e., less pure) grades of Optimize® and the LCD alone, and for purposes of comparison with these same plant signal molecules at the time of planting. Untreated seed was used as another control. The results, which are expressed in terms of the difference in grain yield, ed in units 0; bushels/acre, show that the methods of the claimed invention ed an increase in soybean yield, relative to non- inventive methods (i.e., seed treated at time of planting and non-treated seed).

Examples 3 and. 4 describe comparative experiments conducted in the greenhouse and. which demonstrate that the claimed invention achieves increases in other plant growth characteristics. Example 3 describes an experiment that entailed. treatment of soybean seed. with. pure LCO-V (Cl8zl, MeFuc) one month and one year before planting. The soybean plants ding roots) were harvested ten days alter planting. Results, which are described in terms or di""erences in root length and volume, show the methods of the present invention achieve dramatic ses in these properties. Lastly, Example 4 bes experiments condJCted with soybean seed treated. with. Optimize® 55 days prior to planting and for purposes of comparison, soybean seed treated 7 days prior to planting and untreated seed. The results, expressed in units of mean e area of first trifoliate leaves, show that the d invention enhances plant grOWth in this respect too.

EXAMPLE 1 A field trial was conducted to evaluate embodiments of the present invention on grain yield when applied on soybean seed. The field trial site was located near Whitewater, WI and characterized by Milford silty clay loam soil. Soil testing, conducted six months prior to planting, showed a soil pH of 6.8, an organic matter t of 5.3%, and orus and potassium contents of 39 ppm and 139 ppm, respectively.

The plant signal leecules used in the trial were Optimize®, a mmercial grade of Optimize® (NZ—5OS—l), pure LCO-V' (Cl8:l, MeFuc)(NI-5OGREN-l) and. a non—commercial grade of LCO-V , MeFuc)( NI-5OS-2CF). The soybean seed used in the study was Stine S2118. The plant signal molecules were sprayed onto seeds with/without dilution at a rate of 4.8 fl oz/cwt.

The study was conducted in a randomized complete block design, with a plot size of 10 feet by 50 feet (0.011 acres), with 7.5-inch row spacing. Four replications were conducted. Seed were d with the plant signal molecules 4.5 or 5 months prior to planting and just prior to planting, and. were planted. at a depth. of 1 inch. and. at a seedling rate of 225,000 seeds per acre using a John Deere 750 NT grain drill. The ides Extreme® and AMPS® were both applied 11 days prior to planting (pre—emergence) at rates or 3.0 pt and 2.5 lb, respectively. Assure ®, Roundup rMax® and AMPS® were all applied 46-days post-planting (post-emergent), at rates of 6.0 oz, 21 oz and 2.5 lb, tively. Plants were harvested. 4 months and. 20 days aloer planting.

The Control seed was treated with an amount (wt) I) water, corresponding to the amount (wt) of the experimental signal molecule composition (signal molecule + carrier). The Control seed was stored under the same conditions as the experimental seed prior to planting and planted at the same time as the experimental seed in the same soil.

Results of the study are shown in Table 1 below.

Table l ENT GROUP GRAIN YIELD @ 13% Control - non-inoculated Optimize — at planting 64.2 Optimize — 5 month NZ—5OGREN—l — At planting 62.2 6 NI-5OS-l - 4.5 month 67.2 7 NZ—5OS—2CF — 4.5 month 69.6 As reflected by the comparison n comparative (non—inventive) Group 2 and inventive Group 3, treatment or the soybean seed with the commercial-grade Optimize® at 5 months anting resulted in an increase in soybean yield of 1.5 bushels of soybean. As reflected by the comparison between Group 4 and inventive Group 5, treatment of soybean seed at 5 months pre-planting with pure LCO-V (C18:1, MeFuc) alone resulted in an increase in soybean yield of 8.3 bushels/acre. As reflected by the comparison between Group 2 and inventive Group 6, treatment of the soybean seeds 4.5 months prior to planting with the non-commercial grade of Optimize® resulted. in an increase in soybean yield of 3.0 bushels/acre. Lastly, as shown by the comparison between Group 4 and inventive Group 7, treatment of soybean seeds with the non-commercial grade of LCO—V' (C18:1, MeFuc) alone 4.5 months anting increased soybean yield by 7.4 bushels/acre. Grain yield measurements were taken at a 13% seed moisture level.

EXAMPLE 2 A n trial was conducted to evaluate embodiments of the present ion on grain yield when applied on soybean seed. The field trial site was located near Whitewater, WI and characterized by Milford silty clay loam soil. Soil testing, conducted six months prior to planting, showed a soil pH of 6.6, an organic matter content of 4.8%, and phosphorus and potassium contents of 41 ppm and 131 ppm, respectively.

The plant signal es used in the trial were same as in Example 1. The soybean seed used in the study was Stine S2118. The plant signal leecules were sprayed onto seeds ithout dilution at a rate of 4.8 fl oz/cwt.

The study was conducted. in a randomized te block design, with a plot size of 10 feet by 50 feet (0.011 acres), with 7.5-inch row spacing. Four replications were conducted. Seed were treated with the plant signal molecules 4.5 or 5 months prior to ng and just prior to planting, and. were planted. at a depth. of 1 inch. and. at a seedling rate of 225,000 seeds per acre using a John Deere 750 NT grain drill. The pesticides e® and AMPS® were both applied 10 days prior to planting (pre—emergence) at rates of 3.0 pt and 2.5 lb, respectively. Assure II®, Roundup WeatherMax® and AMPS® were all applied 45-days post-planting (post-emergent), at rates of 6.0 oz, 21 oz and 2.5 lb, respectively. Plants were ted 4 Inonths and 21 days after planting.

The Control seed was treated with an amount (wt) of water, corresponding to the amount (wt) of the experimental signal molecule composition (signal le + carrier). The Control seed was stored under the same conditions as the experimental seed prior to planting and planted at the same time as the experimental seed in the same soil.

Results of the study are shown in Table 2 below.

Table 2 TREATMENT GROUP GRAIN YIEL: Control - non—inoculated .4 Optimize — at planting Optimize — 5 month REN—1 — At planting NI-50GREN-__ - 5 month 2—508- 1 4. 5 month 69.

N_-5OS- 2CF - 4. 5 month As reflected by the comparison between comparative (non—inventive) Group 2 and inventive Group 3, treatment of the soybean seed with the cial—grade Optimize® at 5 months pre—planting resulted in an increase in n yield 0: 4.5 bushels of soybean. As reflected by the comparison between Group 2 and inventive Group 6, treatment or the soybean seeds 4.5 months prior to planting with the non— commercial grade of Optimize® ed in an increase in n yield of 5.3 bushels/acre. As shown by the comparison between Group 4 and inventive Group 7, treatment 0: soybean seeds with the non-commercial grade of LCO-V (Cl8zl, MeFuc) alone 4.5 months pre-planting increased soybean yield by 0.8 bushels/acre. The only negative response as ted by the comparison between non-inventive Group 4 and inventive Group 5, showed that treatment of soybean seed at 5 months pre—planting with the pure LCO alone ed in a decrease in 1.8 bushels/acre, a result attributable to unexplained variability associated with field trials. Grain yield measurements were taken at a 13% seed moisture level.

Greenhouse ments EXAMPLE 3 Soybean seeds treated with 10—34 pure LCO-V (C18:1, MeFuc) and stored at 15°C. Treated seeds and non-treated seeds (control) were planted. 1 and. 12 months after treatment in greenhouse pots containing sandzperlite (1:; e).

Seedlings were grown for 10 days after. seed. plantings then seedlings were harvested, their roots cleaned and ed on the Winrhizo® scanner. The Control seed was treated with an amount (wt) of water, corresponding to the amount (wt) of the experimental signal molecule composition (signal molecule + carrier). The Control seed was stored under the same conditions as the experimental seed. prior to planting and planted at the same time as the experimental seed in the same soil. The results are shown in Table 3. :ter Treatment 1 month after treatment treatHent Root length Root Vol. Root Vol. (cm) (cm3) (cmB) Control 128 0.455 . 0.403 LCO 135* 0.468 . 0.540* increase The results achieved by both inventive ments (seed treated. with LCD at 1 month. and. 12 months prior to planting), and ularly' the results obtained after the 1-year atment, are dramatic, considering that it is known in the art that soybean seed are prone to deteriorate over that length of time.

EXAMPLE 4 Soybean seeds treated. with. OptimizeC) were kept at °C 111 a refrigerator. Seeds were planted 7 (7 dpp) and 55 (55dpp) days after treatment in root boxes containing a peatzperlite mix. Their leaf surface area (cm?) were taken from the firs, iate after 19 days. As illustrated in Fig. 3 and shown in Table 4, the leaves generated from seed treated in accordance with. the present invention. had. a 50% greater mean increase in leaf surface area compared. to the non—inventive embodiment (42% versus 28%).

TABLE 4 7dpp mean 187.05 29. 8215 40.81 _ 55dpp mean 207.18 20. 5278 60.93 Since it is known that the bacterial (Bradyrhizobium japonicum) count on seed docroasos over time, the increase in mean surface area shown in plants ted from seed treated 55 days prior to planting may be utable to the rhizobial LCO.

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

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

Claims (45)

CLAIMS :
1. Claim 1. A method of enhancing plant growth, comprising ng a seed at least one month prior to planting with a chitooligosaccharide (CO) wherein, upon harvesting, the plant exhibits at least one of increased plant yield measured in terms of s/acre, increased root number, increased root length, increased root mass, increased root volume and increased leaf area, as compared to a plant harvested from a control seed.
2. Claim 2. The method of claim 1, wherein the control seed is treated with the CO just prior to or within a week or less of planting.
3. Claim 3. The method of claim 1 or claim 2, wherein the CO is represented by the structure:
4. Claim 4. The method of claim 1 or claim 2, wherein the CO is ented by the structure:
5. Claim 5. The method of any one of claims 1 to 4, wherein the CO is synthetic.
6. Claim 6. The method of any one of claims 1 to 4, wherein the CO is recombinant. 11016451_1
7. Claim 7. The method of any one of claims 1 to 6, wherein the seed is treated with the CO at least 9 months prior to planting.
8. Claim 8. The method of any one of claims 1 to 6, wherein the seed is treated with the CO at least 12 months prior to planting.
9. Claim 9. The method of any one of claims 1 to 6, wherein the seed is treated with the CO at least 2 years prior to planting.
10. Claim 10. The method of any one of claims 1 to 9, further sing treating the seed with at least one phosphate solubilising rganism.
11. Claim 11. The method of any one of claims 1 to 9, further comprising treating the seed with one or more strains of Penicillium.
12. Claim 12. The method of any one of claims 1 to 9, further comprising treating the seed with one or more strains of P. bilaiae.
13. Claim 13. The method of claim 12, wherein the seed is treated with: the strain having the deposit accession number NRRL 50162; the strain having the deposit accession number NRRL 50169; the strain having the t accession number ATCC 20851; the strain having the deposit accession number ATCC 22348; and/or the strain having the deposit accession number ATCC 18309.
14. Claim 14. The method of any one of claims 1 to 13, further comprising treating the seed with one or more strains of P.gaestrivorus.
15. Claim 15. The method of claim 14, wherein the seed is treated with the strain having the deposit accession number NRRL 50170. 11016451_1
16. Claim 16. The method of any one of claims 1 to 15, further comprising treating the seed with one or more strains of Rhizobium.
17. Claim 17. The method of any one of claims 1 to 15, further comprising ng the seed with one or more strains of R. leguminosarum.
18. Claim 18. The method of any one of claims 1 to 17, further comprising treating the seed with one or more s of Sinorhizobium.
19. Claim 19. The method of any one of claims 1 to 17, further comprising treating the seed with one or more strains of S. meliloti.
20. Claim 20. The method of any one of claims 1 to 19, further comprising treating the seed with one or more strains of Bradyrhizobium.
21. Claim 21. The method of any one of claims 1 to 19, further comprising treating the seed with one or more strains of B. japonicum.
22. Claim 22. The method of any one of claims 1 to 21, further comprising treating the seed with a fungicide and/or an insecticide.
23. Claim 23. The method of claim 22, wherein the seed is treated with xyl, clothianidin, xam, fludioxonil, thoxam and/or imidacloprid.
24. Claim 24. The method of claim 22, wherein the seed is treated with mefanoxam, fludioxonil and thiamethoxam.
25. Claim 25. The method of any one of claims 1 to 24, further comprising treating the seed with a chitin and/or a chitosan. 11016451_1
26. Claim 26. The method of any one of claims 1 to 25, further comprising treating the seed with a flavonoid.
27. Claim 27. The method of any one of claims 1 to 26, wherein the CO is applied to the seed at a concentration of about 10-14 to about 10-5 Molar.
28. Claim 28. The method of any one of claims 1 to 26, wherein the CO is applied to the seed at a concentration of about 10-11 to about 10-5 Molar.
29. Claim 29. The method of any one of claims 1 to 26, wherein the CO is applied to the seed at a concentration of about 10-8 to about 10-7 Molar.
30. Claim 30. The method of any one of claims 1 to 29, wherein the CO is applied to the seed in an amount ranging from about 1 to about 400 µg / hundred weight seed (cwt).
31. Claim 31. The method of any one of claims 1 to 29, n the CO is applied to the seed in an amount ranging from about 2 to about 70 µg / cwt.
32. Claim 32. The method of any one of claims 1 to 29, wherein the CO is d to the seed in an amount ranging from about 2.5 to about 3.0 µg / cwt.
33. Claim 33. The method of any one of claims 1 to 32, wherein the seed is leguminous.
34. Claim 34. The method of any one of claims 1 to 32, wherein the seed is a pea seed.
35. Claim 35. The method of any one of claims 1 to 32, wherein the seed is a lentil seed.
36. Claim 36. The method of any one of claims 1 to 32, wherein the seed is a bean seed.
37. Claim 37. The method of any one of claims 1 to 32, wherein the seed is a soybean seed. 11016451_1
38. Claim 38. The method of any one of claims 1 to 32, wherein the seed is non-leguminous.
39. Claim 39. The method of any one of claims 1 to 32, wherein the seed is a wheat seed.
40. Claim 40. The method of any one of claims 1 to 32, n the seed is a barley seed.
41. Claim 41. The method of any one of claims 1 to 32, wherein the seed is a cotton seed.
42. Claim 42. The method of any one of claims 1 to 32, wherein the seed is a canola seed.
43. Claim 43. The method of any one of claims 1 to 32, wherein the seed is a corn seed.
44. Claim 44. A seed treated according to the method of any one of claims 1 to 43.
45. Claim 45. A plant germinated from the seed of claim 44. Novozymes BioAg A/S By the Attorneys for the Applicant SPRUSON & FERGUSON Per: 11016451_1