NZ622042B2 - 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 lipo-chitooligosaccharide (LCO), 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 treated with the LCO just prior to or within a week or less of planting. increased root length, increased root mass, increased root volume and increased leaf area, as compared to a plant harvested from a control seed treated with the LCO just prior to or within a week or less of planting.

Description

SEED TREATMENT METHODS AND COMPOSITIONS BACKGROUND OF THE INVENTION The symbiosis between the gram-negative soil bacteria, Rhizobiaceae and hizobiaceae, and legumes such as soybean, is well documented. The Zbiochemica' basis for these relationships includes an exchange of molecular signaling, wherein the plant-to-bacteria signal compounds include flavones, vones and flavanones, and the bacteria-to-plant signal compounds, which include the end products of the expression of the Bradyrhizobial and Rhizobial nod genes, known as lipo—chitooligosaccharides (LCOs). The symbiosis between these bacteria and th legumes onablos tho legume to fix atmospheric en and thereby grow in soil that has low lable nitrogen levels, 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 increasing the use of en-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 suitable carrier and applying the ition in the immediate vicinity A: o_ a seed or seedling in an effective amount for enhancing seed ation of ng emergence in ison 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, icide, or combination thereof, to e 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), seedling, root or plant.

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

A number of other publications describe the t 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,” Microbiol 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) -487.

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

BRIEF SUMMARY OF THE INVENTION 0006A] In a first aspect, the present invention provides a method of enhancing plant growth, comprising treating a seed at least one month prior to planting with a lipochitooligosaccharide (LCO), 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 l seed treated with the LCO just prior to or within a week or less of ng. (10266535_1):MGH [0006B] In a second aspect, the present invention es 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 growthenhancing agent) to the seed contemporaneously with planting. The t invention is based, in part, on the discovery that ent 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 number, length and mass, compared to plants harvested (10266535_1):MGH from both untreated seeds. The present invention also provides methods of enhancing plant growth and crop production in which additional improvements may be ed over plants crops produced from seeds treated just prior to or within a week or less of planting.

A first aspect of the present invention is directed to a method of enhancing plant growth, sing 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 d in ance with the present method at 2 months prior to planting, at least 3 months prior to planting, 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, sed root volume and sed lea: area, compared to plants harvested from untreated seed. In particular embodiments, the treatment may be used to produce a plant (crop) that ts 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 invention, the plant signal le 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. Rhizobium sp., Bradyrhizobium Sp., e.g., Bradyrhizobium japonicum, Sinorhizobium Sp. and Azorhizobium sp, or from an arbuscular mycorrhizal fungus.

In other embodiments, the plant signal molecule 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 ments, the CO is ed from a microorganism as per LCO's.

In other embodiments, the plant signal molecule is a flavonoid. In other embodiments, the plant signal molecule is ic acid, linoleic acid, linolenic acid or a derivative thereof. In other embodiments, the plant signal molecule is a karrikin.

Combinations of two or more ent 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., roph (Rhizobial inoculant), mycorrhizal fungi, a phosphate solubilizing agent, herbicide, insecticide or a fungicide. In some ments, the treating entails spraying' a composition comprising the plant signal le onto the seed, and. in some other‘ embodiments, the treating entails dripping the composition onto the seeds.

The method of the present 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 upwards of 3 years prior to planting.

The present invention also relates to seeds d 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 planted, 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 ments, even 3 years prior to planting.

A d aspect of the present invention is ed to a package comprising the treated seeds according to the present invention for purposes of ng subsequent to the treatment.

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

As further trated in the working examples, which include comparative experiments conducted under both greenhouse and. field. conditions, embodiments o: the present invention that entailed treatment of soybean seed with an LCD from Rradyrhizobium cum 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 al structures 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 n 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 es of the t invention, the term "plant signal molecule", which. may' be used interchangeably with "plant growth—enhancing agent" broadly .C reiers to any agent, both naturally' occurring' in plants or microbes, and synthetic (and which may be non-naturally ing) that directly or indirectly activates a plant biochemical pathway, resulting in increased plant , measureable at least in terms of 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. Representative examples of plant signal molecules that may be useful in the practice of the present invention include lipo-chitooligosaccharide compounds (LCO's), chito- oligosaccharides (COs), chitinous compounds, flavonoids, jasmonic acid, linoleic acid and linolenic acid and their derivatives, and ins.

The plant signal molecule may be isolated and/or purified component. The term. “isolated” means the signal molecule is removed from its natural state and separated from other molecules lly associated with it. The term “purified" means that the concentration of the signal le 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 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 ucing sugar residues. An example of an LCO is presented below as formula I CH2OR3 0R4 G NH-R7 in which: G is a hexosamine which can be tuted, for example, 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, ent 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., ium sp., Bradyrhizobium sp., Sinorhizobium sp. and Azorhizobium sp. LCO structure is teristic for each such bacterial species, and each strain may produce multiple LCO's with different structures. For example, specific LCOs from S. meliloti have also been described. in 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 R=CH3CO--), they become AcNodRM- 1, and AcNodRM-B, respectively (U.S. Patent 5,545,718).

LCOs from Bradyrhizobium japonicum are described in U.S. Patents 5,175,149 and 011. Broadly, they are pentasaccharide phytohormones sing 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 recovered from bacterial strains that e LCO's, such as strains of Azorhizobium, Bradyrhizobium (including B. japonicum), Mesorhizobium, Rhizobium (including R. leguminosarum), Sinorhizobium (including S. meliloti), and bacterial strains genetically ered to produce LCO's.

LCO's are the primary determinants of host specificity in legume symbiosis (Diaz, et al., Mol. Plant- Microbe Interactions 13:268—276 (2000)). Thus, within the legume family, specific genera and 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 (1997), 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‘ trifolii/red. clover. Hungria also lists the effective flavonoid Nod gene inducers of the rhizobial s, and the ic LCO structures that are produced by the ent rhizobial s. However, LCO specificity is only required to establish nodulation in legumes. In the practice 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, sed 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 ng. Thus, by way of example, an LCD obtained 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 gumes too.

Also encompassed by the present invention is use of LCOs obtained (e.g., isolated and/or ed) from arbuscular mycorrhizal fungi, such as fungi of the group Glomerocycota, e.g., Glomus intraradicus. The structures 0: representative LCOs ed from these fungi are described in and WO 49751 (the LCOs described therein also referred to as "Myc factors"). r encompassed by the present invention is use of synthetic LCO compounds, such as those described in WO2005/063784, and inant LCO's produced through genetic engineering. The basic, naturally occurring LCO structure 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 D'Haeze, et al., Glycobiology 12:79R—105R (2002). Precursor oligosaccharide molecules (COs, which as described. below, are also useful as plant signal les in the present invention) for the construction of LCOs may also be synthesized by genetically engineered organisms, e.g., as in Samain, et al., Carb. Res. 302:35-42 (1997).

LCO's may be utilized in various forms 0: purity and may be used alone or in the form of a culture 0: oducing bacteria or fungi. For example, OPTIM Zfi® rcially avai'able from Novozymes BioAg Limited) cortains a culture Oi B. japonicum that produces an LCO (LCO-V(Cl8:l, MeFuc), MOR”6) that is illustrated in Fig. 2. Methods to e substantially pure LCO's include simply removing the microbial cells from a mixture of LCOs and the microbe, or continuing to isolate and purify the LCO molecules h LCO solvent phase separation followed by HPLC chromatography as bed, for example, in U.S. Patent 5,549,718. Purification can be enhanced by repeated HPLC, and the purifed LCO molecules 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 compounds include , (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 chitosan, (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 ), Pochanavanich, et al., Lett. Appl. Microbiol. 35:17— 21 (2002) (preparation from fungal cell walls), and U.S.

Patent 5,965,545 (preparation from crab shells and hydrolysis of commercial chitosan). Deacetylated chitins and chitosans may be obtained that range from less than 35% to r than 90% deacetylation, and. cover‘ a broad. spectruH1 of lar 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 e, fitfiXA® (Plant Defense Boosters, Inc.) and m (Agrihouse, Inc.).

Yet other ous nds that are 'e for use in the present invention include COs (e.g., isolated and/or purified). COs are known in the art as 0—1—4 linked N actyl glucosamine structures identified as chitin oligomers, also as N—acetylchitooligosaccharides. CO's have unique and different side chain decorations which make them different from chitin molecules N05)n, CAS No. 13984], and chitosan 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 Opinion in Structural Biology, 11:608—616 (2001); , 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 Advances in mental Medicine and Biology, Springer Science; Wan, et al., Plant Cell 21:1053—69 (2009); PCT/F100/00803 (9/21/2000); and Demont-Caulet, et al., Plant Physiol. 120(1):83-92 (1999).

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 precursors 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. 7(4):311-7 (2005) and Samain, et al., J. Biotechnol. 72:33-47 (1999).

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

Classes of flavonoids include chalcones, anthocyanidins, coumarins, flavones, flavanols, flavonols, flavanones, and isoflavones. See, Jain, et al., J. Plant 3iochem. & 3io:echnol. 0 (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, onetin, naringenin, hesperetin, in, and apigenin.

Flavonoid compounds are commercially available, e.g., from Natland. Tnternational Corp., Research. Triangle Park, NC; MP Riomedicals, Trvine, CA; LC Laboratories, 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 cally engineered organisms, such as yeast, as described in n, 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, linoleic acid ((Z,Z)—9,12—Octadecadienoic acid) and its derivatives, and nic 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 produced in the course of the biosyntthesis of jasmonic acid. Jasmonates, linoleic acid and linoleic acid (and their derivatives) are reported to be inducers of nod gene expression or LCO tion. by' rhizobacteriad See, e.g., Mabood, Fazli, Jasmonates induce the expression of nod genes in Bradyrhizobium cum, May 17, 2001; and Mabood, Fazli, "Linoleic and linolenic acid induce the expression or nod genes in Bradyrhizobium cum," 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 branched alkyl group, e.g., a methyl, ethyl or propyl group; an alkenyl group, such as a C2-C8 unbranched or branched alkenyl group; an alkynyl group, such as a C2-C8 unbranched or branched l group; an aryl group having, for e, 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 linoleic acid, linolenic acid, or jasmonic acid has been ed with a ——COR group, where R is an NRfig group, in which R2 and R3 are independently: hydrogen; 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 atalyzed nucleophilic on, wherein the carboxylic acid is reacted with an alcohol in the presence of a catalytic amount of a l acid. Amides may also be prepared by known methods, such as by ng the carboxylic acid with the appropriate amine in the presence of a ng agent such as dicyclohexyl carbodiimide (DCC), under neutral conditions. Suitable salts of linoleic acid, linolenic acid, and ic 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 s (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 on of the base. The salt may be precipitated 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., 2H—furo[2,3-c]pyranones including derivatives and analogues thereof, examples of which are represented by the following structure: wherein; Z is O, S or NR5; R2, R2, R3, and R4 are each independently H, alkyl, alkenyl, alkynyl, phenyl, benzyl, hydroxy, hydroxyalkyl, alkoxy, phenyloxy, benzyloxy, CN, CO?“ COOR=, halogen, NR&%, or Iflb; and IQ” R& and. R7 are each independently H, alkyl or alkenyl, 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 hate, phosphate or hydrogen phosphate, e, benzoate, succinate, fumarate, maleate, lactate, citrate, te, gluconate; methanesulphonate, benzenesulphonate and p-toluenesulphonic acid. onal biologically acceptable metal salts may include alkali metal salts, with. bases, examples of which irclude the sodium and potassium salts. Examples of compounds embraced by the structure and which may be le for use in :te t invention include the following: 3—methyl—2 — iLro[2,3-c]pyran—2—one (where Ih=CH3, 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), 3,5-dimethyl-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 molecule in an agriculturally' acceptable carrier, lly' aqueous in nature, and spraying or dripping the composition onto seed via a continuous treating system. (which. is calibrated. to apply treatment at a predefined rate in proportion to the continuous flow 0" seed), such as a drum-type of treater. These methods advantageously employ relatively small vo_umes of r so as to allow for relatively fast drying of the treated seed.

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

In another embodiment, the treatment entails coating seeds. One such process involves coating the inside wall of a round container with the composition, adding seeds, then rotating the container to cause the seeds to contact the wall and the composition, a process known in the art as "container coating". Seeds can be coated by ations 0; coating methods. Soaking typically 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 ive to moisture. Thus, soaking such seeds for an extended period of time may not be desirable, in which case the soaking is typically carried out for about 1 minute to about 20 minutes. t 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 ent and during any part of storage, the signal le may achieve ed effects by a phenomenon known as seed memory' or seed. tiond See, Macchiavelli and. Brelles— Marino, J. Exp. Bot. 55(408):2635—40 (2004) . Applicants also believe that ing' treatment the signal molecule, e.g., the LCD, di""uses 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 containing the plant signal molecule may further contain a sticking or coating agent to assist in nce of the signal molecule to the seed. For aesthetic purposes, the itions 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 weight, the effective amount generally ranges from about 1 to about 400 ug/hundred weight (cwt) seed, and in some ments from about 2 to about 70 ug/cwt, and in some other ments, from about 2.5 to about 3.0 ug/cwt seed. The e "ective amount of the plant signal molecule, r, may be obtained by a suitable dosage se assay, preferably, in a greenhouse and/or field study.

The treatment may also involve 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 cial" refers to agents that when applied to seeds result in enhancement (which. may’ be statistica ly' significant) of plant characteristics such as plant stand, growth, vigor or yield in comparison to non-treated seeds. Representative examples of such agents that may be useful in the practice of the present invention includes, but is not limited to, diazotrophs, mycorrhizal fungi, herbicides, fungicides, insecticides, and phosphate solubilizing agents.

Suitable herbicides include on, orfen, chlorimuron, lactofen, clomazone, fluazifop, glufosinate, glyphosate, sethoxydim, imazethapyr, imazamox, fomesafe, flumiclorac, uin, and. clethodim. Commercial products ning 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 "fungicide" 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 species of fungi if treatment with the fungicide results in killing or growth inhibition of a fungal tion (e.g., in the soil) relative to an untreated population. Effective fungicides in accordance with the invention will suitably exhibit activity against a broad range of pathogens, including but not limited to Phytophthora, Rhizoctonia, um, Pythium, Phomopsis or Selerotinia and Phakopsora and combinations thereof.

Conmercial fungicides may be suitable for use in the present invention. le commercially available fungicides include PROTEGE, RIVAL or ALLEGIANCE FL or LS (Gustafson, '?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 (Nitragin ina, 3uenos Ares, Argentina). Active ingredients in these and other commercial ides e, 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 icide "exhibits activity against" a ular s of insect if treatment with the insecticide results in killing or inhibition of an insect population ve to an untreated population. Effective insecticides in accordance with. the invention will suitably exhibit activity against a broad range of insects including, but not limited to, wireworms, cutworms, grubs, corn rootworm, seed corn maggots, flea. beetles, chinch. bugs, aphids, leaf beetles, and stink bugs. cial insecticides may be suitable for use in the t invention. Suitable commercially-available insecticides include CRUISER (Syngenta, Wilmington, DE), GAUCHO and PONCHO fson, Plano, TX). Active ients in these and other cial insecticides include thiamethoxam, clothianidin, and imidacloprid. Commercial insecticides are most suitably' used. in accordance with the manufacturer's ctions at the recommended concentrations.

As used herein, phosphate solubilizing agents include, but are not limited to, ate solubilizing microorganisms. As used herein, hate solubilizing microorganism” is a microorganism that is able to increase the amount of orous available for a plant. Phosphate solubilizing microorganisms include fungal and bacterial strains. In embodiment, the phosphate solubilizing microorganism is a spore forming microorganism. miting examples of phosphate solubilizing microorganisms include species from a genus selected from the group consisting o: Acinetobacter, bacter, Arthrobotrys, Aspergillus, Azospirillum, Bacillus, Burkholderia, Candida Chryseomonas, bacter, Eupenicillium, Exiguobacterium, Klebsiella, Kluyvera, Microbacterium, MUcor, Paecilomyces, Paenibacillus, Penicillium, Pseudomonas, Serratia, Stenotrophomonas, Streptomyces, Streptosporangium, Swaminathania, Thiobacillus, Torulospora, Vibrio, Xanthobacter, and Xanthomonas.

Non—limiting examples of phosphate solubilizing microorganisms are ed from the group consisting Acinetobacter calcoaceticus, Acinetobacter sp, Arthrobacter sp., Arthrobotrys oligospora, Aspergillus .niger, Aspergillus sp., Azospirillum halopraeferans, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans,Bacillus licheniformis, Bacillus subtilis, Burkholderia cepacia, Burkholderia vietnamiensis, Candida krissii, Chryseomonas luteola, Enterobacter aerogenes, Enterobacter asburiae, Enterobacter sp., Enterobacter taylorae, cillium parvum, Exiguobacterium sp., Klebsiella sp., ra cryocrescens, Microbacterium sp., Mucor ramosissimus, Paecilomyces hepialid, Paecilomyces marquandii, Paenibacillus macerans, Paenibacillus mucilaginosus, Pantoea aglomerans, Penicillium expansum, Pseudomonas corrugate, Pseudomonas fluorescens, Pseudomonas lutea, Pseudomonas poae, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas trivialis, Serratia marcescens, Stenotrophomonas maltophilia, Streptomyces sp., Streptosporangium sp., Swaminathania salitolerans, Thiobacillus xidans, Torulospora globosa, Vibrio proteolyticus, Xanthobacter agilis, and Xanthomonas campestris. ably, 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 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. ivorus, .P. glabrum, £2 griseofulvum, .P. implicatum, £2 janthinellum, P. num, P. minioluteum, P. montanense, P. nigricans, P. oxalicum, P. pinetorum, P. pinophilum, P. purpurogenum, P. radicans, P. radicum, P. raistrickii, P. rugulosum, P. simplicissimum, P. solitum, P. ile, P. velutinum, .P. catunu .P. m, .P. fussiporus, and 1% expansum.

More preferably, the phosphate solubilizing microorganism Penicillium species is P. bilaiae, P. gaestrivorus, and/or a combination f. Most preferably, the P. e 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.).

According 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, cillium, Exiguobacterium, Klebsiella, ra, Microbacterium, MUcor, omyces, acillus, Penicillium, Pseudomonas, Serratia, Stenotrophomonas, Streptomyces, osporangium, Swaminathania, Thiobacillus, Torulospora, , Xanthobacter, and xanthomonas, including one species selected from the following group: Acinetobacter calcoaceticus, Acinetobacter sp, Arthrobacter sp., Arthrobotrys pora, Aspergillus .nigery Aspergillus sp., Azospirillum halopraeferans, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans,Bacillus licheniformis, Bacillus subtilis, Burkholderia cepacia, Burkholderia vietnamiensis, Candida krissii, Chryseomonas a, Enterobacter aerogenes, Enterobacter asburiae, Enterobacter sp., bacter taylorae, Eupenicillium parvum, Exiguobacterium sp., Klebsiella sp., Kluyvera cryocrescens, Microbacterium sp., Mucor ramosissimus, Paecilomyces hepialid, Paecilomyces marguandii, Paenibacillus macerans, Paenibacillus mucilaginosus, Pantoea aglomerans, Penicillium expansum, monas corrugate, Pseudomonas fluorescens, Pseudomonas lutea, Pseudomonas poae, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas trivialis, Serratia marcescens, rophomonas maltophilia, Streptomyces sp., Streptosporangium sp., Swaminathania salitolerans, Thiobacillus ferrooxidans, Torulospora globosa, Vibrio proteolyticus, Xanthobacter , and Xanthomonas campestris.

Diazotrophs are bacteria and archaea that fix atmospheric nitrogen gas into a more usable form such as ammonia. Examples of diazotrophs include ia from the genera Rhizobium spp. (e.g., R. cellulosilyticum, R. daejeonense, R. etli, R. galegae, R. gallicum, R. giardinii, R. hainanense, R. ense, R. indigoferae, leguminosarum, R. loessense, R. lupini, R. lusitanum, meliloti, R. mongolense, R. miluonense, R. sullae, tropici, R. undicola, and/or R. yanglingense), Bradyrhizobi 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. , S. fredii, S. indiaense, S. kostiense, S. kummerowiae, S. medicae, S. meliloti, S. mexicanus, S. nse, S. saheli, S. terangae, and/or S. xinjiangense), Mesorhizobium spp., (M. albiziae, M. amorphae, M. chacoense, M. ciceri, M. i, 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 ting of B. japonicum, R leguminosarum R meliloti, S. meliloti, and ations thereof. In another embodiment, the diazotroph is B. japonicum. In another embodiment, the diazotroph is R leguminosarum. In another embodiment, the diazotroph is R meliloti. In another embodiment, the diazotroph is S. meliloti.

Mycorrhizal fungi form. tic ations 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 hizal fungi include 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 embodiment, the endomycorrhizae is a strain of Glomus atum, Glomus brasilianum, Glomus clarum, Glomus deserticola, Glomus etunicatum, Glomus fasciculatum, Glomus intraradices, Glomus orum, or Glomus mosseae, 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, Pisolithus tinctorius, Rhizopogon amylopogon, Rhizopogon fulvigleba, Rhizopogon luteolus, ogon villosuli, Scleroderma cepa, Scleroderma citrinum, or a combination thereof.

The mycorrhizal fungi include ecroid mycorrhizae, arbutoid mycorrhizae, or monotropoid hizae. Arbuscular 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, preferably of the phylum 3asidiomyco:a. In yet another ment, the mycorrhiza may be a Hmnotripoid mycorrhiza, preferably of the phylum 3asidiomyco:a. In still yet another ment, the mycorrhiza may be an orchid. mycorrhiza, preferably 0" she genus Rhizoctonia.

The methods of the present ion are applicable to leguminous seed, entative es of which include n, alfalfa, peanut, pea, lentil, bean and clover. The methods of the present ion are also applicable to non- leguminous seed, e.g., Poaceae, itaceae, eae.

Asteraceae, Chenopodiaceae and Solonaceae. Representative examples of non—leguminous seed e field crops such as corn, cereals such as rice, barley and wheat, cotton and canola, 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. techniques.

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 herein, a year shall mean 365 days. Whereas soybean seed. may' have to be planted the following , 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 agriculturally' acceptable carrier‘ may be used, for example, a solid carrier, olid carrier, an aqueous-based liquid carrier, a non—aqueous based liquid r, a suspension, an emulsion or an liable concentrate. Agriculturally—acceptable carriers may include, e.g., adjuvants, inert components, dispersants, surlacuants, iers, binders, stabilizing agents, and/or polymers.

The seed treatment ition may furuher e one or more agriculturally/agronomically beneficial agents (that is in addition to the signal molecule), such as, one or more diazotrophs, mycorrhizal fungi, herbicides, ides, insecticides, and/or ate solubilizing agents.

The present invention will now be bed 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 ative field experiments using soybean seed that demonstrate 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 combination 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, measured in units 0; bushels/acre, show that the methods of the claimed invention achieved an increase in soybean yield, ve 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 ion 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 (including roots) were ted 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 increases in these properties. Lastly, e 4 bes experiments condJCted with soybean seed treated. with. ze® 55 days prior to planting and for purposes of comparison, soybean seed treated 7 days prior to planting and untreated seed. The results, sed in units of mean surface area of first trifoliate leaves, show that the claimed invention enhances plant grOWth in this respect too.

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

The plant signal leecules used in the trial were Optimize®, a non-commercial grade of Optimize® S—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 les 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 treated 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 pesticides 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, respectively. 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 le + carrier). The Control seed was stored under the same conditions as the mental 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 TREATMENT GROUP GRAIN YIELD @ 13% Control - non-inoculated Optimize — at planting 64.2 Optimize — 5 month REN—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 between comparative (non—inventive) Group 2 and inventive Group 3, treatment or the soybean seed with the commercial-grade ze® at 5 months pre—planting resulted in an increase in n yield of 1.5 bushels of soybean. As reflected by the comparison between Group 4 and inventive Group 5, ent of soybean seed at 5 months anting with pure LCO-V , 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 ison 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 pre—planting increased soybean yield by 7.4 bushels/acre. Grain yield measurements were taken at a 13% seed moisture level.

EXAMPLE 2 A soybean trial was conducted to evaluate embodiments of the t invention on grain yield when applied on soybean seed. The field trial site was d 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 leecules 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 d 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 g. Four replications were conducted. Seed were treated 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 pesticides Extreme® 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 s post-planting (post-emergent), at rates of 6.0 oz, 21 oz and 2.5 lb, respectively. Plants were harvested 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 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 2 below.

Table 2 TREATMENT GROUP GRAIN YIEL: l - oculated .4 Optimize — at planting Optimize — 5 month NZ—50GREN—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 nventive) Group 2 and inventive Group 3, treatment of the soybean seed with the commercial—grade Optimize® at 5 months anting 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® resulted in an increase in soybean 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 se as reflected 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 resulted in a decrease in 1.8 bushels/acre, a result utable to unexplained variability associated with field trials. Grain yield measurements were taken at a 13% seed moisture level.

Greenhouse ments EXAMPLE 3 n 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 ent in greenhouse pots containing sandzperlite (1:; mixture).

Seedlings were grown for 10 days after. seed. plantings then seedlings were harvested, their roots cleaned and measured 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 ions 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 embodiments (seed treated. with LCD at 1 month. and. 12 months prior to ng), and particularly' the results obtained after the 1-year pretreatment, are dramatic, ering 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, Lrifoliate after 19 days. As illustrated in Fig. 3 and shown in Table 4, the leaves generated from seed treated in ance 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 sos over time, the increase in mean surface area shown in plants generated from seed treated 55 days prior to planting may be attributable 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 ted to be incorporated by reference.

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

Claims (59)

1. Claim 1. A method of enhancing plant , comprising treating a seed at least one month prior to planting with a lipo-chitooligosaccharide (LCO), wherein, upon harvesting, the plant exhibits at least one of increased plant yield measured in terms of bushels/acre, increased root number, increased root length, sed root mass, increased root volume and increased leaf area, as compared to a plant harvested from a control seed treated with the LCO just prior to or within a week or less of planting.
2. Claim 2. The method of claim 1, wherein the LCO is represented by the formula: in which R represents H or CH3 CO and n is equal to 2 or 3.
3. Claim 3. The method of any one of claims 1 to 2, n the LCO is represented by the structure: 11070485_1
4. Claim 4. The method of any one of claims 1 to 3, wherein the LCO is represented by the structure:
5. Claim 5. The method of any one of claims 1 to 4, n the LCO is synthetic.
6. Claim 6. The method of any one of claims 1 to 4, wherein the LCO is recombinant.
7. Claim 7. The method of any one of claims 1 to 6, wherein the seed is leguminous.
8. Claim 8. The method of any one of claims 1 to 6, wherein the seed is a pea seed.
9. Claim 9. The method of any one of claims 1 to 6, wherein the seed is a lentil seed.
10. Claim 10. The method of any one of claims 1 to 6, n the seed is a bean seed.
11. Claim 11. The method of any one of claims 1 to 6, wherein the seed is a soybean seed.
12. Claim 12. The method of any one of claims 1 to 6, wherein the seed is non-leguminous.
13. Claim 13. The method of any one of claims 1 to 6, wherein the seed is a wheat seed.
14. Claim 14. The method of any one of claims 1 to 6, wherein the seed is a barley seed.
15. Claim 15. The method of any one of claims 1 to 6, wherein the seed is a cotton seed.
16. Claim 16. The method of any one of claims 1 to 6, wherein the seed is a canola seed.
17. Claim 17. The method of any one of claims 1 to 6, wherein the seed is a corn seed.
18. Claim 18. The method of any one of claims 1 to 17, wherein the seed is treated with the LCO at least 9 months prior to planting.
19. Claim 19. The method of any one of claims 1 to 17, wherein the seed is treated with the LCO at least 12 months prior to planting. 11070485_1
20. Claim 20. The method of any one of claims 1 to 17, wherein the seed is treated with the LCO at least 2 years prior to planting.
21. Claim 21. The method of any one of claims 1 to 20, wherein the LCO is isolated and/or purified from a species of Rhizobia selected from the group consisting of Bradyrhizobium spp., Mesorhizobium spp., Rhizobium spp., Sinorhizobium spp. and zobium spp.
22. Claim 22. The method of any one of claims 1 to 20, wherein the LCO is isolated and/or purified from Bradyrhizobium japonicum.
23. Claim 23. The method of any one of claims 1 to 20, wherein the LCO is isolated and/or purified from Sinorhizobium meliloti.
24. Claim 24. The method of any one of claims 1 to 20, wherein the LCO is isolated and/or purified from Rhizobium leguminosarum.
25. Claim 25. The method of any one of claims 1 to 20, wherein the LCO is isolated and/or purified from an arbuscular mycorrhizal .
26. Claim 26. The method of any one of claims 1 to 20, wherein the LCO is isolated and/or purified from a species of Glomerocycota.
27. Claim 27. The method of any one of claims 1 to 20, n the LCO is isolated and/or purified from Glomus intraradices.
28. Claim 28. The method of any one of claims 1 to 27, further comprising ting the seed with at least one phosphate lising microorganism.
29. Claim 29. The method of any one of claims 1 to 27, r comprising contacting the seed with one or more strains of Penicillium.
30. Claim 30. The met hod of any one of claims 1 to 27, further comprising contacting the seed with one or more strains of P. e.
31. Claim 31. The method of claim 30, wherein the seed is contacted with: the strain having the deposit accession number NRRL 50162; the strain having the deposit accession number NRRL 50169; the strain having the deposit accession number ATCC 20851; the strain having the deposit accession number ATCC 22348; and/or the strain having the deposit accession number ATCC 18309.
32. Claim 32. The method of any one of claims 1 to 31, further comprising contacting the seed with one or more strains of P. gaestrivorus.
33. Claim 33. The method of claim 32, wherein the seed is contacted with the strain having the deposit accession number NRRL 50170.
34. Claim 34. The method of any one of claims 1 to 33, further comprising ting the seed with one or more strains of Rhizobium. 11070485_1
35. Claim 35. The method of any one of claims 1 to 33, r comprising contacting the seed with one or more strains of R. leguminosarum.
36. Claim 36. The method of any one of claims 1 to 35, further comprising contacting the seed with one or more strains of Sinorhizobium.
37. Claim 37. The method of any one of claims 1 to 35, further comprising contacting the seed with one or more strains of S. meliloti.
38. Claim 38. The method of any one of claims 1 to 37, further comprising contacting the seed with one or more s of Bradyrhizobium.
39. Claim 39. The method of any one of claims 1 to 37, further sing contacting the seed with one or more strains of B. japonicum.
40. Claim 40. The method of any one of claims 1 to 39, further comprising contacting the seed with a fungicide and/or an insecticide.
41. Claim 41. The method of any one of claims 1 to 39, wherein the seed is contacted with metalaxyl, anidin, xam, fludioxonil, thiamethoxam and/or imidacloprid.
42. Claim 42. The method of any one of claims 1 to 39, wherein the seed is contacted with mefanoxam, fludioxonil and thiamethoxam.
43. Claim 43. The method of any one of claims 1 to 42, further comprising contacting the seed with a chitooligosaccharide (CO).
44. Claim 44. The method of any one of claims 1 to 43, further comprising contacting the seed with a chitin and/or a chitosan.
45. Claim 45. The method of any one of claims 1 to 44, further comprising contacting the seed with a flavonoid.
46. Claim 46. The method of any one of claims 1 to 45, wherein the LCO is d to the seed at a concentration of about 10-14 to about 10-5 Molar.
47. Claim 47. The method of any one of claims 1 to 45, wherein the LCO is applied to the seed at a concentration of about 10-11 to about 10-5 Molar.
48. Claim 48. The method of any one of claims 1 to 45, wherein the LCO is applied to the seed at a concentration of about 10-8 to about 10-7 Molar.
49. Claim 49. T he method of any one of claims 1 to 48, wherein the LCO is applied to the seed in an amount ranging from about 1 to about 400 µg / hundred weight seed (cwt).
50. Claim 50. The method of any one of claims 1 to 48, wherein the LCO is applied to the seed in an amount ranging from about 2 to about 70 µg / cwt.
51. Claim 51. The method of any one of claims 1 to 48, n the LCO is applied to the seed in an amount ranging from about 2.5 to about 3.5 µg / cwt. 11070485_1
52. Claim 52. The method of any one of claims 43 to 51, wherein the CO is applied to the seed at a concentration of about 10-14 to about 10-5 Molar.
53. Claim 53. The method of any one of claims 43 to 51, wherein the CO is applied to the seed at a concentration of about 10-11 to about 10-5 Molar.
54. Claim 54. T he method of any one of claims 43 to 51, wherein the CO is applied to the seed at a concentration of about 10-8 to about 10-7 Molar.
55. Claim 55. The method of any one of claims 43 to 54, wherein the CO is applied to the seed in an amount ranging from about 1 to about 400 µg / hundred weight seed (cwt).
56. Claim 56. The method of any one of claims 43 to 54, wherein the CO is applied to the seed in an amount ranging from about 2 to about 70 µg / cwt.
57. Claim 57. The method of any one of claims 43 to 54, wherein the CO is applied to the seed in an amount ranging from about 2.5 to about 3.5 µg / cwt.
58. Claim 58. A seed treated according to the method of any one of claims 1 to 57.
59. Claim 59. A plant germinated from the seed of claim 58. mes Biologicals gs, Inc. By the Attorneys for the Applicant SPRUSON & ON Per: 11070485_1