NZ622560B2 - Combinations of lipo-chitooligosaccharides and methods for use in enhancing plant growth - Google Patents

Abstract

Disclosed herein are methods of enhancing plant growth, comprising treating plant seed or the plant that germinates from the seed with an effective amount of at least two lipo-chitooligosaccharides, wherein upon harvesting the plant exhibits at least one of increased plant yield measured in terms of bushels/acre, increased root number, increased root length, increased root mass, increased root volume and increased leaf area, compared to untreated plants or plants harvested from untreated seed. Preferably the combination includes one of the pairs shown in the abstract drawing. bushels/acre, increased root number, increased root length, increased root mass, increased root volume and increased leaf area, compared to untreated plants or plants harvested from untreated seed. Preferably the combination includes one of the pairs shown in the abstract drawing.

Description

COMBINATIONS OF LIPO-CHITOOLIGOSACCHARIDES AND METHODS FOR USE IN ENHANCING PLANT GROWTH BACKGROUND OF THE ION The symbiosis between the gram—negative soil bacteria, Rhizobiaceae and hizobiaceae, and legumes such as soybean, is well documented. The biochemical basis for these relationships includes an exchange of molecular signaling, wherein the plant-to-bacteria signal compounds include flavones, isoflavones and flavanones, and the ia-to-plant signal compounds, which include the end products of the expression of the bradyrhizobial and ial nod genes, known as |ipo—chitooligosaccharides . The symbiosis between these bacteria and the legumes enables the legume to fix atmospheric en for plant , thus obviating a need for nitrogen fertilizers. Since nitrogen fertilizers can significantly increase the cost of crops and are associated with a number of polluting effects, the agricultural industry continues its efforts to exploit this biological relationship and develop new agents and methods for improving plant yield without increasing the use of nitrogen-based fertilizers.

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

Further development on this concept is taught in , 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), 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 r 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 ent of seeds just prior to planting.

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

There is, however, still a need for systems for improving or enhancing plant BRIEF SUMMARY OF THE INVENTION Disclosed herein is a method of enhancing plant growth, comprising a) treating (e.g., applying to) plant seed or a plant that germinates from the seed, with an effective amount of at least two lipo-chitooligosaccharides (LCO's), wherein upon harvesting the plant exhibits at least one of increased plant yield measured in terms of bushels/acre, increased root number, increased root , increased root mass, increased root volume and increased leaf area, compared to untreated plants or plants harvested from ted seed.

] According to a first aspect of the t invention, there is provided a method of enhancing plant growth, comprising treating a plant with an effective amount of at least two lipo-chitooligosaccharides (LCO's) wherein the at least two LCO's comprise (11613579_1):EOR or wherein the at least two LCO's comprise wherein upon harvesting, the plant exhibits at least one of increased plant yield measured in terms of s/acre, increased root number, increased root , increased root mass, increased root volume and increased leaf area, compared to untreated plants or plants ted from untreated plants or plants harvested from untreated seed.

As is clear in context, the two LCO's are different from each other. In some embodiments, treatment of the seed includes direct application of the at least two LCO's onto the seed, which may then be planted or stored for a period of time prior to planting. (11463791_2) Treatment of the seed may also include indirect treatment such as by introducing the at least two LCO's into the soil (known in the art as row application). In yet other ments, the at least two LCO's may be applied to the plant that germinates from the seed, e.g., via foliar spray. The methods may further include use of other agronomically beneficial agents, such as micronutrients, plant signal les (such as lipo-chitooligosaccharides, chitinous compounds (e.g., COs), flavonoids, jasmonic acid, linoleic acid and linolenic acid and their derivatives, and karrikins), herbicides, fungicides and insecticides, phosphate-solubilizing microorganisms, diazotrophs (Rhizobial inoculants), and/or mycorrhizal fungi.

The methods of the present invention are applicable to legumes and non-legumes alike. In some embodiments, the nous seed is soybean seed. (11463791_2) In some other embodiments, the seed that is treated is non-leguminous seed such as a field crop seed, (9.9., a cereal such as corn, or a vegetable crop seed such as potato.

BRIEF DESCRIPTION OF THE DRAWINGS Figs. fa and 2a show the chemical structures of two lipo-chitooligosaccharides compounds useful in the practice of the present invention.

Figs. 1b and 2b show the chemical structures of the corresponding chitooligosaccharide nds (00's) that correspond to the LCO's in Figs. 1a and 2a, and which are also useful in the practice of the t invention.

Figs. 3a and 4a show the chemical structures of other LCO's (Myc factors) useful in the practice of the present invention.

Figs. 3b and 4b show the chemical structures of the corresponding Myc CO's, also useful in the practice of the present invention.

Fig. 5 shows the chemical structure of a lipo-chitooligosaccharide useful in the practice of the present invention.

Fig. 6 is a bar graph that illustrates the effect of inventive combinations of LCO's treated on seeds of Macroptilium atropurpureum, compared to a control, expressed in terms of seedling length (root plus shoot in mm).

Figs. 7 and 8 are bar graphs that illustrate the effect of an inventive combination of LCO's, compared to a single LCD and a control, treated on tilium atropurpureum plants, expressed in terms of leaf ess.

Fig. 9 is a bar graph that illustrates the effect of an inventive ation of LCO's, compared to a single LCD and a control, treated on tilium atropurpureum plants, expressed in terms of number of total flowers per treatment.

Fig. 10 is a bar graph that illustrates the effect of an inventive combination of LCO's, compared to a single LCD and a control, treated on Macropti/ium atropurpureum plants, sed in terms of total number of fruits per treatment.

Fig. 11 is a bar graph that illustrates the effect of an inventive combination of LCO's, ed to a single LCD and a control, treated on Macroptilium atropurpureum , expressed in terms of average fruit number per plant.

Fig. 12 is a bar graph that illustrates the effect of an inventive combination of LCO's, compared to a single LCD and a control, treated on Macroptilium atropurpureum plants, sed in terms of total number of average yield (in grams) per plant.

Fig. 13 is a bar graph that illustrates the effect of various ive combinations of LCO's, compared to single LCO's and a control (water), treated on tomato seeds, sed in terms of average root length.

DETAILED DESCRIPTION Lipo-chitooligosaccharide compounds (LCO's), also known in the art as symbiotic Nod signals or Nod s, consist of an oligosaccharide backbone of B-I,4-Iinked N—acetyl—D—glucosamine ("GlcNAc") residues with an N-linked fatty acyl chain condensed at the non—reducing end. LCO's differ in the number of GlcNAc residues in the ne, in the length and degree of saturation of the fatty acyl chain, and in the substitutions of reducing and non-reducing sugar residues. See, e.g., Denarie, et al., Ann. Rev. Biochem. 65:503-35 (1996), Hamel, et al., Planta 232:787-806 (2010)(e.g., Fig. 1 therein which shows structures of chitin, chitosan, CO's and corresponding Nod factors (LCO's)); Prome, et al., Pure & Appl. Chem. 70(1):55-60 (1998). An example of an LCO is presented below as formula I CHZORS NH-R7 in which: G is a hexosamine which can be substituted, for example, by an acetyl group on the nitrogen, a sulfate group, an acetyl group and/or an ether group on an oxygen, R1, R2, R3, R5, R6 and R7, which may be identical or ent, represent H, CH3 CO--, CX Hy CO-- where x is an r n 0 and 17, and y is an integer between 1 and 35, or any other acyl group such as for example a carbamoyl, R4 represents a mono-, di- or triunsaturated aliphatic chain containing at least 12 carbon atoms, and n is an integer between 1 and 4.

LCOs may be obtained (isolated and/or purified) from bacteria such as Rhizobia, e.g., Rhizobium sp., Bradyrhizobium sp., Sinorhizobium sp. and Azorhizobium sp. LCO structures are teristic for each such bacterial species, and each strain may produce multiple LCO's with different structures. For example, specific LCOs from S. me/i/oti have also been described in US. Patent 718 as having the formula II: in which R represents H or CH3 CO-- and n is equal to 2 or 3.

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

LCOs from Bradyrhizobium japonicum are described in US. s 5,175,149 and 5,321,011. Broadly, they are pentasaccharide phytohormones comprising fucose. A number of these B. cum-derived LCOs are described: BjNod-V (C184); BjNod—V (Ac, C181), BjNod-V (C161); and BjNod-V (Ac, C161,), with "V" indicating the presence of five N-acetylglucosamines; "Ac" an acetylation; the number following the "C" ting the number of carbons in the fatty acid side chain; and the number following the the number of double bonds.

LCO's used in embodiments of the invention may be obtained (i.e., ed and/or purified) from bacterial strains that produce LCO's, such as strains of Azorhizobium, Bradyrhizobium (including B. japonicum), Mesorhizobium, Rhizobium (including R. leguminosarum), Sinorhizobium (including S. meliloti), and bacterial strains genetically engineered to produce LCO's. Combinations of two or more LCO's obtained from these rhizobial and bradyrhizobial microorganisms are included within the scope of the present invention.

LCO's are the y determinants of host specificity in legume symbiosis (Diaz, et al., Moi. Plant—Microbe ctions 13:268-276 (2000)). Thus, within the legume family, specific genera and species of rhizobia develop a symbiotic nitrogen-fixing relationship with a specific 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 8. 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 different rhizobial s.

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

Thus, by way of further examples, LCO's and non-naturally occurring tives thereof that may be useful in the practice of the present invention are represented by the following formula: \0 0., OH o 0 O R40 0 0 0 R30 R100 R90 R7 H H o/ / 0 0 wherein R1 represents 014:0, 3OH-014:0, iso-015:0, 016:0, 3-OH-016:0, iso- 015:0, 016:1, 016:2, 016:3, 7:0, iso-017:1, 018:0, 3OH-018:0, 018:0/3-OH, 018:1, OH-018:1, 018:2, 018:3, 018:4, 019:1 carbamoyl, 020:0, 020:1, 3-OH- 020:1, 020:1/3-OH, 020:2, 020:3, 022:1, and (w-1)-OH (which according to D'Haeze, et al., iology 12:79R—105R (2002), includes 018, 020, 022, 024 and 026 hydroxylated species and 016:1A9, 016:2 (A2,9) and 016:3 9)); R2 represents hydrogen or ; R3 represents hydrogen, acetyl or oyl; R4 represents hydrogen, acetyl or carbamoyl; R5 represents hydrogen, acetyl or carbamoyl; R6 represents hydrogen, arabinosyl, fucosyl, acetyl, sulfate ester, 3-0—S— 2-0—MeFuc, 2-0—MeFuc, and Fuc; R7 represents hydrogen, mannosyl or glycerol; R8 ents hydrogen, methyl, or —0H20H; R9 represents hydrogen, osyl, or fucosyl; R10 represents hydrogen, acetyl or fucosyl; and n represents 0, 1, 2 or 3. The structures of the naturally occurring Rhizobial LCO's embraced by this structure are described in D'Haeze, et al., supra.

By way of even further additional examples, an LCD obtained from B. japonicum, illustrated in Fig. 1a, may be used to treat leguminous seed other than soybean and non-leguminous seed such as corn. As another example, the LCD obtainable from Rhizobium leguminosarum biovar viciae illustrated in Fig. 2a (designated LCO-V (018:1), SP104) can be used to treat leguminous seed other than pea and non-legumes too. Thus, in some embodiments, the combination of the two LCO's illustrated in Figs. 1a and 2a are used in the methods of the present invenfion.

Also encompassed by the present invention is use of LCO's obtained (i.e., isolated and/or purified) from a mycorrhizal fungi, such as fungi of the group Glomerocycota, e.g., Glomus intraradicus. The ures of representative LCOs obtained from these fungi are described in and (the LCOs described therein also referred to as "Myc factors"). Representative mycorrhizal fungi-derived LCO's and non-naturally occurring derivatives f are represented by the ing structure: wherein n = 1 or 2; R1 represents 016, 016:0, 016:1, 016:2, 018:0, 018:1A92 or 018:1A11Z; and R2 represents hydrogen or SO3H. In some embodiments, the LCO's are produced by the mycorrhizal fungi which are illustrated in Figs. 3a and 4a.

In some embodiments, these LCO's are used in the methods of the present invenflon.

In some other embodiments, one of the two LCO's used in the methods of the present invention is obtained from S. meliloti, and is illustrated in Fig.

. Thus, in some embodiments of the present invention, the LCO's include at least two of the LCO's rated in Figs. 1a, 2a, 3a, 4a and 5. Broadly, the present invention includes use of any two or more LCO‘s, including naturally occurring (e.g., rhizobial, bradyrhizobial and fungal), recombinant, synthetic and non-naturally occurring derivatives thereof. In some embodiments, both of the at least two LCO's are inant.

Further encompassed by the present invention is use of tic LCO compounds, such as those described in , and recombinant LCO's produced through genetic engineering. The basic, naturally occurring L00 structure may contain modifications or substitutions found in lly occurring LCO's, such as those bed in Spaink, 0rit. Rev. Plant Sci. 54:257-288 (2000) and D'Haeze, supra. Precursor oligosaccharide molecules (00s, which as bed below, are also useful as plant signal molecules in the present ion) for the construction of LCOs may also be synthesized by genetically engineered organisms, e.g., as described in Samain, et al., Carbohydrate Res. 302:35-42 (1997); Cottaz, et al., Meth. Eng. 7(4):311-7 (2005) and Samain, et al., J. hnol. 72:33-47 (1999)(e.g., Fig. 1 n which shows ures of CO's that can be made recombinantly in E. coli harboring different combinations of genes L). Thus, in some embodiments, combinations of at least two LCO's include combinations of the LCO's selected from the LCO's illustrated in Figs. 1a, 2a, 3a, 4a, and 5.

LCO's may be utilized in various forms of purity and may be used alone or in the form of a culture of LCO-producing bacteria or fungi. For example, OPTIMIZE® (commercially available from mes BioAg Limited) ns a culture of B. japonicum that produces an LCO (LCO-V(C18:1, MeFuc), MOR116) that is illustrated in Fig. 1a. Methods to provide substantially pure LCO's include simply removing the microbial cells from a mixture of LCOs and the e, or continuing to isolate and purify the LCO les through LCO solvent phase separation followed by HPLC tography as described, for example, in US.

Patent 5,549,718. Purification can be enhanced by ed HPLC, and the purified LCO molecules can be freeze-dried for long—term storage. Chitooligosaccharides (COs) as described above, may be used as starting materials for the tion of synthetic LCOs. For the purposes of the present invention, recombinant LCO's suitable for use in the present invention are least 60% pure, e.g., at least 60% pure, at least 65% pure, at least 70% pure, at least 75% pure, at least 80% pure, at least 85% pure, at least 90% pure, at least 91% pure, at least 92% pure, at least 93% pure, at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99% pure, up to 100% pure.

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

These methods advantageously employ relatively small volumes of carrier so as to allow for relatively fast drying of the 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 apparatus for performing these processes are cially available from numerous suppliers, e.g., Bayer CropScience (Gustafson).

In another embodiment, the treatment entails coating seeds with the at least two LCO's. 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 of g 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 sensitive to moisture. Thus, soaking such seeds for an extended period of time may not be desirable, in which case the g is typically carried out for about 1 minute to about 20 minutes.

In those ments that entail storage of seed after application of the at least two LCO's, adherence of the LCO's to the seed over any portion of time of the e period is not critical. Without intending to be bound by any particular theory of ion, 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 LCO's may achieve their intended effect by a phenomenon known as seed memory or seed perception. See, Macchiavelli, et al., J. Exp. Bot. 55(408):1635-40 (2004). Applicants also believe that ing ent the LCO's e toward the young developing radicle and activates symbiotic and developmental genes which results in a change in the root architecture of the plant. Notwithstanding, to the extent desirable, the compositions containing the LCO's may further contain a sticking or coating agent. For aesthetic purposes, the compositions may further contain a coating polymer and/or a colorant.

In some embodiments, the at least two LCO's are applied to seed (directly or indirectly) or to the plant via the same composition (that is, they are formulated together). In other embodiments, they are formulated separately, wherein both LCO compositions are applied to seed or the plant, or in some embodiments, one of the LCO's is d to seed and the other is applied to the plant. _10_ The total amount of the at least two LCO's is effective to enhance growth such that upon harvesting the plant exhibits at least one of increased plant yield measured in terms of bushels/acre, sed root number, increased root length, increased root mass, increased root volume and increased leaf area, compared to untreated plants or plants harvested from untreated seed (with either ). The effective amount of the at least two LCO's used to treat the seed, expressed in units of concentration, generally ranges from about 10'5 to about 10'14 M (molar concentration), and in some embodiments, from about 10'5 to about 10'11 M, and in some other embodiments from about 10'7 to about 10'8 M. Expressed in units of weight, the effective amount generally ranges from about 1 to about 400 ug/hundred weight (cwt) seed, and in some embodiments from about 2 to about 70 ug/cwt, and in some other embodiments, from about 2.5 to about 3.0 ug/cwt seed.

For purposes of treatment of seed ctly, i.e., in-furrow treatment, the effective amount of the at least two LCO's generally ranges from 1 ug/acre to about 70 ug/acre, and in some embodiments, from about 50 ug/acre to about 60 ug/acre. For purposes of application to the plants, the effective amount of the LCO's generally ranges from 1 ug/acre to about 30 ug/acre, and in some embodiments, from about 11 ug/acre to about 20 ug/acre.

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

Other Agronomically Beneficial Agents The present invention may further include treatment of the seed or the plants that germinate from the seed with at least one agriculturally/agronomically _ll_ beneficial agent. As used herein and in the art, the term "agriculturally or agronomically beneficial" refers to agents that when applied to seeds or plants results in enhancement (which may be statistically significant) of plant characteristics such as plant stand, growth (e.g., as defined in connection with LCO's), or vigor in ison to non-treated seeds or plants. These agents may be formulated together with the at least two LCO's or applied to the seed or plant via a separate formulation. Representative examples of such agents that may be useful in the practice of the present ion include micronutrients (e.g., vitamins and trace minerals), plant signal molecules (other than LCO's), herbicides, fungicides and insecticides, ate-solubilizing microorganisms, diazotrophs (Rhizobial inoculants), and/or hizal fungi. utrients Representative vitamins that may be useful in the ce of the present invention include calcium henate, folic acid, biotin, and vitamin C.

Representative examples of trace minerals that may be useful in the practice of the present invention include boron, chlorine, manganese, iron, zinc, copper, molybdenum, nickel, selenium and sodium.

The amount of the at least one micronutrient used to treat the seed, expressed in units of concentration, generally ranges from 10 ppm to 100 ppm, and in some embodiments, from about 2 ppm to about 100 ppm. Expressed in units of weight, the effective amount generally ranges in one embodiment from about 180 ug to about 9 dred weight (cwt) seed, and in some embodiments from about 4 ug to about 200 ug/plant when applied on foliage. In other words, for purposes of treatment of seed the effective amount of the at least one micronutrient generally ranges from 30 pg/acre to about 1.5 mg/acre, and in some embodiments, from about 120 mg/acre to about 6 g/acre when applied ly.

Plant signal molecules The present invention may also include treatment of seed or plant with a plant signal le other than an LCO. For purposes of the present invention, the term "plant signal molecule", which may be used interchangeably with "plant growth-enhancing agent" broadly refers to any agent, both naturally occurring in plants or microbes, and tic (and which may be non-naturally occurring) that directly or indirectly activates a plant biochemical pathway, resulting in increased _12_ plant growth, 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, compared to untreated plants or plants harvested from untreated seed. Representative es of plant signal molecules that may be useful in the practice of the present invention include chitinous compounds, flavonoids, jasmonic acid, linoleic acid and linolenic acid and their derivatives (supra), and karrikins.

Chitooligosaccharides C03 are known in the art as [34 linked N-acetyl glucosamine ures identified as chitin oligomers, also as N-acetylchitooligosaccharides. CO's have unique and different side chain decorations which make them ent from chitin molecules [(C8H13NO5)n, CAS No. 1398—61-4], and chitosan molecules [(C5H11NO4)n, CAS No. 9012—76—4]. The CO's of the present invention are also relatively water-soluble compared to chitin and an, and in some embodiments, as described hereinbelow, are pentameric. Representative ture describing the structure and production of ODS that may be suitable for use in the present ion is as follows: Muller, et al., Plant Physiol. 124:733-9 (2000); Van der Holst, et al., Current Opinion in Structural y, 11:608-616 (2001)(e.g., Fig. 1 therein); Robina, et al., Tetrahedron 58:521-530 (2002); D'Haeze, et al., Glycobiol. 12(6):79R-105R (2002); Hamel, et al., Planta 232:787-806 (2010)(e.g., Fig. 1 which shows structures of chitin, chitosan, CO's and corresponding Nod factors )); Rouge, et al. Chapter 27, "The Molecular logy of x Carbohydrates" in Advances in Experimental Medicine and Biology, Springer Science; Wan, etal., Plant Cell 21:1053-69 (2009); PCT/F100/00803 (9/21/2000); and Demont-Caulet, et al., Plant Physiol. 120(1):83-92 (1999).

CO's differ from LCO's in terms of structure mainly in that they lack the pendant fatty acid chain. Rhizobia-derived CO‘s, and non-naturally ing synthetic tives thereof, that may be useful in the practice of the present invention may be represented by the following formula: _l3_ o 0 0 R40 0 o o R30 R100 R90 R7 H H H o/ _R2 n / 0 0 wherein R1 and R2 each independently represents hydrogen or methyl; R3 represents hydrogen, acetyl or carbamoyl; R4 represents hydrogen, acetyl or carbamoyl; R5 represents hydrogen, acetyl or carbamoyl; R5 represents hydrogen, arabinosyl, fucosyl, , sulfate ester, 3—0—8—2—0—MeFuc, 2-0—MeFuc, and 4-0— AcFuc; R7 represents hydrogen, mannosyl or glycerol; R8 ents hydrogen, methyl, or ; R9 represents hydrogen, arabinosyl, or fucosyl; R10 represents hydrogen, acetyl or fucosyl; and n represents 0, 1, 2 or 3. The structures of corresponding Rhizobial LCO's are described in D'Haeze, et al., supra.

Two CO's suitable for use in the present invention are illustrated in Figs. 1b and 2b. They correspond to LCO's produced by Bradyrhizobium japonicum and R. leguminosarum biovar viciae respectively, which ct tically with soybean and pea, respectively, but lack the fatty acid chains.

The structures of yet other CO's that may be suitable for use in the practice of the present invention are easily derivable from LCOs obtained (i.e., isolated and/or purified) from a mycorrhizal fungi, such as fungi of the group ocycota, e.g., Glomus intraradices. See, e.g., and Maillet, et al., Nature 469:58—63 (2011) (the LCOs bed therein also referred to as "Myc factors"). Representative mycorrhizal fungi—derived CO's are represented by the following structure: _l4_ wherein n = 1 or 2; R1 represents hydrogen or methyl; and R2 represents hydrogen or SO3H. Two other CO's suitable for use in the present invention, one of which is sulfated, and the other being non-sulfated, are rated in Figs. 3b and 4b respectively. They correspond to aforementioned two different LCO's produced by the hizal fungi Glomus intraradices, and which are illustrated in Figs. 3a and 4a.

The COs may be synthetic or recombinant. Methods for preparation of synthetic CO's are described, for e, in Robina, supra. Methods for producing recombinant CO's e.g., using E. coli as a host, are known in the art. See, e.g., Dumon, et al., ChemBioChem 7:359-65 (2006), Samain, et al., Carbohydrate Res. 302:35-42 (1997); Cottaz, et al., Meth. Eng. 7(4):311-7 (2005) and Samain, et al., J.

Biotechnol. 72:33-47 (e.g., Fig. 1 therein which shows structures of CO's that can be made recombinantly in E. coli harboring different combinations of genes nodBCHL). For the purposes of the t invention, the recombinant CO's are at least 60% pure, e.g., at least 60% pure, at least 65% pure, at least 70% pure, at least 75% pure, at least 80% pure, at least 85% pure, at least 90% pure, at least 91% pure, at least 92% pure, at least 93% pure, at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99% pure, up to 100% pure.

Other chitinous compounds include chitins and chitosans, which are major ents of the cell walls of fungi and the exoskeletons of insects and crustaceans, are also composed of GIcNAc residues. Chitinous compounds include chitin, (IUPAC: N—[5—[[3—acetylamino—4,5—dihydroxy(hydroxymethyl)oxan- 2yl]methoxymethyl]—2—[[5—acety|amino—4,6—dihydroxy—2-(hydroxy methyl)oxan yl]methoxymethyl]—4-hydroxy(hydroxymethyl)oxanys]ethanamide), and chitosan, (IUPAC: 5—amino—6—[5-amino[5-amino-4,6-dihydroxy- _15_ 2(hydroxymethyl)oxanyl]oxyhydroxy—2-(hydroxymethyl)oxanyl]oxy- 2(hydroxymethyl)oxane-3,4-diol). These compounds may be obtained commercially, e.g., from Sigma-Aldrich, or prepared from insects, crustacean shells, or fungal cell walls. Methods for the preparation of chitin and chitosan are known in the art, and have been bed, for example, in US. Patent 207 (preparation from crustacean ), Pochanavanich, et al., Lett. Appl. Microbiol. 35:17-21 (2002) ration from fungal cell , and US. Patent 5,965,545 (preparation from crab shells and hydrolysis of commercial an). See, also, Jung, et al., Carbohydrate Polymers 672256-59 (2007); Khan, et al., Photosynthetica 40(4):621-4 (2002). Deacetylated chitins and chitosans may be obtained that range from less than 35% to greater than 90% deacetylation, and cover a broad spectrum of molecular weights, e.g., low molecular weight chitosan oligomers of less than 15kD and chitin oligomers of 0.5 to 2kD; "practical grade" chitosan with a molecular weight of about 150kD; and high molecular weight an of up to 700kD. Chitin and chitosan compositions formulated for seed treatment are also commercially ble. Commercial products include, for example, ELEXA® (Plant e Boosters, Inc.) and T'V' (Agrihouse, Inc.).

Flavonoids are phenolic compounds having the l structure of two aromatic rings connected by a three-carbon bridge. Flavonoids are produced by plants and have many functions, e.g., 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 Biochem. & Biotechnol. 11:1-10 (2002); Shaw, et al., Environmental Microbiol. 11:1867-80 (2006).

Representative flavonoids that may be useful in the practice of the present invention include genistein, in, formononetin, naringenin, etin, luteolin, and apigenin. Flavonoid compounds are commercially available, e.g., from Natland International Corp., Research Triangle Park, NC; MP Biomedicals, Irvine, CA; LC Laboratories, Woburn MA. Flavonoid compounds may be ed from plants or seeds, e.g., as described in US. Patents 5,702,752; 5,990,291; and 6,146,668. Flavonoid compounds may also be produced by cally engineered organisms, such as yeast, as described in Ralston, et al., Plant Physiology 137:1375-88 (2005). _l6_ Jasmonic acid (JA, [1 R-[10,2B(Z)]]—3-OXO (pentenyl)cyclopentaneacetic acid) and its derivatives (which include linoleic acid and linolenic acid (which are bed above in connection with fatty acids and their derivatives), may be used in the practice of the present invention. Jasmonic acid and its methyl ester, methyl jasmonate (MeJA), collectively known as jasmonates, are octadecanoid-based compounds that occur lly in . Jasmonic acid is produced by the roots of wheat seedlings, and by fungal microorganisms such as Botryodip/odia 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 thesis of jasmonic acid. Like linoleic acid and linolenic acid, jasmonates (and their derivatives) are reported to be rs of nod gene expression or LCD production by rhizobacteria.

See, e.g., Mabood, Fazli, Jasmonates induce the expression of nod genes in Bradyrhizobium cum, May 17, 2001.

Useful derivatives ofjasmonic acid, linoleic acid and linolenic acid that may be useful in the practice of the t invention include esters, , glycosides and salts. Representative esters are compounds in which the carboxyl group of jasmonic acid, linoleic acid and linolenic 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 alkynyl group; an aryl group having, for example, 6 to 10 carbon atoms; or a aryl group having, for example, 4 to 9 carbon atoms, wherein the heteroatoms in the heteroaryl group can be, for example, N, O, P, or 8. Representative amides are compounds in which the carboxyl group of jasmonic acid, linoleic acid and linolenic acid has been replaced with a ——COR group, where R is an NRZR3 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 l group, such as a 02-08 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 , 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 _l7_ known methods, such as acid-catalyzed nucleophilic on, wherein the carboxylic acid is reacted with an alcohol in the presence of a catalytic amount of a mineral acid. Amides may also be prepared by known methods, such as by reacting the carboxylic acid with the appropriate amine in the ce of a coupling agent such as ohexyl carbodiimide (DCC), under neutral conditions. Suitable salts of jasmonic acid, linoleic acid and linolenic acid include 6.9., base addition salts. The bases that may be used as reagents to prepare lically 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 may be readily prepared by mixing together a solution of linoleic acid, linolenic acid, orjasmonic acid with a solution 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.

Karrikins are vinylogous 4H—pyrones e.g., o[2,3-c]pyranones including tives and analogues thereof. Examples of these compounds are represented by the following structure: wherein; Z is O, S or NR5; R1, R2, R3, and R4 are each independently H, alkyl, alkenyl, alkynyl, phenyl, benzyl, hydroxy, hydroxyalkyl, alkoxy, phenyloxy, benzyloxy, CN, CORa, COOR=, halogen, NR6R7, or N02; and R5, R6, and R7 are each independently H, alkyl or l, or a biologically acceptable salt thereof. Examples of biologically able salts of these nds may include acid addition salts formed with biologically acceptable acids, examples of which include hydrochloride, romide, sulphate or bisulphate, phosphate or hydrogen ate, acetate, benzoate, succinate, fumarate, maleate, e, citrate, tartrate, gluconate; methanesulphonate, benzenesulphonate and p—toluenesulphonic acid. Additional biologically acceptable metal salts may include alkali metal salts, with bases, examples of which include the sodium and potassium salts. Examples of _l8_ compounds embraced by the structure and which may be suitable for use in the present invention include the following: 3-methyl-2H-furo[2,3-c]pyranone (where R1=CH3, R2, R3, R4=H), 2H-furo[2,3-c]pyran-2—one (where R1, R2, R3, R4=H), 7- methyl-2H-furo[2,3-c]pyran-2—one (where R1, R2, R4=H, R3=CH3), 5-methyl-2H- furo[2,3-c]pyran-2—one (where R1, R2, R3=H, R4=CH3), 3,7-dimethyl-2H-furo[2,3- c]pyranone (where R1, , R2, R4=H), 3,5-dimethyl—2H-furo[2,3-c]pyran-2— one (where R1, R4=CH3, R2, R3=H), 3,5,7-trimethyl-2H—furo[2,3—c]pyran-2—one (where R1, R3, R4=CH3, R2=H), oxymethyl-3—methyl-2H-furo[2,3—c]pyran—2—one (where R1=CH3, R2, R3=H, R4=CH2OCH3), 4-bromo-3,7-dimethyl-2H-furo[2,3-c]pyranone (where R1, R3=CH3, R2=Br, R4=H), 3-methylfuro[2,3-c]pyridin-2(3H)—one (where Z=NH, R1=CH3, R2, R3, R4=H), 3,6—dimethylfuro[2,3—c]pyridin-2(6H)-one (where Z=N- -CH3, R1=CH3, R2, R3, R4=H). See, US. Patent 7,576,213. These molecules are also known as karrikins. See, Halford, supra.

The amount of the at least one plant signal molecule used to treat the seed, expressed in units of concentration, generally ranges from about 10'5 to about '14 M (molar concentration), and in some embodiments, from about 10'5 to about '11 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 embodiments from about 2 to about 70 ug/cwt, and in some other embodiments, from about 2.5 to about 3.0 ug/cwt seed.

For purposes of treatment of seed indirectly, i.e., in-furrow treatment, the effective amount of the at least one plant signal molecule generally ranges from 1 ug/acre to about 70 ug/acre, and in some embodiments, from about 50 ug/acre to about 60 e. For purposes of application to the plants, the effective amount of the at least one plant signal molecule generally ranges from 1 ug/acre to about pg/acre, and in some embodiments, from about 11 ug/acre to about 20 ug/acre. ides, Fungicides and Insecticides Suitable ides e bentazon, acifluorfen, chlorimuron, lactofen, one, fluazifop, glufosinate, glyphosate, sethoxydim, imazethapyr, imazamox, fomesafe, flumiclorac, uin, and clethodim. Commercial products containing each of these compounds are y available. Herbicide concentration _19_ in the composition will generally correspond to the labeled use rate for a particular ide.

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 ular species of fungi if treatment with the fungicide results in killing or growth inhibition of a fungal population (e.g., in the soil) ve to an untreated population.

Effective fungicides in accordance with the invention will suitably exhibit ty against a broad range of pathogens, including but not d to Phytophthora, Rhizoctonia, Fusarium, Pythium, Phomopsis or Se/erotinia and Phakopsora and combinations thereof. cial fungicides may be suitable for use in the present invention. Suitable commercially available fungicides e PROTEGE, RIVAL or ALLEGIANCE FL or LS (Gustafson, Plano, TX), WARDEN RTA (Agrilance, St. Paul, MN), APRON XL, APRON MAXX RTA or RFC, MAXIM 4FS or XL (Syngenta, Wilmington, DE), CAPTAN (Arvesta, Guelph, Ontario) and PROTREAT (Nitragin Argentina, Buenos Ares, Argentina). Active ingredients in these and other commercial fungicides include, but are not limited to, fludioxonil, mefenoxam, azoxystrobin and xyl. Commercial ides 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 s in killing or inhibition of an insect population relative 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.

Commercial insecticides may be suitable for use in the present invention. Suitable commercially-available insecticides e CRUISER (Syngenta, Wilmington, DE), GAUCHO and PONCHO (Gustafson, Plano, TX). Active ingredients in these and other commercial insecticides e thiamethoxam, clothianidin, and imidacloprid. Commercial icides are most suitably used in accordance with the manufacturer's instructions at the recommended concentrations. _20_ Phosphate Solubilizing Microorganisms, Diazotrophs (Rhizobial inoculants), and/or Mycorrhizal fungi.

The present invention may further include treatment of the seed with a phosphate solubilizing microorganism. As used herein, “phosphate solubilizing microorganism” is a microorganism that is able to se the amount of phosphorous available for a plant. ate solubilizing microorganisms include fungal and bacterial strains. In embodiment, the ate solubilizing microorganism is a spore g microorganism.

Non-limiting examples of phosphate lizing microorganisms include s from a genus selected from the group consisting of obacter, Arthrobacter, Arthrobotrys, Aspergillus, Azospirillum, Bacillus, Burkho/deria, Candida Chiyseomonas, Enterobacter, Eupenicillium, Exiguobacterium, K/ebsiella, Kluyvera, Microbacterium, Mucor, Paecilomyces, Paenibacillus, Penicillium, Pseudomonas, Serratia, Stenotrophomonas, Streptomyces, osporangium, athania, Thiobacillus, Torulospora, Vibrio, Xanthobacter, and Xanthomonas.

Non-limiting es of phosphate solubilizing microorganisms are selected from the group consisting Acinetobacter ceticus, Acinetobacter sp, Arthrobacter sp., Arthrobotrys oligospora, Aspergillus niger, Aspergillus sp., Azospirillum halopraeferans, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans,Baci/lus licheniformis, Bacillus subtilis, Burkholderia cepacia, Burkholderia vietnamiensis, Candida krissii, Chryseomonas luteola, Enterobacter aerogenes, Enterobacter ae, bacter sp., Enterobacter taylorae, Eupenicillium parvum, Exiguobacterium sp., Klebsiella sp., Kluyvera Ciyocrescens, Microbacterium sp., Mucor ramosissimus, Paecilomyces hepialid, Paecilomyces marquandii, Paenibacillus macerans, Paenibacillus mucilaginosus, Pantoea rans, Penicillium um, Pseudomonas corrugate, Pseudomonas fluorescens, Pseudomonas lutea, Pseudomonas poae, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas trivia/is, Serratia marcescens, Stenotrophomonas maltophilia, Streptomyces sp., osporangium sp., Swaminathania salitolerans, Thiobacilius ferrooxidans, Torulospora globosa, Vibrio proteolyticus, Xanthobacter agilis, and Xanthomonas campestris In a particular embodiment, the phosphate solubilizing microorganism is a strain of the fungus Penicillium. Strains of the fungus Penicillium that may be _21_ useful in the practice of the t invention include P. bilaiae (formerly known as P. bilaii), P. albidum, P. aurantiogriseum, P. chrysogenum, P. citreonigrum, P. citrinum, P. digitatum, P. frequentas, P. fuscum, P. gaestrivorus, P. glabrum, P. griseofulvum, P. implicatum, P. janthinellum, P. lilacinum, P. minioluteum, P. montanense, P. nigricans, P. oxalicum, P. pinetorum, P. pinophilum, P. purpurogenum, P. rad/cans, P. radicum, P. raistrickii, P. sum, P. cissimum, P. m, P. variabile, P. velutinum, P. viridicatum, P. glaucum, P. fussiporus, and P. expansum.

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

In some embodiments, more than one ate lizing microorganism is used, such as, at least two, at least three, at least four, at least five, at least 6, including any combination of the obacter, Arthrobacter, Arthrobotrys, Aspergillus, Azospirillum, Bacillus, Burkholderia, Candida Chryseomonas, Enterobacter, Eupenicillium, Exiguobacterium, Klebsiella, Kluyvera, acterium, Mucor, Paecilomyces, Paenibacillus, Penicillium, Pseudomonas, Serratia, Stenotrophomonas, Streptomyces, Streptosporangium, Swaminathania, Thiobacillus, Torulospora, Vibrio, bacter, and Xanthomonas, including one species selected from the following group: Acinetobacter calcoaceticus, Acinetobacter sp, Arthrobacter sp., Arthrobotrys oligospora, Aspergillus niger, Aspergillus sp., Azospirillum ha/opraeferans, us amyloliquefaciens, us atrophaeus, Bacillus circulans,Baci/Ius licheniformis, Bacillus subtilis, Burkholderia cepacia, Burkho/deria vietnamiensis, Candida krissii, Chryseomonas luteola, bacter aerogenes, Enterobacter asburiae, Enterobacter sp., Enterobacter taylorae, ci/lium pan/um, Exiguobacterium sp., K/ebsie/la sp., K/uyvera cryocrescens, Microbacterium sp., Mucor ramosissimus, Paecilomyces hepialid, Paecilomyces marquandii, Paenibacillus ns, Paenibaci/lus mucilaginosus, Pantoea ag/omerans, Penicillium expansum, Pseudomonas corrugate, Pseudomonas scens, Pseudomonas lutea, Pseudomonas poae, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas trivia/is, Serratia _22_ marcescens, Stenotrophomonas maltophilia, omyces sp., Streptosporangium sp., Swaminathania salitolerans, cillus ferrooxidans, Torulospora globosa, Vibrio proteolyticus, Xanthobacter agilis, and Xanthomonas campestris In some embodiments, two ent strains of the same s may also be combined, for e, at least two different strains of Penicillium are used.

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

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

In an ment, the phosphate solubilizing microorganism is a llium fungus. The Penicillium fungus according to the invention can be grown using solid state or liquid fermentation and a suitable carbon source. Penicillium es may be grown using any suitable method known to the person skilled in the art. For example, the fungus may be cultured on a solid growth medium such as potato dextrose agar or malt extract agar, or in flasks containing suitable liquid media such as Czapek—Dox medium or potato dextrose broth. These culture methods may be used in the preparation of an inoculum of Penicillium spp. for treating (e.g., coating) seeds and/or application to an agronomically acceptable r to be applied to soil. The term "inoculum" as used in this specification is ed to mean any form of phosphate solubilizing microorganism, fungus cells, _23_ mycelium or spores, bacterial cells or bacterial spores, which is capable of propagating on or in the soil when the conditions of temperature, moisture, etc., are favorable for fungal growth.

Solid state production of Penicillium spores may be achieved by inoculating a solid medium such as a peat or vermiculite—based substrate, or grains including, but not limited to, oats, wheat, barley, or rice. The sterilized medium (achieved through autoclaving or irradiation) is inoculated with a spore sion (1x102-1x107 cfu/ml) of the appropriate Penicillium spp. and the moisture adjusted to to 50%, depending on the substrate. The material is incubated for 2 to 8 weeks at room temperature. The spores may also be produced by liquid fermentation (Cunningham et al., 1990. Can J Bot. 682270—2274). Liquid production may be achieved by cultivating the fungus in any suitable media, such as potato dextrose broth or e yeast extract media, under appropriate pH and temperature conditions that may be determined in accordance with standard procedures in the art.

The resulting material may be used directly, or the spores may be harvested, concentrated by centrifugation, formulated, and then dried using air drying, freeze drying, or fluid bed drying techniques (Friesen, et al., 2005, Appl.

Microbiol. hnol. -404) to produce a wettable powder. The wettable powder is then ded in water, applied to the e of seeds, and allowed to dry prior to planting. The le powder may be used in conjunction with other seed treatments, such as, but not limited to, chemical seed treatments, carriers (e.g., talc, clay, kaolin, silica gel, kaolinite) or polymers (e.g., methylcellulose, polyvinylpyrrolidone). Alternatively, a spore suspension of the appropriate Penicillium spp. may be applied to a suitable ompatible carrier (e.g., peat-based powder or granule) to appropriate final re content. The al may be incubated at room temperature, typically for about 1 day to about 8 weeks, prior to use.

Aside from the ingredients used to cultivate the phosphate solubilizing microorganism, including, e.g., ingredients referenced above in the cultivation of Penicillium, the phosphate lizing microorganism may be formulated using other agronomically acceptable carriers. As used herein in connection with "carrier", the term omically able" refers to any material which can be used to deliver the actives to a seed, soil or plant, and preferably which carrier can be added (to the _24_ seed, soil or plant) without having an adverse effect on plant growth, soil structure, soil drainage or the like. Suitable carriers comprise, but are not limited to, wheat chaff, bran, ground wheat straw, ased powders or granules, gypsum-based granules, and clays (e.g., kaolin, bentonite, montmorillonite). When spores are added to the soil a granular formulation will be preferable. Formulations as liquid, peat, or wettable powder will be le for coating of seeds. When used to coat seeds, the material can be mixed with water, applied to the seeds and allowed to dry. Example of yet other carriers include moistened bran, dried, sieved and applied to seeds prior coated with an adhesive, e.g., gum arabic. In embodiments that entail formulation of the actives in a single composition, the agronomically acceptable carrier may be aqueous.

The amount of the at least one phosphate solubilizing microorganism varies depending on the type of seed or soil, the type of crop plants, the amounts of the source of phosphorus and/or micronutrients present in the soil or added thereto, etc. A le amount can be found by simple trial and error experiments for each particular case. Normally, for Penicillium, for example, the application amount falls into the range of 0.001-1.0 Kg fungal spores and mycelium (fresh weight) per hectare, or 102-106 colony forming units (cfu) per seed (when coated seeds are used), or on a granular carrier applying between 1x106 and 1x1011 colony forming units per hectare. The fungal cells in the form of e.g., spores and the carrier can be added to a seed row of the soil at the root level or can be used to coat seeds prior to In embodiments, for example, that entail use of at least two strains of a ate solubilizing microorganism, such as, two strains of Penicillium, cial fertilizers may be added to the soil instead of (or even as well as) natural rock ate. The source of phosphorous may contain a source of phosphorous native to the soil. In other embodiments, the source of orous may be added to the soil. In one embodiment the source is rock phosphate. In another ment the source is a manufactured fertilizer. Commercially available manufactured phosphate fertilizers are of many types. Some common ones are those containing monoammonium phosphate (MAP), triple super phosphate (TSP), diammonium phosphate, ordinary superphosphate and ammonium polyphosphate. All of these fertilizers are ed by chemical sing of insoluble natural rock phosphates _25_ in large scale fertilizer-manufacturing facilities and the t is expensive. By means of the present ion it is possible to reduce the amount of these fertilizers applied to the soil while still maintaining the same amount of orus uptake from the soil.

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

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

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

Diazotrophs are bacteria and archaea that fix atmospheric nitrogen gas into a more usable form such as ammonia. Examples of rophs include bacteria from the genera Rhizobium spp. (e.g., R. osilyticum, R. daejeonense, R. etli, R. e, R. gallicum, R. nii, R. hainanense, R. huautlense, R. indigoferae, R. leguminosarum, R. loessense, R. lupini, R. lusitanum, R. meliloti, R. mongolense, R. miluonense, R. sullae, R. tropici, R. undicola, and/or R. yanglingense), Bradyrhizobium spp. (e.g., B. bete, B. canariense, B. elkanii, B. iriomotense, B. cum, B. e, B. liaoningense, B. pachyrhizi, and/or B. yuanmingense), Azorhizobium spp. (e.g., A. caulinodans and/or A. doebereinerae), Sinorhizobium spp. (e.g., S. abri, S. adhaerens, S. americanum, S. aboris, S. fredii, S. nse, S. kostiense, S. kummerowiae, S. medicae, S. meliloti, S. mexicanus, S. morelense, S. saheli, S. terangae, and/or S. xinjiangense), Mesorhizobium spp., (M. albiziae, M. amorphae, M. chacoense, M. ciceri, M. huakuii, M. loti, M. mediterraneum, M. pluifarium, M. septentrionale, M. temperatum, and/or M. tianshanense), and combinations thereof. In a particular embodiment, the diazotroph is selected from the group consisting of B. japonicum, R leguminosarum, R meliloti, S. meliloti, and combinations thereof. In another embodiment, the diazotroph is B. japonicum. In another embodiment, the diazotroph is R _26_ leguminosarum. In another embodiment, the diazotroph is R meliloti. In another embodiment, the diazotroph is S. meliloti.

Mycorrhizal fungi form symbiotic associations with the roots of a vascular plant, and e, e.g., absorptive capacity for water and mineral nutrients due to the comparatively large surface area of mycelium. Mycorrhizal fungi include corrhizal fungi (also called lar arbuscular mycorrhizae, VAMs, arbuscular mycorrhizae, or AMs), an ectomycorrhizal fungi, or a combination thereof.

In one embodiment, the mycorrhizal fungi is an endomycorrhizae of the phylum Glomeromycota and genera Glomus and Gigaspora. In still a further embodiment, the endomycorrhizae is a strain of Glomus aggregatum, Glomus brasilianum, Glomus clarum, Glomus deserticola, Glomus etunicatum, Glomus fasciculatum, Glomus intraradices, Glomus monosporum, or Glomus mosseae, Gigaspora margarita, or a combination thereof.

Examples of mycorrhizal fungi include ectomycorrhizae of the phylum Basidiomycota, cota, and Zygomycota. Other examples include a strain of Laccaria bicolor, Laccaria laccata, Pisolithus tinctorius, Rhizopogon amylopogon, Rhizopogon fulvigleba, Rhizopogon luteolus, Rhizopogon villosuli, Scleroderma cepa, Scleroderma um, or a combination thereof.

The mycorrhizal fungi include ecroid mycorrhizae, arbutoid mycorrhizae, or opoid mycorrhizae. Arbuscular and ectomycorrhizae form ericoid hiza 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, ably of the phylum Ascomycota, such as Hymenoscyphous ericae or Oidiodendron sp. In another ment, the mycorrhiza also may be an arbutoid mycorrhiza, preferably of the phylum Basidiomycota. In yet r embodiment, the mycorrhiza may be a monotripoid mycorrhiza, preferably of the phylum Basidiomycota. In still yet another embodiment, the mycorrhiza may be an orchid mycorrhiza, preferably of the genus Rhizoctonia.

The methods of the t invention are able to leguminous seed, representative es of which include soybean, alfalfa, peanut, pea, lentil, bean and clover. The s of the present invention are also applicable to non-leguminous seed, e.g., Poaceae, Cucurbitaceae, Malvaceae,. Asteraceae, _27_ odiaceae and Solonaceae. Representative examples of non-leguminous seed include field crops such as corn, rice, oat, rye, barley and wheat, cotton and canola, and vegetable crops such as potatoes, tomatoes, cucumbers, beets, e and cantaloupe.

The invention will now be described in terms of the following non- ng examples. Unless indicated to the ry, water was used as the control (indicated as "control".

Examples Greenhouse Experiments Example 1: Siratro seedling growth in vitro enhanced by LCO combinations Siratro (Macropti/ium atropurpureum) seeds were surface-sterilized with 10% bleach solution for 10 minutes followed by 3 rinses with sterilized distilled water. Seed were then placed in test tubes containing 15 ml sterile solidified agar medium supplemented with the LCOs illustrated in Figs. 1a and 2a (and which are referred to in the examples as the "soybean LCD" and the "pea LCD") (with total of '8M concentration either alone or in combination). Two other LCOs, i.e., pea LCD or the LCD illustrated in Fig. 5 (which is also referred to in the es as the "alfalfa LCO") was added to soybean LCO to study the effect of their combinations.

Seeds were grown for 7 days under grow light at 20°C with 16/8 h day/night cycle and then harvested for seedling length.

As ted by the comparison between soy LCO ed with another LCO (inventive embodiment) and soy LCO alone (non-inventive and comparable), the combination of soy and alfalfa LCO was more effective than soy LCO alone or its combination with pea LCO (Fig.6). Soybean LCO combined with alfalfa LCO produced the tallest seedling when total root and shoot length were summed. This difference was significant.

EXAMPLE 2: LCO foliar application on cherry tomato Based on the findings from the soybean LCD and the alfalfa LCO combination in Siratro (example 1), further investigation was conducted on tomato.

Florida petite cherry tomato plants were grown from seeds in greenhouse plastic containers and sprayed with soy LCO or its ation with a LCO during the initiation of flower buds at 5 ml/plant application rate. A second spry was also _28_ applied one week after the first application. At different maturity, leaf greenness, flower number, fruit number and final fruit fresh weight were measured.

The results achieved by the inventive ment (soy LCO + alfalfa LCO) showed that there was a slight increase in leaf greenness with LCD ation as compared to non-inventive and comparable soy LCO (Figs. 7 and 8).

In terms of total flower formed over a five-day period, LCO combination was significantly higher than non-inventive soy LCO. Similarly, when fruit numbers were counted over a six-day period, inventive soy and alfalfa LCO combination turned out to be significantly higher than soy LCO (Figs. 9 and 10). At the end of harvest, the average fruit number per plant was significantly higher for non-inventive soy LCD and inventive soy—alfalfa LCO combination as compared to control ent.

However, the average fresh—weight yield of cherry tomatoes was only significant for soy-alfalfa LCO combination over control and soy LCO (Figs. 11 and 12).

EXAMPLE 3: LCOs and their combinations on tomato seedling root growth Tomato seeds of var. Royal Mounty were placed in lates containing moist (soaked with treatment solutions) germination paper. Treatment solutions were prepared with four different LCOs, namely Pea LCO AC (acylated), Pea LCO NAC (non-acylated), Alfalfa LCD and Soybean LCD. The total LCO concentration used to make a water-based treatment solution was maintained at 10'9 M. Petriplates were then placed in dark at room temperature for ation. Eight days after ation, seedlings were measured with a hand held ruler for their root length.

Results obtained from this ment indicated that all dual LCO types enhanced tomato seedling root length as compared to control but only certain LCO combinations i.e. pea NAC and soybean LCO, pea AC plus soybean LCD and pea NAC plus a LCO generated significant root enhancement as compared to non-inventive and comparable single LCO types (Fig. 8). From the experiment, it appeared to be that for tomato seedlings, pea NAC and soybean LCO combination was the best of all combinations. The s also indicate that combinations of certain LCOs was more beneficial for tomato seedlings than others and it may be ruled out that combination of all four LCOs was better.

All patent and non—patent publications cited in this specification are indicative of the level of skill of those d in the art to which this invention _29_ pertains. All these publications are herein incorporated by nce to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is ore to be tood 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 ion as defined by the appended claims. _30_ WE

Claims (1)

CLAIM :

1. A method of enhancing plant growth, sing treating a plant with an effective amount of at least two lipo-chitooligosaccharides (LCO's) wherein the at least two LCO's comprise or wherein the at least two LCO's comprise (11613579_1):EOR