NZ723076B2 - 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 of LCOs includes the two shown in the 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 of LCOs includes the two shown in the drawing.

Description

COMBINATIONS OF LIPO-CHITOOLIGOSACCHARIDES AND METHODS FOR USE IN ENHANCING PLANT GROWTH BACKGROUND OF THE INVENTION The symbiosis between the gram—negative soil ia, iaceae and Bradyrhizobiaceae, and legumes such as soybean, is well nted. 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 bacteria-to-plant signal compounds, which include the end products of the expression of the bradyrhizobial and rhizobial nod genes, known as |ipo—chitooligosaccharides (LCOs). The sis between these bacteria and the legumes enables the legume to fix atmospheric nitrogen for plant growth, thus obviating a need for nitrogen izers. Since nitrogen fertilizers can significantly increase the cost of crops and are associated with a number of polluting s, 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 immediate vicinity of a seed or seedling in an effective amount for enhancing seed ation of seedling emergence in ison to an untreated seed or seedling.

Further development on this concept is taught in , ed to combinations of at least one plant inducer, namely an LCD, in combination with a fungicide, insecticide, 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), ng, root or plant.

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

More recently, Halford, "Smoke Signals," in Chem. Eng. News (April 12, 201 0), at pages 37-38, reports that karrikins or butenolides which are contained in smoke act as growth ants 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 213.

There is, however, still a need for systems for improving or enhancing plant growth.

BRIEF SUMMARY OF THE INVENTION [0006A] In a first aspect, the present invention es a method of enhancing plant growth, comprising treating plant seed or the plant that germinates from the seed with an effective amount of at least two distinct lipo-chitooligosaccharides (LCO’s), said at least two distinct LCO’s comprising the following LCO’s: 12081409_1 - 2a - n said at least two LCO’s synergistically enhance said plant growth, and 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 ted from untreated seed.

The present invention relates to 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 length, increased root mass, increased root volume and sed leaf area, compared to 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, ent 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.

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 in-furrow ation). In yet other embodiments, 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 molecules (such as lipo-chitooligosaccharides, chitinous compounds (e.g., COs), flavonoids, jasmonic acid, ic acid and nic acid and their derivatives, and ins), herbicides, fungicides and insecticides, phosphate-solubilizing microorganisms, diazotrophs (Rhizobia! inoculants), and/or mycorrhizal fungi.

The methods of the present invention are applicable to legumes and gumes alike. In some embodiments, the leguminous seed is soybean seed. 12081409_1 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 ce of the present invention.

Figs. 1b and 2b show the chemical structures of the corresponding chitooligosaccharide compounds (00's) that correspond to the LCO's in Figs. 1a and 2a, and which are also useful in the ce of the present invention.

Figs. 3a and 4a show the al structures of other LCO's (Myc factors) useful in the practice of the t ion.

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 ive 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 rate the effect of an inventive combination of LCO's, compared to a single LCD and a control, treated on Macroptilium atropurpureum plants, expressed in terms of leaf greenness.

Fig. 9 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, 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, expressed 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, compared to a single LCD and a l, treated on Macroptilium atropurpureum plants, sed 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, expressed in terms of total number of average yield (in grams) per plant.

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

ED DESCRIPTION Lipo-chitooligosaccharide compounds (LCO's), also known in the art as tic Nod signals or Nod factors, consist of an oligosaccharide backbone of B-I,4-Iinked N—acetyl—D—glucosamine Ac") 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 backbone, 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. m. 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 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, R2, R3, R5, R6 and R7, which may be identical or different, represent H, CH3 CO--, CX Hy CO-- where x is an integer between 0 and 17, and y is an integer between 1 and 35, or any other acyl group such as for e a carbamoyl, R4 ents a mono-, di- or triunsaturated aliphatic chain containing at least 12 carbon atoms, and n is an integer n 1 and 4.

LCOs may be ed (isolated and/or purified) from bacteria such as Rhizobia, e.g., Rhizobium sp., Bradyrhizobium sp., Sinorhizobium sp. and Azorhizobium sp. LCO structures are characteristic 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 5,549,718 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.

Patents 5,175,149 and 5,321,011. y, they are pentasaccharide phytohormones comprising methylfucose. A number of these B. japonicum-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., isolated 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 ial and hizobial microorganisms are included within the scope of the present invention.

LCO's are the primary determinants of host specificity in legume symbiosis (Diaz, et al., Moi. Microbe Interactions 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. -830 (1997), Examples of these bacteria/legume symbiotic partnerships include 8. meliloti/alfalfa and sweet clover; R. leguminosarum biovar viciae/peas and s; 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 specific LCO structures that are produced by the different rhizobial s.

However, LCO specificity is only required to ish nodulation in legumes. In the practice of the present invention, use of a given LCD is not d to treatment of seed of its symbiotic legume partner, in order to achieve 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, compared to plants ted from untreated seed, or ed to plants harvested from seed treated 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 derivatives 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, iso-017:0, 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 018-26(w-1)-OH (which according to D'Haeze, et al., Glycobiology 12:79R—105R (2002), includes 018, 020, 022, 024 and 026 hydroxylated species and 016:1A9, 016:2 (A2,9) and 016:3 (A2,4,9)); R2 represents en or methyl; R3 represents hydrogen, acetyl or carbamoyl; R4 represents en, acetyl or carbamoyl; R5 represents hydrogen, acetyl or carbamoyl; R6 represents hydrogen, osyl, l, acetyl, sulfate ester, 3-0—S— 2-0—MeFuc, 2-0—MeFuc, and 4-0—AcFuc; R7 represents hydrogen, mannosyl or glycerol; R8 represents hydrogen, methyl, or —0H20H; R9 represents hydrogen, arabinosyl, or l; 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 e, et al., supra.

By way of even further onal 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 rated in Figs. 1a and 2a are used in the methods of the present invenfion.

Also encompassed by the present ion 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 structures of representative LCOs obtained from these fungi are described in WO 49751 and (the LCOs described n also referred to as "Myc factors"). Representative hizal fungi-derived LCO's and non-naturally occurring derivatives thereof are represented by the following structure: wherein n = 1 or 2; R1 represents 016, 016:0, 016:1, 016:2, 018:0, 018:1A92 or 018:1A11Z; and R2 ents 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 ments, 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 illustrated in Figs. 1a, 2a, 3a, 4a and 5. Broadly, the present invention includes use of any two or more LCO‘s, ing 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 recombinant. r encompassed by the present invention is use of synthetic 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 naturally occurring LCO's, such as those bed in Spaink, 0rit. Rev. Plant Sci. -288 (2000) and D'Haeze, supra. Precursor oligosaccharide molecules (00s, which as described below, are also useful as plant signal molecules in the present invention) for the construction of LCOs may also be synthesized by genetically ered organisms, e.g., as described in Samain, et al., Carbohydrate Res. 302:35-42 (1997); Cottaz, et al., Meth. Eng. 11-7 (2005) and , et al., J. Biotechnol. 72:33-47 (1999)(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). 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® rcially available from mes BioAg d) 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 microbe, or continuing to isolate and purify the LCO molecules through LCO solvent phase separation followed by HPLC chromatography as described, for example, in US.

Patent 718. Purification can be enhanced by repeated HPLC, and the purified LCO molecules can be freeze-dried for long—term e. Chitooligosaccharides (COs) as described above, may be used as starting als 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 treated 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 ive amount of the at least two LCO's in an lturally 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 commercially available from numerous ers, e.g., Bayer CropScience (Gustafson).

In another embodiment, the treatment entails g seeds with the at least two LCO's. One such process involves g the inside wall of a round container with the composition, adding seeds, then rotating the ner to cause the seeds to contact the wall and the composition, a process known in the art as "container coating". Seeds can be coated by combinations of coating methods.

Soaking lly entails use of an aqueous solution containing the plant growth enhancing agent. For example, seeds can be soaked for about 1 minute to about 24 hours (e.g., for at least 1 min, 5 min, 10 min, 20 min, 40 min, 80 min, 3 hr, 6 hr, 12 hr, 24 hr). Some types of seeds (e.g., soybean seeds) tend to be sensitive to moisture. Thus, soaking such seeds for an extended period of time may not be desirable, in which case the g is typically carried out for about 1 minute to about 20 minutes.

In those embodiments 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 storage period is not critical. Without intending to be bound by any particular theory of operation, Applicants believe that even to the extent that the treating may not cause the plant signal molecule to remain in contact with the seed surface after treatment and during any part of storage, the 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). ants also believe that following treatment 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. hstanding, 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 ments, 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 e growth such that 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, compared to ted plants or plants harvested from untreated seed (with either active). The ive 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 indirectly, i.e., in-furrow treatment, the effective amount of the at least two LCO's lly ranges from 1 ug/acre to about 70 ug/acre, and in some ments, 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 e 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 embodiments, 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 ners, in accordance with standard techniques. 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 storage conditions which are known in the art. s 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 comparison 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 es of such agents that may be useful in the practice of the present invention 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 mycorrhizal fungi.

Micronutrients Representative ns that may be useful in the practice of the present invention include calcium pantothenate, folic acid, biotin, and n C.

Representative examples of trace minerals that may be useful in the practice of the present invention include boron, chlorine, ese, 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 ive amount generally ranges in one embodiment from about 180 ug to about 9 mg/hundred 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 ments, from about 120 mg/acre to about 6 g/acre when applied foliarly.

Plant signal molecules The present invention may also include treatment of seed or plant with a plant signal molecule 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 lly occurring in plants or es, and synthetic (and which may be non-naturally occurring) that directly or indirectly activates a plant mical 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 sed leaf area, compared to untreated plants or plants harvested from untreated seed. Representative examples of plant signal molecules that may be useful in the practice of the present invention include chitinous compounds, oids, jasmonic acid, linoleic acid and linolenic acid and their derivatives ), and ins.

Chitooligosaccharides C03 are known in the art as [34 linked N-acetyl glucosamine structures identified as chitin oligomers, also as N-acetylchitooligosaccharides. CO's have unique and ent side chain decorations which make them different 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 chitosan, and in some embodiments, as described below, are pentameric. Representative literature describing the structure and production of ODS that may be suitable for use in the present invention is as follows: Muller, et al., Plant Physiol. 124:733-9 (2000); Van der Holst, et al., t Opinion in Structural Biology, -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 (LCO's)); Rouge, et al. Chapter 27, "The lar Immunology of Complex Carbohydrates" in Advances in Experimental Medicine and Biology, Springer Science; Wan, etal., Plant Cell 3-69 (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 t fatty acid chain. Rhizobia-derived CO‘s, and non-naturally occurring synthetic derivatives thereof, that may be useful in the practice of the present invention may be ented 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 ents hydrogen, acetyl or carbamoyl; R4 ents hydrogen, acetyl or carbamoyl; R5 represents hydrogen, acetyl or oyl; R5 represents hydrogen, arabinosyl, fucosyl, acetyl, e 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 —CH20H; R9 represents hydrogen, arabinosyl, or fucosyl; R10 represents hydrogen, acetyl or fucosyl; and n ents 0, 1, 2 or 3. The ures 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 interact symbiotically 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 described 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 illustrated in Figs. 3b and 4b respectively. They correspond to aforementioned two different LCO's ed by the mycorrhizal fungi Glomus intraradices, and which are illustrated in Figs. 3a and 4a.

The COs may be tic or recombinant. Methods for preparation of synthetic CO's are described, for example, 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., oChem 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 (1999)(e.g., Fig. 1 therein which shows structures of CO's that can be made recombinantly in E. coli harboring different combinations of genes L). For the purposes of the present 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 components 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 hoxymethyl]—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 described, for example, in US. Patent 4,536,207 (preparation from crustacean shells), Pochanavanich, et al., Lett. Appl. Microbiol. 35:17-21 (2002) (preparation from fungal cell walls), and US. Patent 5,965,545 (preparation from crab shells and hydrolysis of commercial chitosan). 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 lar weights, e.g., low molecular weight chitosan ers of less than 15kD and chitin oligomers of 0.5 to 2kD; "practical grade" an with a molecular weight of about 150kD; and high molecular weight chitosan of up to 700kD. Chitin and chitosan compositions formulated for seed treatment are also commercially ble. cial products include, for example, ELEXA® (Plant Defense Boosters, Inc.) and T'V' (Agrihouse, Inc.).

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

Representative flavonoids that may be useful in the practice of the present invention e genistein, daidzein, formononetin, naringenin, hesperetin, luteolin, and apigenin. Flavonoid compounds are commercially available, e.g., from Natland International Corp., ch Triangle Park, NC; MP Biomedicals, Irvine, CA; LC Laboratories, Woburn MA. Flavonoid compounds may be isolated from plants or seeds, e.g., as described in US. Patents 5,702,752; 5,990,291; and 6,146,668. Flavonoid nds may also be produced by genetically 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 tives (which e linoleic acid and linolenic acid (which are described above in connection with fatty acids and their derivatives), may be used in the ce of the t invention. 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 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 biosynthesis of jasmonic acid. Like linoleic acid and linolenic acid, jasmonates (and their derivatives) are reported to be inducers of nod gene expression or LCD production by acteria.

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

Useful derivatives ofjasmonic acid, linoleic acid and linolenic acid that may be useful in the practice of the present invention include esters, amides, 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 heteroaryl group having, for example, 4 to 9 carbon atoms, n the atoms 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 nic acid has been replaced with a ——COR group, where R is an NRZR3 group, in which R2 and R3 are ndently: hydrogen; an alkyl group, such as a C1-C8 ched or branched alkyl group, e.g., a methyl, ethyl or propyl group; an alkenyl group, such as a 02-08 unbranched or branched alkenyl group; an l 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 aryl group can be, for example, N, O, P, or S. Esters may be prepared by _l7_ known methods, such as acid-catalyzed nucleophilic addition, 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 ylic acid with the appropriate amine in the presence of a coupling agent such as dicyclohexyl carbodiimide (DCC), under neutral conditions. Suitable salts of jasmonic acid, linoleic acid and linolenic acid include 6.9., base on salts. The bases that may be used as ts to e metabolically acceptable base salts of these compounds e those d 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., 2H-furo[2,3-c]pyranones including derivatives and ues thereof. Examples of these compounds are represented by the ing structure: wherein; Z is O, S or NR5; R1, R2, R3, and R4 are each independently H, alkyl, alkenyl, l, , benzyl, hydroxy, hydroxyalkyl, alkoxy, phenyloxy, benzyloxy, CN, CORa, COOR=, halogen, NR6R7, or N02; and R5, R6, and R7 are each independently H, alkyl or alkenyl, or a biologically acceptable salt thereof. Examples of biologically acceptable salts of these nds may include acid addition salts formed with biologically acceptable acids, examples of which include hydrochloride, hydrobromide, sulphate or bisulphate, phosphate or hydrogen phosphate, acetate, benzoate, succinate, fumarate, maleate, 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 e the following: yl-2H-furo[2,3-c]pyranone (where R1=CH3, R2, R3, R4=H), o[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, R3=CH3, 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), 5-methoxymethyl-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 tration, generally ranges from about 10'5 to about '14 M (molar tration), 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 dred 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 one plant signal molecule lly 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 , 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.

Herbicides, Fungicides and Insecticides Suitable herbicides include bentazon, acifluorfen, chlorimuron, en, clomazone, fluazifop, glufosinate, glyphosate, sethoxydim, imazethapyr, imazamox, fomesafe, flumiclorac, imazaquin, and clethodim. Commercial products containing each of these compounds are readily available. Herbicide concentration _19_ in the composition will generally correspond to the labeled use rate for a particular 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 s 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 ion will ly exhibit activity against a broad range of pathogens, including but not limited to Phytophthora, Rhizoctonia, Fusarium, Pythium, Phomopsis or Se/erotinia and Phakopsora and combinations thereof.

Commercial fungicides may be suitable for use in the present invention. Suitable commercially available fungicides include 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, gton, DE), CAPTAN (Arvesta, Guelph, Ontario) and PROTREAT (Nitragin Argentina, Buenos Ares, ina). Active ingredients in these and other commercial fungicides include, but are not limited to, xonil, mefenoxam, azoxystrobin and metalaxyl. Commercial fungicides are most suitably used in accordance with the manufacturer's ctions at the recommended concentrations.

As used herein, an insecticide "exhibits activity against" a particular species of insect if treatment with the insecticide results in killing or inhibition of an insect population relative to an untreated population. Effective insecticides in accordance with the invention will suitably exhibit activity 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 icides may be suitable for use in the present invention. Suitable commercially-available icides include CRUISER nta, Wilmington, DE), GAUCHO and PONCHO (Gustafson, Plano, TX). Active ingredients in these and other commercial insecticides include thiamethoxam, clothianidin, and imidacloprid. cial insecticides are most suitably used in accordance with the cturer's instructions at the recommended concentrations. _20_ Phosphate Solubilizing Microorganisms, Diazotrophs (Rhizobial inoculants), and/or Mycorrhizal fungi.

The present ion 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 increase the amount of phosphorous available for a plant. Phosphate solubilizing microorganisms include fungal and bacterial strains. In ment, the phosphate solubilizing rganism is a spore forming microorganism.

Non-limiting examples of phosphate solubilizing microorganisms include species from a genus selected from the group consisting of Acinetobacter, Arthrobacter, Arthrobotrys, Aspergillus, Azospirillum, Bacillus, Burkho/deria, Candida Chiyseomonas, Enterobacter, Eupenicillium, Exiguobacterium, ella, Kluyvera, Microbacterium, Mucor, Paecilomyces, Paenibacillus, Penicillium, Pseudomonas, Serratia, Stenotrophomonas, Streptomyces, Streptosporangium, Swaminathania, Thiobacillus, spora, Vibrio, Xanthobacter, and Xanthomonas.

Non-limiting es of phosphate solubilizing microorganisms are selected from the group ting Acinetobacter calcoaceticus, Acinetobacter sp, Arthrobacter sp., Arthrobotrys oligospora, Aspergillus niger, Aspergillus sp., Azospirillum halopraeferans, us amyloliquefaciens, Bacillus atrophaeus, Bacillus ans,Baci/lus licheniformis, Bacillus subtilis, Burkholderia a, Burkholderia vietnamiensis, Candida krissii, Chryseomonas luteola, bacter aerogenes, Enterobacter asburiae, Enterobacter sp., bacter taylorae, Eupenicillium parvum, Exiguobacterium sp., Klebsiella sp., Kluyvera Ciyocrescens, Microbacterium sp., Mucor ramosissimus, Paecilomyces hepialid, Paecilomyces marquandii, Paenibacillus macerans, Paenibacillus ginosus, Pantoea rans, Penicillium expansum, Pseudomonas corrugate, Pseudomonas fluorescens, Pseudomonas lutea, Pseudomonas poae, monas putida, Pseudomonas stutzeri, Pseudomonas trivia/is, Serratia marcescens, Stenotrophomonas hilia, Streptomyces sp., Streptosporangium 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 present invention include P. bilaiae (formerly known as P. bilaii), P. albidum, P. aurantiogriseum, P. chrysogenum, P. citreonigrum, P. citrinum, P. tum, P. frequentas, P. fuscum, P. gaestrivorus, P. m, 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. rugulosum, P. simplicissimum, P. sol/tum, P. variabile, P. velutinum, P. viridicatum, P. glaucum, P. fussiporus, and P. expansum.

In one ular embodiment, the Penicillium species is P. bi/aiae. In another particular embodiment 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). In another particular embodiment the Penicillium species is P. gaestrivorus, e.g., NRRL 50170 (see, n, supra).

In some embodiments, more than one phosphate lizing rganism is used, such as, at least two, at least three, at least four, at least five, at least 6, ing any combination of the Acinetobacter, Arthrobacter, Arthrobotrys, Aspergillus, Azospirillum, Bacillus, Burkholderia, Candida Chryseomonas, Enterobacter, Eupenicillium, Exiguobacterium, Klebsiella, Kluyvera, Microbacterium, Mucor, Paecilomyces, Paenibacillus, Penicillium, Pseudomonas, Serratia, Stenotrophomonas, Streptomyces, Streptosporangium, Swaminathania, Thiobacillus, Torulospora, Vibrio, Xanthobacter, and Xanthomonas, including one species selected from the following group: Acinetobacter ceticus, Acinetobacter sp, Arthrobacter sp., Arthrobotrys oligospora, Aspergillus niger, Aspergillus sp., Azospirillum ha/opraeferans, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans,Baci/Ius licheniformis, Bacillus subtilis, Burkholderia cepacia, Burkho/deria vietnamiensis, Candida krissii, omonas luteola, Enterobacter aerogenes, Enterobacter asburiae, Enterobacter sp., Enterobacter taylorae, ci/lium pan/um, Exiguobacterium sp., K/ebsie/la sp., K/uyvera cryocrescens, Microbacterium sp., Mucor ramosissimus, omyces hepialid, omyces marquandii, Paenibacillus macerans, Paenibaci/lus mucilaginosus, Pantoea ag/omerans, Penicillium expansum, Pseudomonas corrugate, Pseudomonas scens, Pseudomonas lutea, Pseudomonas poae, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas /is, Serratia _22_ marcescens, Stenotrophomonas maltophilia, Streptomyces sp., Streptosporangium sp., Swaminathania salitolerans, cillus ferrooxidans, Torulospora globosa, Vibrio proteolyticus, bacter agilis, and Xanthomonas campestris In some embodiments, two different strains of the same s may also be combined, for example, 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 y containing insoluble (or sparingly soluble) ates, the use of the combined fungal s will result in an se in the amount of phosphorus available for plant uptake compared to the use of only one Penicillium 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 s 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 prepared in the form of a stable spore.

In an embodiment, the phosphate solubilizing microorganism is a Penicillium fungus. The Penicillium fungus according to the invention can be grown using solid state or liquid tation and a le carbon source. Penicillium isolates 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 ning 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 carrier to be applied to soil. The term "inoculum" as used in this specification is intended to mean any form of ate solubilizing microorganism, fungus cells, _23_ mycelium or spores, bacterial cells or ial spores, which is capable of ating 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 suspension (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 se broth or sucrose 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 , or fluid bed drying techniques (Friesen, et al., 2005, Appl.

Microbiol. Biotechnol. 68:397-404) to produce a wettable powder. The wettable powder is then suspended in water, applied to the surface of seeds, and allowed to dry prior to planting. The wettable powder may be used in conjunction with other seed ents, such as, but not limited to, chemical seed treatments, rs (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 soil—compatible carrier (e.g., ased powder or granule) to appropriate final moisture content. The material may be incubated at room temperature, lly for about 1 day to about 8 weeks, prior to use.

Aside from the ingredients used to cultivate the phosphate solubilizing microorganism, ing, e.g., ingredients referenced above in the cultivation of Penicillium, the phosphate solubilizing microorganism may be formulated using other mically acceptable carriers. As used herein in tion with "carrier", the term "agronomically acceptable" refers to any al 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, peat—based 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 suitable 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 rs include ned 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 ate 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 suitable amount can be found by simple trial and error ments 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 um (fresh weight) per hectare, or 6 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 planting.

In embodiments, for example, that entail use of at least two strains of a phosphate solubilizing microorganism, such as, two strains of Penicillium, cial izers may be added to the soil instead of (or even as well as) natural rock phosphate. The source of phosphorous may contain a source of phosphorous native to the soil. In other embodiments, the source of phosphorous may be added to the soil. In one embodiment the source is rock phosphate. In r embodiment 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 hosphate and ammonium polyphosphate. All of these fertilizers are produced by chemical processing of ble natural rock phosphates _25_ in large scale fertilizer-manufacturing facilities and the product is expensive. By means of the present invention it is possible to reduce the amount of these izers applied to the soil while still maintaining the same amount of phosphorus uptake from the soil.

In a further embodiment, the source or phosphorus is organic. An organic izer refers to a soil amendment derived from natural sources that guarantees, at least, the minimum tages of nitrogen, phosphate, and potash.

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

Other fertilizers, such as nitrogen sources, 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 en gas into a more usable form such as ammonia. Examples of diazotrophs include bacteria from the genera ium spp. (e.g., R. cellulosilyticum, R. daejeonense, R. etli, R. galegae, R. gallicum, R. giardinii, R. hainanense, R. ense, R. ferae, R. leguminosarum, R. loessense, R. lupini, R. lusitanum, R. meliloti, R. mongolense, R. miluonense, R. sullae, R. tropici, R. undicola, and/or R. yanglingense), Bradyrhizobium spp. (e.g., B. bete, B. canariense, B. elkanii, B. iriomotense, B. japonicum, B. jicamae, B. ngense, 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. ti, S. mexicanus, S. morelense, S. , S. ae, 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 ations with the roots of a vascular plant, and provide, e.g., absorptive capacity for water and mineral nutrients due to the comparatively large e area of mycelium. Mycorrhizal 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 endomycorrhizae of the phylum Glomeromycota and genera Glomus and Gigaspora. In still a further ment, the corrhizae is a strain of Glomus aggregatum, Glomus brasilianum, Glomus clarum, Glomus icola, Glomus atum, Glomus fasciculatum, Glomus intraradices, Glomus monosporum, or Glomus mosseae, ora margarita, or a combination thereof.

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

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

The methods of the present invention are applicable to nous seed, representative examples of which include soybean, alfalfa, peanut, pea, lentil, bean and clover. The methods of the present invention are also applicable to non-leguminous seed, e.g., Poaceae, Cucurbitaceae, Malvaceae,. Asteraceae, _27_ Chenopodiaceae 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, lettuce and cantaloupe.

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

Examples Greenhouse Experiments Example 1: Siratro seedling growth in vitro ed by LCO combinations Siratro (Macropti/ium atropurpureum) seeds were e-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 e 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 examples as the fa LCO") was added to n 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 reflected by the comparison between soy LCO combined with another LCO tive 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 t seedling when total root and shoot length were summed. This ence 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 ners and d with soy LCO or its combination with alfalfa 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 embodiment (soy LCO + alfalfa LCO) showed that there was a slight increase in leaf greenness with LCD combination 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 ation was significantly higher than non-inventive soy LCO. rly, when fruit numbers were counted over a six-day , 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 treatment.

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

E 3: LCOs and their combinations on tomato seedling root growth Tomato seeds of var. Royal Mounty were placed in petriplates containing moist (soaked with treatment solutions) germination paper. Treatment solutions were prepared with four different LCOs, namely Pea LCO AC (acylated), Pea LCO NAC cylated), 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 germination. Eight days after germination, seedlings were measured with a hand held ruler for their root length.

Results obtained from this experiment indicated that all individual LCO types ed tomato seedling root length as compared to control but only n LCO combinations i.e. pea NAC and soybean LCO, pea AC plus soybean LCD and pea NAC plus alfalfa LCO generated significant root enhancement as compared to non-inventive and comparable single LCO types (Fig. 8). From the ment, it appeared to be that for tomato seedlings, pea NAC and n LCO combination was the best of all combinations. The results also indicate that ations 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 skilled 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 dually 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 therefore to be understood that numerous cations 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_

Claims (37)

CLAIMS :

1. A method of enhancing plant growth, comprising ng plant seed or the plant that germinates from the seed with an effective amount of at least two distinct lipo-chitooligosaccharides (LCO’s), said at least two distinct LCO’s comprising the following LCO’s: wherein said at least two LCO’s synergistically e said plant growth, and upon harvesting the plant exhibits at least one of increased plant yield measured in terms of bushels/acre, increased root number, sed root length, increased root mass, increased root volume and increased leaf area, compared to untreated plants or plants harvested from untreated seed.

2. The method of claim 1, wherein the at least two LCO's comprise an LCO obtained from a strain of rhizobia. (11632444_1):EOR

3. The method of claim 2, wherein the strain of ia is selected from the group consisting of Rhizobium sp., Bradyrhizobium sp., Sinorhizobium sp. and Azorhizobium sp.

4. The method of claim 1, wherein the at least two LCO's comprise an LCO obtained from B. japonicum.

5. The method of claim 1, wherein the at least two LCO's comprise an LCO obtained from R. leguminosarum biovar viciae

6. The method of claim 1, wherein the at least two LCO's comprise an LCO obtained from S. meliloti.

7. The method of claim 1, n the at least two LCO's comprise an LCO obtained from Glomus intraradicus.

8. The method of claim 1, wherein the at least two LCO's comprise a recombinant LCO.

9. The method of claim 8, n the recombinant LCO has a purity of at least 60%.

10. The method of claim 8, wherein the recombinant LCO has a purity of at least 70%.

11. The method of claim 8, wherein the recombinant LCO has a purity of at least 80%.

12. The method of claim 8, wherein the recombinant LCO has a purity of at least 90%.

13. The method of claim 1, wherein the at least two LCO's comprise a synthetic LCO.

14. The method of any one of claims 1 to 13, wherein the at least two LCO's are d to seed prior to planting or at about the time of planting.

15. The method of claim 14, wherein the effective amount of the at least two LCO's are from about 10-5 to about 10-14 Molar. (11632444_1):EOR

16. The method of any one of claims 1 to 13, wherein the at least two LCO's are applied to seed in .

17. The method of claim 16, wherein the effective amount of the at least two LCO's is from 1 μg/acre to about 70 μg/acre.

18. The method of any one of claims 1 to 13, wherein the at least two LCO's are applied to the plant via foliar treatment.

19. The method of claim 14, wherein the effective amount of the at least two LCO's is from 1 μg/acre to about 30 μg/acre.

20. The method of any one of claims 1 to 19, further comprising applying to the plant or seed thereof at least one agronomically beneficial agent.

21. The method of claim 20, wherein the at least one agronomically beneficial agent is a micronutrient.

22. The method of claim 21, wherein the micronutrient is selected from the group consisting of vitamins and trace minerals.

23. The method of claim 20, wherein the at least one agronomically beneficial agent is a plant signal molecule.

24. The method of claim 23, wherein the plant signal molecule is a chitooligosaccharide (CO).

25. The method of claim 23, n the plant signal molecule is selected from the group ting of chitinous compounds; flavonoids; jasmonic acid and derivatives thereof, linoleic acid and derivatives thereof, linolenic acid and derivatives thereof; and karrikins and derivatives thereof.

26. The method of claim 20, wherein the agronomically cial agent is an herbicide, icide, a fungicide or any combination thereof.

27. The method of claim 20, wherein the agronomically beneficial agent is a phosphate solubilising microorganism, roph (Rhizobial ants), and/or mycorrhizal fungi. (11632444_1):EOR

28. The method of claim 27, wherein the at least one phosphate solubilizing microorganism comprises a strain of the fungus Penicillium.

29. The method of claim 27, wherein the at least one phosphate solubilizing rganism comprises a strain of P. bilaiae.

30. The method of claim 29, wherein the strain of P. bilaiae is selected from the group consisting of NRRL 50162, NRRL 50169, ATCC 20851, ATCC 22348, and ATCC 18309.

31. The method of claim 27, wherein the at least one phosphate lizing microorganism comprises a strain of P. gaestrivorus.

32. The method of any one of claims 1 to 31, wherein the plant or seed f is leguminous.

33. The method of claim 32, n the leguminous plant or seed thereof is soybean.

34. The method of any one of claims 1 to 31, wherein the plant or seed thereof is non-leguminous.

35. The method of claim 34, wherein the non-leguminous plant or seed thereof is a field crop plant or seed.

36. The method of claim 35, wherein the field crop plant or seed is corn.

37. The method of claim 34, wherein the non-leguminous plant or seed thereof is a vegetable crop plant or seed. Novozymes BioAg A/S Novozymes Biologicals, Inc. By the Attorneys for the Applicant SPRUSON & FERGUSON Per: (11632444_1):EOR