WO2014049546A2

WO2014049546A2 - Compositions and methods for enhancing plant growth and development

Info

Publication number: WO2014049546A2
Application number: PCT/IB2013/058879
Authority: WO
Inventors: Violaine HERRBACH; Sandra BENSMIHEN
Original assignee: Institut National De La Recherche Agronomique; Centre National De La Recherche Scientifique
Priority date: 2012-09-27
Filing date: 2013-09-26
Publication date: 2014-04-03
Also published as: WO2014049546A3

Abstract

Disclosed are methods of enhancing plant growth, comprising treating plant seed or the plant that germinates from the seed with a signal molecule and a phytohormone comprising an auxin or a synthetic analog thereof, wherein the enhanced growth comprises increased lateral root formation compared to an untreated plant or a plant harvested from untreated seed, and wherein the signal molecule and the auxin or synthetic analog thereof used for the treating are effective to produce a greater than additive effect on lateral root formation.

Description

COMPOSITIONS AND METHODS FOR ENHANCING PLANT GROWTH AND

DEVELOPMENT

BACKGROUND OF THE INVENTION

Plant growth depends at least in part on interactions between the plant and microorganisms that habitate the surrounding soil. For example, the symbiosis between the gram-negative soil bacteria, Rhizobiaceae and

Bradyrhizobiaceae, 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 bacteria-to-plant signal compounds, which include the end products of the expression of the bradyrhizobial and rhizobial nod genes, known as lipo-chitooligosaccharides (LCOs). The symbiosis between these bacteria and the legumes enables the legume to fix atmospheric nitrogen for plant growth, 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.

Another known and well studied symbiotic association between plants and soil microorganisms involves arbuscular mycorrhizal (AM) fungi. This group of fungi, recently renamed Glomeromycota, is widely distributed distributed throughout the plant kingdom including angiosperms, gymnosperms, pteridophytes and some bryophytes (Smith and Read, 2008) . Among the angiosperms, at least 80% of the species can form AM symbioses, the only major exceptions being Brassicaceae and Chenopodiaceae . Arbuscular mycorrhizal fungi are able to transfer rare or poorly soluble mineral nutrients such as phosphorus, zinc and copper from the soil to the plant, which in turn provides carbohydrates to the fungus. This exchange of nutrients can be of critical importance when soil fertility and water availability are low, conditions that severely limit agricultural production in most parts of the world (Smith, et al., Mycorrhizal symbiosis. 787 pp., Academic Press. (2008) ) .

Continuing efforts are made to exploit these symbiotic relationships with the goal of increasing plant growth and yield.

BRIEF SUMMARY OF THE INVENTION

In plants, the formation of lateral roots is a crucial step in the development of the root system. Lateral roots provide stable anchorage of the plant and contribute to efficient water use and uptake of nutrients from the soil. The main regulator of lateral root development is the phytohormone auxin, which is believed to play a central role in the initiation and outgrowth of lateral roots.

A first aspect of the present invention is directed to a method of enhancing plant growth, comprising treating plant seed or the plant that germinates from the seed with a signal molecule and a phytohormone comprising an auxin or a synthetic analog thereof, wherein the enhanced growth comprises increased lateral root formation compared to an untreated plant or a plant harvested from untreated seed, and wherein the signal molecule and the auxin or synthetic analog thereof used for the treating are effective to produce a greater than additive effect on lateral root formation. The signal molecule and the auxin are collective referred to herein as the "actives" or "active agents".

A second aspect of the present invention is directed to plant seed coated with a signal molecule and a phytohormone comprising an auxin or a synthetic analog thereof, wherein the signal molecule and the auxin or synthetic analog are coated onto the seed each in amounts that are effective to produce a greater than additive effect on lateral root formation of a plant that germinates from the seed. A third aspect of the present invention is directed to a composition for enhancing plant growth, comprising a signal molecule, a phytohormone comprising an auxin or a synthetic analog thereof, and an agronomically acceptable carrier, wherein the signal molecule and the auxin or synthetic analog are present in amounts effective to produce a greater than additive effect on lateral root formation.

The density of the lateral roots is important in plant development and plant biomass production. Thus, by achieving a synergistic increase in the number of lateral roots, use of the present invention may lead to enhanced lateral branching of the root system, a more stable anchorage in the soil, improved nutrient uptake and a concomitant increase in biomass production.

Aside from an increase in lateral root formation, which may be measured in terms of the increased number of lateral roots, increased plant growth may also be manifested in terms of at least one other auxin-mediated mechanism by which plants develop, including for example, new leaf formation, vascular tissue development, adventitious root/tissue development, increased fruiting and floral primordial initiation .

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the structure of Nod and Myc symbiotic factors, with a chitin tetramer (rarely pentamer) of N-acetyl- glucosamine and an acyl chain. Figure la shows the structure of a lipo-chitooligosaccharide (also known as a nod factor) , which is the major nod factor produced by the bacterium, Sinorhizobium meliloti, with a sulfate group, an acetate group and a C16:2 fatty acid, and which is useful in the practice of the present invention; Figure lb shows the structure of a Myc factor which is one of the major Myc factors produced by the fungus Glomus intraradices, with or without a sulfate group, and a C18:l or a C16:0 fatty acid, and which is useful in the practice of the present invention. Figure 2 is a bar graph that shows the synergistic effect on lateral root induction in Medicago truncatula plants treated with the S. meliloti nod factor (illustrated in Figure la) and the synthetic auxin, 1-naphthalene acetic acid (NAA), measured in days after germination. Bars show the mean percentage of new lateral root formed, as compared to the mock treatment. NAA treatments only (ranging from 10^~8 to 1CT⁶ M) are shown in dark grey (columns A, B and C) , and NF+/- NAA treatments are shown in light grey (columns 1, 2 and 3). Data are mean of percentages calculated on 50 plants and 5 successive days. Bars show SEM (N=250) . Statistical test: GLM (treatment effect: p<0.001; A, B, C and 1, 2, 3: statistically different groups) .

Figure 3 is a bar graph showing the synergistic effect on the induction of lateral root formation in M. truncatula plants treated with NAA and a combination of the Myc factor from G. intraradices (illustrated in Figure lb) and another Myc factor that differs from the Myc factor illustrated in Figure lb in that the C18:1 fatty acid is replaced with a C16:0 fatty acid. Bars show the mean percentage of new lateral root formed, as compared to the mock treatment. Data are mean of percentages calculated on 30 plants and 4 successive days. Bars show SEM (N=120). Statistical test: GLM (p<0.05; a, b, c: statistically different groups) .

Figure 4 is a bar graph that shows that combined treatment with the S. meliloti nod factor (illustrated in Figure la) and NAA does not have a stronger effect than auxin alone on primary root length of M. truncatula plants. Combined NF treatment does not modify NAA effect of primary root length. Bars show the mean primary root length, as compared to the mock treatment. NAA treatments only (ranging from 10^-8 to 10^-6 M) are shown in dark grey and NF+/- NAA treatments are shown in light grey. Data are mean of percentages calculated on 55 plants at day 4. Bars show SEM (N=55) . Statistical test: Kruskal-Wallis (p<0.05; a, b, c, d, e: statistically different groups; same letter shows no statistical difference) . DETAILED DESCRIPTION

Plant Signal Molecules

For purposes of the present invention, the term "plant signal molecule", which for purposes of the present invention, may be used interchangeably with "plant growth-enhancing agent," broadly refers to any agent, both naturally occurring in plants or microbes, and synthetic (and which may be non-naturally occurring) that directly or indirectly activates a plant biochemical pathway, resulting in increased plant growth, 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 lipo-chitooligosaccharide compounds (LCO's), flavonoids, jasmonic acid, and karrikins. The present invention includes use of two or more signal molecules of the same type, e.g., a plurality of LCOs, as well as use of two or more different types of signal molecules, e.g., an LCO and a flavanoid. Yet other signal molecules that may be suitable for use in the present invention include chitinous compounds (e.g., chitooligosaccharides ) , and linoleic acid and linolenic acid and their derivatives.

Lipo-chitooligosaccharides

Lipo-chitooligosaccharide compounds (LCO's), also known in the art as symbiotic Nod signals or Nod factors, consist of an oligosaccharide backbone of β-1,4-linked ΛΓ-acetyl-D-glucosamine ("GIcNAc") residues with an N-linked fatty acyl chain condensed at the non-reducing end. LCO's differ in the number of GIcNAc residues in the backbone, in the length and degree of saturation of the fatty acyl chain, and in the substitutions of reducing and non-reducing sugar residues. See, e.g., Denarie, et al., Ann. Rev. Biochem. 5:503-35 (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 fi;: 55-60 (1998) . An example of an LCO is presented below as formula I

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,

Ri, R2, R3, R5, Re and R₇, which may be identical or different, represent H, CH₃ CO--, C_x H_y 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 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 characteristic for each such bacterial species, and each strain may produce multiple LCO's with different structures. For example, specific LCOs from S. meliloti have also been described in U.S. Patent 5,549,718 as having the formula II:

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 AcNodR -1, and AcNodRM-3, respectively (U.S. Patent 5,545,718).

LCOs from Bradyrhizobium japonicum are described in U.S. Patents 5,175,149 and 5,321,011. Broadly, they are pentasaccharide phytohormones comprising methylfucose . A number of these B . japonicum-derxved LCOs are described: BjNod-V (Ci8:i) BjNod-V (Ac, C_18:i) , BjNod-V (Ci_6:i) ; and BjNod-V (A_c, Ci6:o)/ with "V" indicating the presence of five N-acetylglucosamines; "Ac" an acetylation; the number following the "C" indicating the number of carbons in the fatty acid side chain; and the number following the ":" the number of double bonds .

LCO's used in embodiments of the invention 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 rhizobial and bradyrhizobial 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., Mol. Plant-Microbe Interactions 23:268-276 (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 S. meli Joti/alfalfa and sweet clover; R . leguminosarum biovar viciae/peas and lentils; R. leguminosarum biovar phaseoli/heans ; Brady hizobium japonicum/soybeans ; and R. leguminosarum biovar trifolii/red clover. The Hungria publication also lists the effective flavonoid Nod gene inducers of the rhizobial species, and the specific LCO structures that are produced by the different rhizobial species. However, LCO specificity is only required to establish nodulation in legumes. In the practice of the present invention, use of a given LCO is not limited to treatment of seed of its symbiotic legume partner, in order to achieve an increase in plant growth, including a synergistic increase in lateral root formation.

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:

wherein R₂ represents C14:0, 3OH-Cl4:0, iso-C15:0, C16:0, 3-OH-Cl6:0, iso-C15:0, C16:l, C16:2, C16:3, iso-C17:0, iso-C17:l, C18:0, 3OH-C18.-0, C18.-0/3-OH, C18:l, OH-C18.-1, C18:2, C18:3, C18:4, C19:l carbamoyl, C20:0, C20:l, 3-OH-C20:l, C20:l/3-OH, C20:2, C20:3, C22:l, and C18-26 (ω-l ) -OH (which according to D'Haeze, et al., Glycobiology J2.-79R-105R (2002), includes C18, C20, C22, C24 and C26 hydroxylated species and C16:1A9, C16:2 (Δ2,9) and C16:3 (Δ2,4,9)); R₂ represents hydrogen or methyl; R₃ represents hydrogen, acetyl or carbamoyl; R represents hydrogen, acetyl or carbamoyl; R₅ represents hydrogen, acetyl or carbamoyl; R₆ represents hydrogen, arabinosyl, fucosyl, acetyl, sulfate ester, 3-O-S-2-0-MeFuc, 2-0-MeFuc, and 4-0-AcFuc; R₇ represents hydrogen, mannosyl or glycerol; R₈ represents hydrogen, methyl, or -CH₂OH; R₉ represents hydrogen, arabinosyl, or fucosyl; Rio 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 LCO obtained from B. japonicum may be used to treat leguminous seed other than soybean and non-leguminous seed such as corn. As another example, an LCO obtainable from Rhizobium leguminosarum biovar viciae (designated LCO-V (C18:l), SP104) can be used to treat leguminous seed other than pea and non-legumes too.

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 structures of representative LCOs obtained from these fungi are described in WO 2010/049751 and WO 2010/049751 (the LCOs described therein also referred to as "Myc factors"). Representative mycorrhizal fungi-derived LCO ' s and non-naturally occurring derivatives thereof are represented by the following structure:

wherein n = 1 or 2; Ri represents C16, C16:0, C16:l, C16:2, 018:0, 018:1Δ9Ζ or 018:1Δ11Ζ; and R₂ represents hydrogen or S0₃H. 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 invention. Other Myc factors that may be useful in the practice of the present invention are described in U.S. Patent Application Publication 2011/0301032 Al .

In some other embodiments, an LCO useful in the present invention is obtained from S. meliloti. Broadly, the present invention includes use of any LCO, including naturally occurring (e.g., rhizobial, bradyrhizobial and fungal), recombinant, synthetic and non-naturally occurring derivatives thereof.

Further encompassed by the present invention is use of synthetic LCO compounds, such as those described in WO 2005/063784, and recombinant LCO's produced through genetic engineering. The basic, naturally occurring LCO structure may contain modifications or substitutions found in naturally occurring LCO's, such as those described in Spaink, Crit. Rev. Plant Sci. 54:257-288 (2000) and D'Haeze, supra. Precursor oligosaccharide molecules (COs, which as described below, are also useful as plant signal molecules in the present invention) for the construction of LCOs may also be synthesized by genetically engineered organisms, e.g., as described in Samain, et al., Carbohydrate Res. 502:35-42 (1997); Cottaz, et al., Meth. Eng. 7(4) :311-Ί (2005) and Samain, et al., J. Biotechnol. 72:33-47 (1999) .

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 Novozymes BioAg Limited) contains a culture of B. japonicum that produces an LCO (LCO-V (C18 : 1, MeFuc) , MORI 16) . 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 U.S. Patent 5,549,718. Purification can be enhanced by repeated HPLC, and the purified LCO molecules can be freeze-dried for long-term storage. Chitooligosaccharides (COs) may be used as starting materials for the production 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.

Flavonoids

Flavonoids are phenolic compounds having the general structure of two aromatic rings connected by a three-carbon bridge. Flavonoids are produced by plants and have many functions, e.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. 12:1867-80 (2006).

Representative flavonoids that may be useful in the practice of the present invention include genistein, daidzein, forraononetin, naringenin, hesperetin, 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 isolated from plants or seeds, e.g., as described in U.S. Patents 5,702,752; 5,990,291; and 6,146,668. Flavonoid compounds may also be produced by genetically engineered organisms, such as yeast, as described in Ralston, et al., Plant Physiology 137:1375-88 (2005) .

Jasmonic Acid and Derivatives

Jasmonic acid (JA, [1R- [la, 2β (Z) ] ] -3-oxo-2-

(pentenyl ) cyclopentaneacetic acid) and its derivatives (which include linoleic acid and linolenic acid (which are described 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 naturally in plants. Jasmonic acid is produced by the roots of wheat seedlings, and by fungal microorganisms such as Botryodiplodia theobromae and Gibbrella fuj ikuroi , 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 LCO production by rhizobacteria. See, e.g., Mabood, Fazli, Jasmonates induce the expression of nod genes in Bradyrhizobium japonicum, May 17, 2001.

Useful derivatives of jasmonic 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_r 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, wherein the heteroatoms in the heteroaryl group can be, for example, N, 0, P, or S . 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 NR²R³ group, in which R² and R³ are independently: hydrogen; an alkyl group, such as a C₂-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, wherein the heteroatoms in the heteroaryl group can be, for example, N, 0, P, or S. Esters may be prepared by 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 carboxylic 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 e.g., base addition salts. The bases that may be used as reagents to prepare metabolically acceptable base salts of these compounds include those derived from cations such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium). These salts may be readily prepared by mixing together a solution of linoleic acid, linolenic acid, or jasmonic 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

Karrikins are vinylogous 4H-pyrones e.g., 2H-furo [2 , 3-c] pyran-2-ones including derivatives and analogues thereof. Examples of these compounds are represented by the following structure:

wherein; Z is 0, S or NR₅; Ri, R₂, R3 and R4 are each independently H, alkyl, alkenyl, alkynyl, phenyl, benzyl, hydroxy, hydroxyalkyl, alkoxy, phenyloxy, benzyloxy, CN, C0R₆, C00R=, 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 compounds may include acid addition salts formed with biologically acceptable acids, examples of which include hydrochloride, hydrobromide, sulphate or bisulphate, phosphate or hydrogen phosphate, acetate, benzoate, succinate, fumarate, maleate, lactate, citrate, tartrate, gluconate; methanesulphonate , benzenesulphonate and p-toluenesulphonic acid. Additional biologically acceptable metal salts may include alkali metal salts, with bases, examples of which include the sodium and potassium salts. Examples of 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] pyran-2-one (where Ri=CH₃, R₂, R3, R₄=H) , 2H-furo [2, 3-c] pyran-2-one (where R_if R₂, R₃, R4=H) , 7-methyl-2H-furo [2, 3-c] yran-2-one (where Ri, R₂, R=H, R3=CH₃) , 5-methyl-2H-furo [2, 3-c] pyran-2-one (where R_if R₂, R3=H, R₄=CH₃) , 3, 7 -dimethyl-2H-furo [2, 3-c] pyran-2-one (where R_x, R₃=CH_3f R₂, R₄=H) , 3, 5-dimethyl-2H-furo [2, 3-c] pyran-2-one (where R_x, R₄=CH₃, R₂, R₃=H) , 3, 5, 7-trimethyl-2H-furo [2, 3-c] pyran-2-one (where Ri, R₃, R₄=CH₃, R2=H) , 5-methoxymethyl-3-methyl-2H-furo [2 , 3-c] pyran- 2-one (where Ri=CH₃, R₂, R₃=H, R₄=CH₂OCH₃) , 4 -bromo-3 , 7-dimethyl- 2H-furo[2,3-c]pyran-2-one (where R_lf R₃=CH₃, R₂=Br, R₄=H) , 3- methylfuro [2 , 3-c] pyridin-2 ( 3H) -one (where Z=NH, Ri=CH₃, R₂, R₃, R₄=H) , 3, 6-dimethylfuro [2 , 3-c] pyridin-2 ( 6H) -one (where Z= — CH₃, Ri=CH₃, R₂, R₃, R₄=H) . See, U.S. Patent 7, 576, 213. These molecules are also known as karrikins. See, Halford, supra.

Chitoollgosaccharldes

COs are known in the art as β-1-4 linked N-acetyl glucosamine structures identified as chitin oligomers, also as N-acetylchitooligosaccharides . CO's have unique and different side chain decorations which make them different from chitin molecules [ (C₈H₁₃N0₅) _n, CAS No. 1398-61-4], and chitosan molecules [ (C₅H_uN0₄)„, 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 hereinbelow, are pentameric. Representative literature describing the structure and production of COs that may be suitable for use in the present invention is as follows: Muller, et al., Plant Physiol. 224:733-9 (2000); Van der Hoist, et al., Current Opinion in Structural Biology, 12:608-616

(2001) (e.g., FIG. 1 therein); Robina, et al., Tetrahedron 55:521-530 (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 Molecular Immunology of Complex Carbohydrates" in Advances in Experimental Medicine and Biology, Springer Science; Wan, et al., Plant Cell 22:1053-69 (2009); PCT/FlOO/00803 (9/21/2000); and Demont-Caulet, et al., Plant Physiol. 220(2J :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 occurring synthetic derivatives thereof, that may be useful in the practice of the present invention may be represented by the following formula:

wherein P._! and R₂ each independently represents hydrogen or methyl; R₃ represents hydrogen, acetyl or carbamoyl; R₄ represents hydrogen, acetyl or carbamoyl; R_¾ represents hydrogen, acetyl or carbamoyl; R6 represents hydrogen, arabinosyl, fucosyl, 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 -CH₂OH; 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, efc al., supra.

Two CO's that may be suitable for use in the present invention correspond to LCO's produced by Bradyrhizobium japonicum and R. legu inosar m 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 Glomerocycota, e.g., Glomus intraradices . See, e.g., WO 2010/049751 and Maillet, et al., Nature 465:58-63 (2011) (the LCOs described therein also referred to as "Myc factors") . Representative mycorrhizal fungi-derived CO's are represented by the following structure:

wherein n = 1 or 2 ; R_x represents hydrogen or methyl; and R_∑ represents hydrogen or SO₃H.

The COs may be synthetic 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., ChemBioChem 7:359-65 (2006), Samain, et al., Carbohydrate Res. 302:35-42 (1997); Cottaz, et al., Meth. Eng. 7(4,1:311-7 (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 nodBCHL) . 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 that may be useful in the practice of the present invention 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-6- (hydroxymethyl) oxart-2yl ] methoxymethyl ] -2- [ [ 5-acetylamino-4 , 6- dihydroxy-2- (hydroxy methyl) oxan-3-yI ] methoxymethyl ] -4-hydroxy- 6- (hydroxymethyl ) oxan-3-ys] ethanamide) , and chitosan, (IUPAC: 5-amino-6- [ 5-amino- 6- [5-amino- , 6-dihydroxy- 2 (hydroxymethyl ) oxan-3-yl] oxy-4 -hydroxy-2- (hydroxymethyl ) oxan- 3-yl] oxy-2 (hydroxymethyl) oxane-3, 4-diol) . These compounds may be obtained commercially, e.g., from Sigma-Aldrich, or prepared from insects, crustacean shells, or fungal cell walls. Methods for the preparation of chitin and chitosan are known in the art, and have been described, for example, in U.S. Patent 4,536,207 (preparation from crustacean shells), Pochanavanich, et al., Lett. Appl. Microbiol. 35:17-21 (2002) (preparation from fungal cell walls), and U.S. Patent 5,965,545 (preparation from crab shells and hydrolysis of commercial chitosan). See, also, Jung, et al._r Carbohydrate Polymers 57:256-59 (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 chitosan of up to 700kD. Chitin and chitosan compositions formulated for seed treatment are also commercially available. Commercial products include, for example, ELEXA® (Plant Defense Boosters, Inc.) and BEYOND™ (Agrihouse, Inc.).

Auxins

Naturally occurring auxins and synthetic analogs thereof (also referred to herein as synthetic auxins) may be suitable for use in the present invention. Naturally occurring auxins include indoleacetic acids such as indole-3-acetic acid (IAA), 4-chloro-indole acetic acid, indole-3-acetamide, indole- 3-butyric acid and 2-phenylacetic acid. Synthetic auxins that may be useful in the practice of the present invention include 1-naphthalene acetic acid (NAA) , phenyl-indole-3-acetate, indole-propionic acid, and 3-hydroxy-phenyl-indole-3-acetate . The literature also refers to indole-3-butyric acid as a synthetic auxin. Auxins are commercially available and can be made in accordance with methodology known in the art.

Seeds may be treated with the signal molecule and the auxin in several ways such as spraying or dripping. Spray and drip treatment may be conducted by formulating an effective amount of the signal molecule and the auxin in an agronomically (agriculturally) acceptable carrier (which are known in the art and are 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 the composition or compositions containing the signal molecule and/or the auxin are delivered into a mixer, may also be employed. Systems and apparatus for performing these processes are commercially available from numerous suppliers, e.g., Bayer CropScience (Gustafson) .

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

In embodiments that entail storage of seed after application of the signal molecule and the auxin, adherence of these actives 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, they 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). Notwithstanding, to the extent desirable, the compositions containing the actives 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 signal molecule and the auxin 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 a composition containing the signal molecule and another composition containing the auxin are applied to seed or the plant, or in some embodiments, the signal molecule is applied to seed and the auxin is applied to the plant (and vice versa) .

Amounts of the Actives

The amount of each of the actives, when used together, is effective to achieve a synergistic or greater than additive effective on lateral root development in a plant, compared to plants or plants harvested from treated with either active (but not both) . In the case of LCOs, for example, the effective amount 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 lO^-11 M, and in some other embodiments from about 10^~6 to about lCT⁹ 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 pg/hundred weight (cwt) seed, and in some embodiments from about 2 to about 70 μg/cwt, and in some other embodiments, from about 2.5 to about 3.0 pg/cwt seed. Amounts of LCOs that may be useful in the practice of the present invention are also described in U.S. Patent 6,979,664; WO 2005/062899; and WO 2008/085958. For purposes of the present invention, the term "about" when used in the context of amounts/concentrations of actives, refers to a difference of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or up to 10% of the measured value.

For purposes of treatment of seed indirectly, i.e., in-furrow treatment, the effective amount of the LCO generally ranges from 1 pg/acre to about 70 pg/acre, and in some embodiments, from about 50 pg/acre to about 60 pg/acre. For purposes of application to the plants, the effective amount of the LCO generally ranges from 1 μg/acre to about 30 μg/acre, and in some embodiments, from about 11 μg/acre to about 20 μg/acre. The optimal effective amounts of LCO may vary from plant species to plant species. Determination of these amounts, as well as effective amounts of other signal molecules, is within the level of ordinary skill in the art and may be determined in accordance with standard techniques.

The amount of auxin, also expressed in terms of concentration, should be "non-saturating", which for purposes of the present invention, refers to concentrations that (as illustrated in the working example hereinbelow) do not have a deleterious (negative) effect on primary root growth or length or the ability of the seed to germinate. As shown in the working examples described below, in the case of M. truncatula, the saturating concentration for NAA is 10^-6 M. In general, effective amounts of the auxin used to treat the seed, expressed in units of concentration, generally range from less than 10^-6 M to about 10^-10 M, and in some embodiments, from about 10^-7 to about 10^-10 M, and in some embodiments from about 10^-6 M to about 10^-s M, and in some other embodiments from about 10^-7 to about ICT^-8 M. The optimal effective amounts of the auxin may vary from plant species to plant species. For example, for purposes of treating corn seed or corn plants, an optimal range may be from about 10^~9 M to about ICT¹⁰ M. Determination of these amounts is within the level of ordinary skill in the art and may be determined in accordance with standard techniques.

Seed may be treated with the actives 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 embodiments 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 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. 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 Agronomxcally 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 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 examples of such agents that may be useful in the practice of the present invention include micronutrients (e.g., vitamins and trace minerals), herbicides, fungicides and insecticides.

Micronutrients

Representative vitamins that may be useful in the practice of the present invention include calcium pantothenate, 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 g to about 9 mg/hundred weight (cwt) seed, and in some embodiments from about 4 μg to about 200 g/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 foliarly.

Herbicides, Fungicides and Insecticides

Suitable herbicides include bentazon, acifluorfen, chlorimuron, lactofen, clomazone, fluazifop, glufosinate, glyphosate, sethoxydim, imazethapyr, imazamox, fomesafe, flumiclorac, imazaquin, and clethodim. Commercial products containing each of these compounds are readily available. Herbicide concentration 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 results in killing or growth inhibition of a fungal population (e.g., in the soil) relative to an untreated population. Effective fungicides in accordance with the invention will suitably exhibit activity against a broad range of pathogens, including but not limited to Phytophthora , Rhizoctonia , Fusarium, Pythium, Phomopsis or Selerotinia 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, Piano, TX) , WARDEN RTA (Agrilance, St. Paul, MN) , APRON XL, APRON MAXX RTA or RFC, MAXIM 4FS or XL (Syngenta, Wilmington, DE), CAPTAN (Arvesta, Guelph, Ontario) and PROTREAT (Nitragin Argentina, Buenos Ares, Argentina) . Active ingredients in these and other commercial fungicides include, but are not limited to, fludioxonil, mefenoxam, azoxystrobin and metalaxyl. Commercial fungicides are most suitably used in accordance with the manufacturer's instructions at the recommended concentrations .

As used herein, an insecticide "exhibits activity against" a 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, soybean rootworm, seed soybean maggots, flea beetles, chinch bugs, aphids, leaf beetles, and stink bugs.

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

The methods of the present invention are applicable to leguminous seed and plants, 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 and plants, e.g., Poaceae, Cucurbitaceae, Malvaceae, Asteraceae, 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.

NON-LIMITING WORKING EXAMPLE

Medicago truncatula seeds (A17 or 2HA genotypes) were germinated and transferred to in vitro culture plates containing M medium (G. Becard and J. A. Fortin. New Phytol. 1988 108:211-218), with no sucrose, phytagel at 4g/l as the gelifyng agent and pH adjusted to 5.8, supplemented with a) 10^-8 M S. meliloti Nod factor (Fig. 1A) and NAA at concentrations of 1CT⁸M to 10^-6M NAA, or b) 10^{- 7} M synthetic non- sulphated LCO (as described in Maillet, F. , et al . , 2011 (Nature, 2011. 469(7328): p. 58-63) and NAA 10^-8M, along with 10^-7 M NS LCO alone, 10^-8 M NAA alone, and as controls in each case, acetonitrile and ethanol . The seedlings were grown on those plates for 10 to 12 days, in a growth chamber at 21°C with a 16h light period. The number of emerged lateral roots (LR) was counted daily. Statistical analysis on the mean number of lateral roots obtained in each condition was analysed on a 4 to 6 day kinetics using a Generalized Linear Model (GLM) statistical test with the Centurion Statgraphics software (p value <0.05). The results, graphically illustrated in Figs. 2 and 3 (corresponding to treatments a) and b) , respectively) , show that these embodiments of the present invention achieved a synergistic effect on lateral root formation. As illustrated in Fig. 4, the combined treatment of the Nod factor and NAA did not affect primary root length more than the NAA treatment by itself .

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 modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A method of enhancing plant growth, comprising treating plant seed or a plant that germinates from the seed with a signal molecule and a phytohormone comprising an auxin or a synthetic analog thereof, wherein the enhanced growth comprises increased lateral root formation in the plant compared to an untreated plant or a plant harvested from untreated seed, and wherein the signal molecule and the auxin or synthetic analog thereof used for the treating are effective to produce a greater than additive effect on lateral root formation in the plant .

2. The method of claim 1, wherein the signal molecule comprises a lipo-chitooligosaccharide (LCO) .

3. The method of claim 2, wherein the LCO is derived from S, meliloti .

4. The method of claim 1, wherein the amount of the LCO used to treat the plant seed or plant is less than 10-' Molar.

5. The method of claim 4, wherein the amount of the LCO is about 10^-8 M.

6. The method of claim 1, wherein the signal molecule is a Myc factor derived from a species of Glomerocycota.

7. The method of claim 1, wherein the species of Glomerocycota is Glomus intraradices .

8. The method of claim 1, wherein the signal molecule is a flavonoid.

9. The method of claim 1, wherein the signal molecule comprises jasmonic acid or a derivative thereof.

10. The method of claim 1, wherein the signal molecule comprises a karrikin or a derivative thereof.

11. The method of any preceding claim, wherein the auxin comprises indole-3-acetic acid (IAA) , 4-chloro-indole acetic acid, indole-3-butyric acid, indole-3-acetamide, 2-phenylacetic acid, or a combination of two or more thereof.

12. The method of any one of claims 1-10, wherein the synthetic analog of auxin comprises 1-naphthalene acetic acid, indole-3-acetate, indole-propionic acid, indole-3-butyric acid, 3-hydroxy-phenyl-indole-3-acetate, or a combination of two or more thereof.

13. The method of claim 1, wherein the amount of the auxin or synthetic analog thereof used for the treating is less than about 10^~6 M to about 10^-10 M.

14. The method of claim 13, wherein the amount of the auxin or synthetic analog thereof used for the treating is about 10^~8 M.

15. The method of claim 1, wherein the plant seed or plant is treated with the signal molecule and the auxin prior to planting or at about the time of planting.

16. The method of claim 1, wherein the plant seed is treated with the signal molecule and the auxin in furrow.

17. The method of claim 1, wherein the plant is treated with the signal molecule and the auxin via foliar treatment.

18. The method of claim 1, further comprising treating the seed or plant with at least one agronomically beneficial agent.

19. The method of claim 18, wherein the agronomically beneficial agent is selected from the group consisting of micronutrients, vitamins, trace minerals, herbicides, insecticides, fungicides, and combinations of two or more thereof .

20. The method of claim 1, wherein the seed or plant is leguminous .

21. The method of claim 20, wherein the leguminous seed or plant is a soybean seed or plant.

22. The method of claim 1, wherein the seed or plant is non-leguminous .

23. The method claim 22, wherein the non-leguminous seed or plant is a field crop seed or plant.

24. The method of claim 22, wherein the non-leguminous seed or plant is a corn seed or plant.

25. The method of claim 22, wherein the non-leguminous seed or plant is a vegetable crop plant or seed.

26. Plant seed coated with a signal molecule and a phytohormone comprising an auxin or a synthetic analog thereof, wherein the signal molecule and the auxin or synthetic analog are coated onto the seed each in amounts that are effective to produce a greater than additive effect on lateral root formation in a plant that germinates from the thus treated seed.

27. A composition for enhancing plant growth, comprising a signal molecule, a phytohormone comprising an auxin or a synthetic analog thereof, and an agronomically acceptable carrier, wherein the signal molecule and the auxin or synthetic analog are present in amounts effective to produce a greater than additive effect on lateral root formation in the plant compared to an untreated plant or a plant harvested from untreated seed.