GROWTH AND YIELD

FIELD OF THE DISCLOSURE

The present disclosure relates to formulations and methods of use of synthetic oligoglucosamines for improving plant growth and crop yield.

BACKGROUND

There is a need for cost-effective synthetically-produced

compounds that improve plant health and result in improved plant growth and crop yield. The present disclosure addresses this need.

SUMMARY OF THE DISCLOSURE

The present disclosure provides compositions and methods for improving plant growth and crop yield. More specifically, the present disclosure relates to agricultural compositions comprising synthetic oligoglucosamines of Structure A. These agricultural compositions may be applied to propagating materials, including seeds and other

regenerate plant parts, including cuttings, bulbs, rhizomes and tubers. They may also be applied to foliage, or soil either prior to or following planting of propagating materials. Such applications may be made alone or in combination with fungicides, insecticides, nematicides and other agricultural agents used to improve plant growth and crop yield.

DETAILED DESCRIPTION

The features and advantages of the present disclosure will be more readily understood, by those of ordinary skill in the art from reading the following detailed description. It is to be appreciated that certain features of the disclosure, which are, for clarity, described above and below in the context of separate embodiments, may also be provided in combination in a single element. Conversely, various features of the disclosure that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination. In addition, references to the singular may also include the plural (for example, "a" and "an" may refer to one or more) unless the context specifically states otherwise.

The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both proceeded by the word "about". In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, the disclosure of these ranges is intended as a continuous range including each and every value between the minimum and maximum values.

The disclosure provides agricultural compositions comprising a substantiall pure compound represented by Structure A;

Structure A wherein n is 0, 1 or 2; XR ¹ is an azide group or X is O or S and R ¹ is hydrogen, a linear or branched, saturated or unsaturated hydrocarbon containing from 1 to 20 carbon atoms, aryl, or substituted aryl; each R ² and R ³ are independently hydrogen, an alkyl group having in the range of from 1 to 20 carbon atoms.

In other embodiments the agricultural composition comprises Product 13 and/or Product 14;

Product 13

Product 14

In other embodiments, any one or more of the agricultural compositions may be used, wherein the above substantially pure compounds of Structure A are present at a concentration in the range of from 10 ^"3 moles/liter (M) to 10 ^"12 M, or present in the composition at a concentration of about 10 ^"7 M.

In other embodiments, the substantially pure compounds of Structure A are present in the agricultural composition in the range of from 10 ^"3 M to 10 ^"4 M.

In other embodiments, the substantially pure compounds of Structure A are present in the agricultural composition in the range of from 10 ^"4 M to 10 ^"5 M.

In other embodiments, the substantially pure compounds of Structure A are present in the agricultural composition in the range of from 10 ^"5 M to 10 ^"6 M.

In other embodiments, the substantially pure compounds of Structure A are present in the agricultural composition in the range of from 10 ^"6 M to 10 ^"7 M.

In other embodiments, the substantially pure compounds of Structure A are present in the agricultural composition in the range of from 10 ^"7 M to 10 ^"8 M.

In other embodiments, the substantially pure compounds of Structure A are present in the agricultural composition in the range of from 10 ^"8 M to 10 ^"9 M.

In other embodiments, the substantially pure compounds of Structure A are present in the agricultural composition in the range of from 10 ^"9 M to 10 ^"10 M. In other embodiments, the substantially pure compounds of

Structure A are present in the agricultural composition in the range of from 10 ^"10 M to 10 ^{"1 1} M.

In other embodiments, the substantially pure compounds of

Structure A are present in the agricultural composition in the range of from 10 ^{"1 1} M to 10 ^"12 M.

In some embodiments, the agricultural composition can further comprise one or more signal molecules.

In other embodiments, the signal molecule can be a

lipochitooligosaccharide.

In other embodiments, the agricultural composition is applied to propagating material of a plant.

In other embodiments, the propagating material is a seed.

In still further embodiments, the propagating material is corn or soybean or a seed potato.

In other embodiments, the agricultural composition is applied to the seed as a seed coating to increase rate of germination, seedling

emergence, radicle growth, early growth, plant height, vigor, plant health, biomass and/or yield.

In other embodiments, the agricultural composition is applied to foliage.

In other embodiments, the agricultural composition is applied to soil either prior to or following planting plant propagating material.

In yet other embodiments, the present disclosure relates to a method for treating a plant, propagating materials, foliage or soil, comprising applying the agricultural composition to the plant, propagating materials, foliage or soil.

In still further embodiments, the method utilizes an agricultural composition comprising one or both of Products 13 and/or 14.

In yet other embodiments, any one or more of the foregoing compositions may be applied to a legume, such as soybean, or to a non- legume, such as corn.

The term "agricultural composition" as used herein comprises one or more substances formulated for at least one agricultural application. Agricultural applications are understood to include, but not be limited to, yield improvement, pest control, disease control and resistance to abiotic environmental stress.

As used herein the term "biologically effective amount" refers to that amount of a substance required to produce the desired effect on a plant, plant propagating material and/or plant part, such as, for example, germination improvement, growth improvement, yield improvement, pest control, disease control and resistance to abiotic environmental stress. Effective amounts of the composition will depend on several factors, including treatment method, plant species, propagating material type and environmental conditions.

Foliage as defined in the present application includes all aerial plant organs, that is, the leaves, stems, flowers and fruit.

As used herein, "percent germination" refers the percentage of seeds that germinate after planting or being placed under conditions otherwise suitable for germination. The term "accelerate the rate of germination" and its equivalents refer to an increase in the percent germination of experimentally treated seeds compared to seeds

designated as experimental controls as a function of time. In the

Examples presented herein, seed germination rates were determined with laboratory-based germination assays conducted under optimum conditions for germination wherein germination percentages were determined at a specified time following initiation of the experiment. General descriptions of seed germination tests can be found in the Handbook of Seed

Technology for Genebanks, Volume I. Principles and Methodology, R.H. Ellis, T.D. Hong and E.H. Roberts, Eds., International Board for Plant Genetic resources, Rome, 1985, pp. 94-120 and the Seed Vigor Testing Handbook, Contribution No. 32 to the Handbook on Seed Testing prepared by the Seed Vigor Test Committee of the Association of Official Seed Analysts, 1983. Examples of seed cold and salt stress germination assays are respectively described in Burris and Navratil, Agronomy Journal, 71 : 985-988 (1979) and Scialabba, et al., Seed Science &

Technology, 27: 865-870 (1999). Plant "growth" as used herein is defined by, but not limited to, measurements of seedling emergence, standability, radicle growth, early growth, plant height, time to flowering, tillering (for grasses), days to maturity, vigor, biomass and yield.

As referred to in the present disclosure and claims, the term

"propagating material" means a seed or regenerable plant part. The term "regenerable plant part" means a part of the plant other than a seed from which a whole plant may be grown or regenerated when the plant part is placed in agricultural or horticultural growing media such as moistened soil, peat moss, sand, vermiculite, perlite, rock wool, fiberglass, coconut husk fiber, tree fern fiber, and the like, or even a completely liquid medium such as water. Regenerable plant parts commonly include rhizomes, tubers (including seed potatoes), bulbs and corms of such geophytic plant species as potato, sweet potato, yam, onion, dahlia, tulip, narcissus, etc. Regenerable plant parts include plant parts that are divided (e.g., cut) to preserve their ability to grow into a new plant. Therefore regenerable plant parts include viable divisions of rhizomes, tubers, bulbs and corms which retain meristematic tissue, such as an eye. Regenerable plant parts can also include other plant parts such as cut or separated stems and leaves from which some species of plants can be grown using horticultural or agricultural growing media. As referred to in the present disclosure and claims, unless otherwise indicated, the term "seed" includes both unsprouted seeds and seeds in which the testa (seed coat) still surrounds part of the emerging shoot and root.

The term "rhizosphere" as defined herein refers to the area of soil that immediately surrounds and is affected by the plant's roots.

As used herein, the term "treating" means applying a biologically effective amount of a substantially pure Structure A compound, or a composition containing a substantially pure Structure A compound, to a seed or other propagating material, foliage or plant rhizosphere; related terms such as "treatment" are defined analogously.

The term "yield" as defined herein refers to the return of crop material per unit area obtained after harvesting a plant crop. An increase in crop yield refers to an increase in crop yield relative to an untreated control treatment. Crop materials include, but are not limited to, seeds, fruits, roots, tubers, leaves and types of crop biomass. Descriptions of field-plot techniques used to evaluate crop yield may be found in W.R. Fehr, Principles of Cultivar Development, McGraw-Hill, Inc., New York, NY, 1987, pp. 261 -286 and references incorporated therein.

"Insect resistant trait" is used herein to refer to a plant containing a toxin that has toxic acitivity against one or more pests, including, but not limited to, members of the Lepidoptera, Diptera, Hemiptera and

Coleoptera orders or the Nematoda phylum or a protein that has homology to such a protein. Pesticidal proteins have been purified from organisms including, for example, Bacillus sp., Pseudomonas sp., Photorhabdus sp., Xenorhabdus sp., Clostridium bifermentans and Paenibacillus popilliae. Pesticidal proteins include but are not limited to: insecticidal proteins from Pseudomonas sp. such as PSEEN3174 (Monalysin; (201 1 ) PLoS

Pathogens 7:1 -13); from Pseudomonas protegens strain CHAO and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386; GenBank Accession No. EU400157); from Pseudomonas Taiwanensis (Liu, et ai, (2010) J. Agric. Food Chem., 58:12343-12349) and from Pseudomonas pseudoalcligenes (Zhang, et al., (2009) Annals of Microbiology 59:45-50 and Li, et ai, (2007) Plant Cell Tiss. Organ Cult. 89:159-168); insecticidal proteins from Photorhabdus sp. and

Xenorhabdus sp. (Hinchliffe, et ai., (2010) The Open Toxicology Journal, 3:101 -1 18 and Morgan, et ai., (2001 ) Applied and Envir. Micro. 67:2062- 2069); US Patent Number 6,048,838, and US Patent Number 6,379,946; a PIP-1 polypeptide of US Patent Publication US20140007292; an AflP-1A and/or AflP-1 B polypeptide of US Patent Publication US20140033361 ; a PHI-4 polypeptide of US patent Publication US20140274885 and PCT Patent Publication WO2014/150914; a PIP-47 polypeptide of PCT Serial Number PCT/US14/51063, a PIP-72 polypeptide of PCT Serial Number PCT/US14/55128, and δ-endotoxins including, but not limited to, the Cry1 , Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry1 1 , Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21 , Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry 28, Cry 29, Cry 30, Cry31 , Cry32, Cry33, Cry34, Cry35,Cry36, Cry37, Cry38, Cry39, Cry40, Cry41 , Cry42, Cry43, Cry44, Cry45, Cry 46, Cry47, Cry49, Cry50, Cry51 , Cry52, Cry53, Cry 54, Cry55, Cry56, Cry57, Cry58, Cry59, Cry60, Cry61 , Cry62, Cry63, Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70, Cry71 , and Cry 72 classes of δ-endotoxin genes and the B. thuringiensis (Bt) cytolytic cytl and cyt2 genes.

In one embodiments, the present disclosure provides an agricultural com osition comprising substantially pure compounds of Structure A;

Structure A,

wherein n is 0, 1 or 2; XR ¹ is an azide group; or X is O or S and R ¹ is hydrogen, a linear or branched, saturated or unsaturated hydrocarbon containing from 1 to 20 carbon atoms, aryl, or substituted aryl; each R ² and R ³ are independently hydrogen or an alkyl group having in the range of from 1 to 20 carbon atoms.

In another embodiment, the substantially pure compound of

Structure A is Product 13

Product 13.

In another embodiments, the substantially pure compound of Structure A is roduct 14;

Product 14. In other embodiments, the agricultural composition comprises the substantially pure compound of Structure A at a concentration of about 10 ^"5 M to 10 ^"12 M.

In still further embodiments, the agricultural composition is applied to propagating material.

In some embodiments, the propagating material is a seed of wheat, durum wheat, barley, oat, rye, maize (corn), sorghum, rice, wild rice, cotton, flax, sunflower, soybean, garden bean, lima bean, broad bean, garden pea, peanut, alfalfa, beet, garden lettuce, rapeseed, cole crop, turnip, leaf mustard, black mustard, tomato, potato, pepper, eggplant, tobacco, cucumber, muskmelon, watermelon, squash, carrot, zinnia, cosmos, chrysanthemum, sweet scabious, snapdragon, gerbera, babys- breath, statice, blazing star, lisianthus, yarrow, marigold, pansy, impatiens, petunia, geranium and coleus.

In other embodiments, the agricultural composition is applied to seed to accelerate the rate of germination, seedling emergence, radicle growth, early growth, plant height, vigor, biomass and/or yield.

In other embodiments, the agricultural composition further comprises one or more insecticides, fungicides, nematocides, bactericides, acaricides, entomopathogenic bacteria, viruses or fungi, plant growth regulators, rooting stimulant, chemosterilant, repellent, attractant, pheromone, feeding stimulant, apocarotenoid, flavonoid, jasmonate or strigolactone.

In some embodiments, the agricultural composition can be applied to foliage.

In other embodiments, the agricultural composition is applied to soil either prior to or following the planting of propagating material.

In other embodiments, the present disclosure relates to a method for treating a plant, propagating materials, foliage or soil, comprising applying the agricultural composition to the plant, propagating materials, foliage or soil.

In some embodiments of the method, the agricultural composition comprises a substantially pure compound having a structure of Product 13;

Product 13.

In other embodiments of the method, the agricultural composition comprises a substantially pure compound having a structure of Product 14

Product 14.

In other embodiments of the method, the agricultural composition is applied as a seed coating.

In still other embodiments of the method, the agricultural composition is applied to foliage.

In other embodiments of the method, the agricultural composition is applied to soil either prior to or following the planting of the propagating material.

In other embodiments of the method, the agricultural composition is applied to a dicot.

In still other embodiments of the method, the agricultural composition is applied to soybean.

In other embodiments of the method, the agricultural composition is applied to a monocot.

In still other embodiments of the method, the agricultural composition is applied to corn.

Compounds having the Structure A, and derivatives thereof, for example, wherein n is greater than or equal to 3 or N-acetylated oligoglucosamines are known and can be produced according to a variety of methods. For example, US Patent Number 7,485,718 describes methods for the production of compounds of Structure A. Another known method is to reduce the molecular weight of chitosan using chemical or enzymatic depolymerization techniques. The depolymerization techniques provides products that have a wide range of molecular weights and numbers of repeat units and require chromatographic separation of the various products to prepare discrete chain lengths. US Patent Number 7,485,718, on the other hand, describes an efficient chain extension coupling of single glucosamine monomers, providing compounds having a defined chain length. This process allows the efficient construction of compounds of Structure A wherein n is a known whole number. In some embodiments, the substantially pure compounds of Structure A are synthetic compounds produced via a step-wise polymerization from a glucosamine monomer or a monomeric glucosamine derivative.

As used herein, the phrases "substantially pure compound of

Structure A" and "substantially pure Structure A compound" mean that the disclosed composition contains less than 10 percent by weight of compounds of Structure A wherein n is greater than or equal to 3, based on the total weight of the compounds of Structure A in the composition. In some embodiments, the disclosed composition contains less than 5 percent by weight of compounds of Structure A wherein n is greater than or equal to 3, and in other embodiments, contains less than 2 percent by weight of compounds of Structure A wherein n is greater than or equal to 3. It should be noted that the phrase "substantially pure" when used to describe compounds of Structure A refers only to those compounds represented by Structure A and having a value of n that is greater than or equal to 0. As one of ordinary skill in the art would know, other reaction impurities (i.e., compounds not having a structure represented by

Structure A) may be present and this phrase does not exclude the possibility of other reaction impurities being present in the disclosed compositions.

Agricultural compositions comprising the substantially pure

compound of Structure A can be applied as a seed treatment formulation, as a seed coating composition, as a foliar formulation, as a sprayable foliar formulation or as a formulation suitable for treating the growing medium. Such formulations typically contain from about 10 ^"3 M to 10 ^"12 M of a substantially pure compound of Structure A. In other embodiments, formulations contain from about 10 ^"6 M to 10 ^"10 M of a substantially pure Structure A compound. The locus of the propagating materials can be treated with a substantially pure Structure A compound by many different methods. All that is needed is for a biologically effective amount of a substantially pure Structure A compound to be applied on or sufficiently close to the propagating material so that it can be absorbed by the propagating material. The substantially pure Structure A compound can be applied by such methods as drenching the growing medium including a propagating material with a solution or dispersion of a substantially pure Structure A compound, mixing a substantially pure Structure A compound with growing medium and planting a propagating material in the treated growing medium (e.g., nursery box treatments), or various forms of propagating material treatments whereby a substantially pure Structure A compound is applied to a propagating material before it is planted in a growing medium. In some embodiments, the agricultural can provide increased plant growth, an increased rate of germination, an increased rate of seedling emergence, an increased rate of radicle growth, an increased rate of early growth, increased pest control, increased disease control, increased plant height, increased vigor, increased resistance to abiotic environmental stress, and increased biomass and/or yield when compared to an untreated control.

In some embodiments, the substantially pure compound of Structure A can be used as a seed treatment formulation with an agriculturally suitable carrier comprising at least one of a liquid diluent, a solid diluent or a surfactant. A wide variety of formulations are suitable, the most suitable types of formulations depend upon the method of application. As is well known to those skilled in the art, the purpose of formulation is to provide a safe and convenient means of transporting, measuring and dispensing the agricultural agent and also to optimize its efficacy.

Depending on the method of application useful formulations include liquids such as solutions (including emulsifiable concentrates),

suspensions, emulsions (including microemulsions and/or suspoemulsions) and the like which optionally can be thickened into gels. Useful formulations further include solids such as dusts, powders, granules, pellets, tablets, films, and the like which can be water-dispersible ("wettable") or water-soluble. The substantially pure compound of

Structure A can be (micro)encapsulated and further formed into a suspension or solid formulation; alternatively the entire formulation comprising the substantially pure compound of Structure A can be encapsulated (or "overcoated"). Encapsulation can control or delay release of the substantially pure compound of Structure A. Sprayable formulations can be extended in suitable media and used at spray volumes from about one to several hundred liters per hectare.

High-strength compositions are primarily used as intermediates for further formulation.

The formulations will typically contain effective amounts of the substantially pure compound of Structure A, diluent and surfactant within the following approximate ranges that add up to 100 percent by weight.

Weight Percent

Compound

of Structure Diluent Surfactant A

Water-Dispersible and 5-90 0-94 1 -15 Water-soluble Granules,

Tablets and Powders.

Suspensions, Emulsions, 5-50 40-95 0-15 Solutions (including

Emulsifiable Concentrates)

Dusts 1 -25 70-99 0-5

Granules and Pellets 0.01 -99 5-99.99 0-15

High Strength Compositions 90-99 0-10 0-2

Typical solid diluents are described in Watkins et al., Handbook of Insecticide Dust Diluents and Carriers, 2nd Ed., Dorland Books, Caldwell, New Jersey. Typical liquid diluents are described in Marsden, Solvents Guide, 2nd Ed., Interscience, New York, 1950. McCutcheon's Emulsifiers and Detergents and McCutcheon's Functional Materials (North America and International Editions, 2001 ), The Manufacturing Confection Publ. Co., Glen Rock, New Jersey, as well as Sisely and Wood, Encyclopedia of Surface Active Agents, Chemical Publ. Co., Inc., New York, 1964, list surfactants and recommended uses. All formulations can contain minor amounts of additives to reduce foam, caking, corrosion, microbiological growth and the like, or thickeners to increase viscosity.

Surfactants include, for example, ethoxylated alcohols, ethoxylated alkylphenols, ethoxylated sorbitan fatty acid esters, ethoxylated amines, ethoxylated fatty acids, esters and oils, dialkyl sulfosuccinates, alkyl sulfates, alkylaryl sulfonates, organosilicones, Λ/,/V-dialkyltaurates, glycol esters, phosphate esters, lignin sulfonates, naphthalene sulfonate formaldehyde condensates, polycarboxylates, and block polymers including polyoxyethylene/polyoxypropylene block copolymers.

Solid diluents can include, for example, clays such as bentonite, montmorillonite, attapulgite and kaolin, starch, sugar, silica, talc, diatomaceous earth, urea, calcium carbonate, sodium carbonate and bicarbonate, and sodium sulfate. Liquid diluents can include, for example, water, or an organic diluent, for example, Λ/,/V-dimethylformamide, dimethyl sulfoxide, ethyl acetate, diethyl ether, formamide, 2-pyrrolidone, N-methylpyrrolidone, /V-alkylpyrrolidone, ethylene glycol, polypropylene glycol, 1 ,3-propane diol, 1 ,3-propane diol polyethers, alkyl and dialkyl ethers of 1 ,3-propane diol, alkyl and dialkyl ethers of 1 ,3-propane diol polyethers, diethylene glycol, diethylene glycol ethers, dipropylene glycol ethers, diglyme, hexamethylene glycol, pentamethylene glycol,

polyethylene glycol, poly hydroxy I ated alkanes, propylene glycol ethers, tetramethylene glycol, tetramethylene glycol ethers, triethylene glycol, triethylene glycol ethers, tripropylene glycol, tripropylene glycol ethers, 1 ,3- butylene glycol, 1 ,3-butylene glycol ethers, butylene carbonate, glycerol, thiodiglycol, propylene carbonate, dibasic esters, paraffins, alkylbenzenes, alkylnaphthalenes, oils of olive, castor, linseed, tung, sesame, corn, peanut, cotton-seed, soybean, rape-seed and coconut, fatty acid esters, ketones such as, for example, acetone, gamma-butyrolactone, methyl ethyl ketone, cyclohexanone, 2-heptanone, isophorone and 4-hydroxy-4- methyl-2-pentanone, and alcohols such as, for example, C1 to C12 aliphatic alcohols, diacetone alcohol, ethanol, furfuryl alcohol,

tetrahydrofurfuryl alcohol polyethylene glycol ether, isopropanol, propanol, methanol, cyclohexanol, decanol, benzyl and tetrahydrofurfuryl alcohol, phosphoric acid esters, sulfolane, tetrahydrofuran or a combination thereof.

In some embodiments, the agricultural composition can comprise in the range of 80 to 100 percent by weight of water, based on the total weight of the liquid diluent. In other embodiments, the liquid diluent can comprise in the range of from 90 to 100 percent water, and, in still further embodiments, in the range of from 95 to 100 percent water, wherein the percentages by weight are based on the total amount of the liquid diluent. The remaining amount of liquid diluent can be one or more of the organic diluents listed above.

Solutions, including emulsifiable concentrates, can be prepared by simply mixing the ingredients. Dusts and powders can be prepared by blending and, usually, grinding as in a hammer mill or fluid-energy mill. Suspensions are usually prepared by wet-milling; see, for example, U.S. 3,060,084. Granules and pellets can be prepared by spraying the active material upon preformed granular carriers or by agglomeration techniques. See Browning, "Agglomeration", Chemical Engineering, December 4, 1967, pp. 147-48, Perry's Chemical Engineer's Handbook, 4th Ed., McGraw-Hill, New York, 1963, pp. 8-57 and following, and PCT

Publication WO 91/13546. Pellets can be prepared as described in U.S. 4,172,714. Water-dispersible and water-soluble granules can be prepared as taught in U.S. 4,144,050, U.S. 3,920,442 and DE 3,246,493. Tablets can be prepared as taught in U.S. 5,180,587, U.S. 5,232,701 and U.S. 5,208,030. Films can be prepared as taught in GB 2,095,558 and U.S. 3,299,566.

For further information regarding the art of formulation, see

T. S. Woods, "The Formulator's Toolbox - Product Forms for Modern Agriculture" in Pesticide Chemistry and Bioscience, The Food- Environment Challenge, T. Brooks and T. R. Roberts, Eds., Proceedings of the 9th International Congress on Pesticide Chemistry, The Royal Society of Chemistry, Cambridge, 1999, pp. 120-133. See also U.S. 3,235,361 , Col. 6, line 16 through Col. 7, line 19 and Examples 10- 41 ; U.S. 3,309,192, Col. 5, line 43 through Col. 7, line 62 and Examples 8, 12, 15, 39, 41 , 52, 53, 58, 132, 138-140, 162-164, 166, 167 and 169-182; U.S. 2,891 ,855, Col. 3, line 66 through Col. 5, line 17 and Examples 1 -4; Klingman, Weed Control as a Science, John Wiley and Sons, Inc., New York, 1961 , pp. 81 -96; and Hance et al., Weed Control Handbook, 8th Ed., Blackwell Scientific Publications, Oxford, 1989.

In order to inhibit or prevent microbial growth, one or more biocides can be added. Suitable biocides can include, for example, 5-chloro-2- methyl-3(2H)-isothiazolone , o-phenylphenol, sodium-o-phenylphenate, cis-1 -(chloroallyl)-3,5,7-triaza-1 -azoniaadamantane chloride, 7-ethyl bicyclooxazolidine, 2,2-dibromo-3-nitrilopropionamide, bronopol, glutaraldehyde, copper hydroxide, cresol, dichlorophen, dipyrithione, fenaminosulf, formaldehyde, hydrargaphen, 8-hydroxyquinoline sulfate, kasugamycin, nitrapyrin, octhilinone, oxolinic acid, oxytetracycline, probenazole, streptomycin, tecloftalam, thimerosal, polyquaternary ammonium chloride, alkylbenzyl dimethyl ammonium chloride, 2-methyl-4- isothiazolone, 2-ethyl-4-isothiazolin-3-one, 2-propyl-4-isothiazolin-3-one, 2-butyl-4-isothiazolin-3-one, 2-amyl-4-isothiazolin-3-one, 5-chloro-2- methyl-4-isothiazolin-3-one, 5-bromo-2-methyl-4-isothiazolin-3-one, 5- iodo-2-methyl-4-isothiazolin-3-one, 5-chloro-2-butyl-4-isothiazolin-3-one, 5-bromo-2-ethyl-4-isothiazolin-3-one, 5-iodo-2-amyl-4-isothiazolin-3-one, 2-n-octyl-4-isothiazolin-3-one, 4,5-dichloro-2-n-octyl-4-isothazolin-3-one, 1 ,2-benzisothiazolin-3-one or a combination thereof.

The agricultural compositions used for treating propagating materials, or plants grown therefrom, according to this disclosure can also comprise (besides the substantially pure Structure A compound) an effective amount of one or more other biologically active compounds or agents. Suitable biologically active compounds or agents include, but are not limited to, insecticides, fungicides, nematocides, bactericides, acaricides, entomopathogenic bacteria, viruses or fungi, plant growth regulators such as rooting stimulants, chemosterilants, repellents, attractants, pheromones, feeding stimulants and other signal compounds including, but not limited to, apocarotenoids, flavonoids, jasmonates and strigolactones (Akiyama, et al., in Nature, 435:824-827 (2005); Harrison, in Ann. Rev. Microbiol., 59:19-42 (2005); Besserer, et al., in PLoS Biol., 4(7):e226 (2006); WO2009049747). Polymeric polyhydroxy acids, such as, ARCUS™ ST, available from FBSciences, Collierville, Tennessee can also be added. Biologically active agents can also comprise

microorganisms that stimulate plant growth. Such microorganisms include, but are not limited to, biologically active species within the bacterial genera Azorhizobium, Bacillus, Bradyrhizobium, Mesorhizobium, Paenibacillus and Rhizobium (Khan, et al., in Bioresource Technology, 99(8): 3016-3023 (2008); Plant Growth and Health Promoting Bacteria (Microbiology

Monographs), D. K. Maheshwari, Ed., Springer-Verlag, Berlin, 2010. Such microorganisms also include, but are not limited to, plant growth promoting species within the fungal genera Cladosporum, Corvularia, Fusarium, Gliocladium, Metarhizium, Penicilliunn and Trichoderma (Kim, et al. in BMC Microbiology, 8:231 (2008); Khan, et al., in World Journal of Microbiology and Biotechnology, 28(4): 1483-1494 (2012), Biotechnology of Microbes and Sustainable Utilization, R. C. Rajak, Ed., Scientific Publishers,

Jodhpur, India, 2002, pp. 1 16-120. These agents can be formulated into the agricultural composition giving an even broader spectrum of

agricultural utility than can be achieved with the substantially pure

Structure A compound alone.

Examples of such biologically active compounds or agents with which compounds can be formulated are: insecticides such as abamectin, acephate, acetamiprid, amidoflumet (S-1955), avermectin, azadirachtin, azinphos-methyl, bifenthrin, binfenazate, buprofezin, carbofuran, chlorantraniliprole, chlorfenapyr, chlorfluazuron, chlorpyrifos, chlorpyrifos- methyl, chromafenozide, clothianidin, cyantraniliprole, cyfluthrin, beta-cyfluthrin, cyhalothrin, lambda-cyhalothrin, cypermethrin, cyromazine, deltamethrin, diafenthiuron, diazinon, diflubenzuron, dimethoate, diofenolan, emamectin, endosulfan, esfenvalerate, ethiprole, fenothicarb, fenoxycarb, fenpropathrin, fenproximate, fenvalerate, fipronil, flonicamid, flucythrinate, flupyradifurone, tau-fluvalinate, flufenerim (UR-50701 ), flufenoxuron, fonophos, halofenozide, hexaflumuron, imidacloprid, indoxacarb, isofenphos, lufenuron, malathion, metaldehyde,

methamidophos, methidathion, methomyl, methoprene, methoxychlor, monocrotophos, methoxyfenozide, nithiazin, novaluron, noviflumuron (XDE-007), oxamyl, parathion, parath ion-methyl, permethrin, phorate, phosalone, phosmet, phosphamidon, pirimicarb, profenofos, pymetrozine, pyridalyl, pyriproxyfen, rotenone, spinosad, spiromesifin (BSN 2060), sulprofos, tebufenozide, teflubenzuron, tefluthrin, terbufos,

tetrachlorvinphos, thiacloprid, thiamethoxam, thiodicarb, thiosultap- sodium, tralomethrin, trichlorfon and triflumuron; fungicides such as acibenzolar, azoxystrobin, benomyl, blasticidin-S, Bordeaux mixture (tribasic copper sulfate), bromuconazole, carpropamid, captafol, captan, carbendazim, chloroneb, chlorothalonil, copper oxychloride, copper salts, cyflufenamid, cymoxanil, cyproconazole, cyprodinil, (S)-3,5-dichloro-/V-(3- chloro-1 -ethyl-1 -methyl-2-oxopropyl)-4-methylbenzamide (RH 7281 ), diclocymet (S-2900), diclomezine, dicloran, difenoconazole, (S)-3,5- dihydro-5-methyl-2-(methylthio)-5-phenyl-3-(phenylamino)-4/- /-imidazol-4- one (RP 407213), dimethomorph, dimoxystrobin, diniconazole,

diniconazole-M, dodine, edifenphos, epoxiconazole, famoxadone, fenamidone, fenarimol, fenbuconazole, fencaramid (SZX0722), fenpidonil, fenpropidin, fenpropimorph, fentin acetate, fentin hydroxide, fluazinam, fludioxonil, flumetover (RPA 403397), flumorf/flumorlin (SYP-L190), fluoxastrobin (HEC 5725), fluquinconazole, flusilazole, flutolanil, fluopyram, flutriafol, folpet, fosetyl-aluminum, furalaxyl, furametapyr (S-82658), hexaconazole, ipconazole, iprobenfos, iprodione,

isoprothiolane, kasugamycin, kresoxim-methyl, mancozeb, maneb, mefenoxam, mepronil, metalaxyl, metconazole,

metominostrobin/fenominostrobin (SSF-126), metrafenone (AC 375839), myclobutanil, neo-asozin (ferric methanearsonate), nicobifen (BAS 510), orysastrobin, oxadixyl, oxathiapiprolin, penconazole, pencycuron, penflufen, penthiopyrad, picoxystrobin, probenazole, prochloraz, propamocarb, propiconazole, proquinazid, prothioconazole (JAU 6476), pyrifenox, pyraclostrobin, pyrimethanil, pyroquilon, quinoxyfen, sedaxane, spiroxamine, sulfur, tebuconazole, tetraconazole, thiabendazole, thifluzamide, thiophanate-methyl, thiram, tiadinil, triadimefon, triadimenol, tricyclazole, trifloxystrobin, triticonazole, validamycin and vinclozolin;

nematocides such as aldicarb, oxamyl and fenamiphos; bactericides such as streptomycin; acaricides such as amitraz, chinomethionat,

chlorobenzilate, cyhexatin, dicofol, dienochlor, etoxazole, fenazaquin, fenbutatin oxide, fenpropathrin, fenpyroximate, hexythiazox, propargite, pyridaben and tebufenpyrad; and biological agents including Bacillus thuringiensis (including ssp. aizawai and kurstaki), Bacillus thuringiensis delta-endotoxin, Bacillus firmus 1-1582, Bacillus simplex, Pasteuria nishizawae, baculoviruses, and entomopathogenic bacteria, viruses, as well as naturally occurring and genetically modified viral insecticides including members of the family Baculoviridae and fungi, including entomophagous fungi. A general reference for these agricultural protectants is The Pesticide Manual, 12th Edition, C. D. S. Tomlin, Ed., British Crop Protection Council, Farnham, Surrey, U.K., 2000.

Combinations of any of the above mentioned can also be used.

Two particular Bacillus strains that can be used are Bacillus amyloliquifaciens 22CP1 (ATCC PTA-6508) and Bacillus amyloliquifaciens 15AP4 (ATCC PTA-6507). On January 12, 2005, Bacillus

amyloliquifaciens 22CP1 was deposited at the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Virginia 201 10- 2209 and given accession number PTA-6508. The deposits were made under the provisions of the Budapest Treaty on the International

Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Also on January 12, 2005, Bacillus amyloliquifaciens 15AP4 was deposited at the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Virginia 201 10-2209 and given accession numbers PTA-6507. The deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

In some embodiments, the agricultural composition can comprise the substantially pure compounds of Structure A and Bacillus

amyloliquifaciens 22CP1 . In other embodiments, the agricultural composition can comprise at least one of the substantially pure

compounds of Structure A and Bacillus amyloliquifaciens 15AP4. The anthranilamide insecticides, which includes chlorantraniliprole and cyantraniliprole, comprises a large class of compounds having insecticidal activity. The agricultural composition can further comprise and one of the compounds of Structure 1 , including N-oxides or salts therefrom;

1

wherein

X is N, CF, CCI, CBr or CI;

R ⁷ is CH ₃ , CI, Br or F;

R ⁸ is H, F, CI, Br or -CN;

R ⁹ is F, CI, Br, C1 to C4 haloalkyl, C1 to C4 haloalkoxy or Q;

R ¹⁰ is NR ¹³ R ¹⁴ , N=S(CH ₃ ) ₂ , N=S(CH ₂ CH ₃ ) ₂ , N=S(CH(CH ₃ ) ₂ )2;

R ¹¹ is H, F, CI or Br;

R ¹² is H, F, CI or Br;

each R ¹³ and R ¹⁴ is independently H, C1 to C6 alkyl, C3 to C6 cycloalkyl, cyclopropyl methyl or 1 -cyclopropylethyl; and

Q is a -CH ₂ -tetrazole radical. Suitable embodiments for Q can include any structure having a formula according to Q-1 to Q-1 1 ;

In other embodiments, the agricultural composition can further comprise any of the known anthranilic diamide insecticides, for example, those described in US 6,747,047, US 8,324,390, US 2010/0048640, WO 2007/006670, WO 2013/024009, WO 2013/024010,

WO 2013/024004, WO 2013/024170 or WO 2013/024003. Specific embodiments from US 8,324,390 can include any of those compounds disclosed as examples 1 through 544. Specific embodiments from US 2010/0048640 can include any of those compounds disclosed in Tables 1 through 68 or compounds represented by Chemical Formula 44 through 1 18. Each of the references to the above patents and

applications are hereby incorporated by reference. In some embodiments, the plant growth regulators for mixing with the substantially pure Structure A compounds used in compositions for treating stem cuttings are 1 H-indole-3-acetic acid, 1 H-indole-3-butanoic acid and 1 -naphthaleneacetic acid and their agriculturally suitable salt, ester and amide derivatives, such as 1 -napthaleneacetamide. Preferred fungicides for mixing with the substantially pure Structure A compounds include fungicides useful as seed treatments such as thiram, maneb, mancozeb and captan.

In some embodiments, microorganisms can be added to the agricultural composition. Suitable examples of microorganisms, can include, for example, 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 one embodiment, the phosphate solubilizing microorganism is a spore forming microorganism.

In some embodiments, the phosphate solubilizing microorganisms can include, for example, species from a genus selected from the group consisting of 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.

In further embodiments, the phosphate solubilizing microorganisms can include, for example, Acinetobacter calcoaceticus, Acinetobacter sp, Arthrobacter sp., Arthrobotrys oligospora, Aspergillus niger, Aspergillus sp., Azospirillum halopraeferans, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans,Bacillus licheniformis, Bacillus subtilis, Burkholderia cepacia, Burkholderia vietnamiensis, Candida krissii,

Chryseomonas luteola, Enterobacter aerogenes, Enterobacter asburiae, Enterobacter sp., Enterobacter taylorae, Eupenicillium parvum,

Exiguobacterium sp., Klebsiella sp., Kluyvera cryocrescens, Microbacterium sp., Mucor ramosissimus, Paecilomyces hepialid,

Paecilomyces marquandii, Paenibacillus macerans, Paenibacillus mucilaginosus, Pantoea aglomerans, Penicillium expansum,

Pseudomonas corrugate, Pseudomonas fluorescens, Pseudomonas lutea, Pseudomonas poae, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas trivialis, Serratia marcescens, Stenotrophomonas maltophilia, Streptomyces sp., Streptosporangium sp., Swaminathania salitolerans, Thiobacillus ferrooxidans, Torulospora globosa, Vibrio proteolytics, 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 useful in the practice of the present disclosure include P. bilaiae (formerly known as P. bilaii), P. albidum, P.

aurantiogriseum, P. chrysogenum, P. citreonigrum, P. citrinum, P.

digitatum, P. frequentas, P. fuscum, P. gaestrivorus, P. glabrum, P.

griseofulvum, P. implicatum, P. janthinellum, P. Iilacinum, P. minioluteum, P. montanense, P. nigricans, P. oxalicum, P. pinetorum, P. pinophilum, P. purpurogenum, P. radicans, P. radicum, P. raistrickii, P. rugulosum, P. simplicissimum, P. solitum, P. variabile, P. velutinum, P. viridicatum, P. glaucum, P. fussiporus, and P. expansum.

In another particular embodiment, the Penicillium species is P.

bilaiae. In still another particular embodiment the P. bilaiae strains are selected from the group consisting of American Type Culture Collection (ATCC) ATCC 20851 , Northern Regional Research Laboratory (NRRL) NRRL 50169, ATCC 22348, ATCC 18309, NRRL 50162 (Wakelin, et al., 2004. Biol Fertil Soils 40:36-43). In another particular embodiment the Penicillium species is P. gaestrivorus, e.g., NRRL 50170 (see, Wakelin, supra.).

In some embodiments, more than one phosphate solubilizing microorganism is used, for example, at least two, at least three, at least four, at least five, at least 6, including, for example, any combination of the Acinetobacter, Arthrobacter, Arthrobotrys, Aspergillus, Azospirillum, Bacillus, Burkholderia, Candida Chryseomonas, Enterobacter,

Eupenicillium, Exiguobacterium, Klebsiella, Kluyvera, Microbacterium, Mucor, Paecilomyces, Paenibacillus, Penicillium, Pseudomonas, Serratia, Stenotrophomonas, Streptomyces, Streptosporangium, Swaminathania, Thiobacillus, Torulospora, Vibrio, Xanthobacter, and Xanthomonas, including one species selected from the following group: Acinetobacter calcoaceticus, Acinetobacter sp, Arthrobacter sp., Arthrobotrys oligospora, Aspergillus niger, Aspergillus sp., Azospirillum halopraeferans, Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans, Bacillus licheniformis, Bacillus subtilis, Burkholderia cepacia, Burkholderia vietnamiensis, Candida krissii, Chryseomonas luteola, Enterobacter aerogenes, Enterobacter asburiae, Enterobacter sp., Enterobacter taylorae, Eupenicillium parvum, Exiguobacterium sp., Klebsiella sp., Kluyvera cryocrescens, Microbacterium sp., Mucor ramosissimus,

Paecilomyces hepialid, Paecilomyces marquandii, Paenibacillus macerans, Paenibacillus mucilaginosus, Pantoea aglomerans, Penicillium expansum, Pseudomonas corrugate, Pseudomonas fluorescens,

Pseudomonas lutea, Pseudomonas poae, Pseudomonas putida,

Pseudomonas stutzeri, Pseudomonas trivialis, Serratia marcescens, Stenotrophomonas maltophilia, Streptomyces sp., Streptosporangium sp., Swaminathania salitolerans, Thiobacillus ferrooxidans, Torulospora globosa, Vibrio proteolytics, Xanthobacter agilis, and Xanthomonas campestris.

In some embodiments, two different strains of the same species 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 already containing insoluble (or sparingly soluble) phosphates, the use of the combined fungal strains will result in an increase in the amount of phosphorus available for plant uptake compared to the use of only one 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 strains are NRRL 50169 and NRRL 50162. In further embodiments, the at least two strains are NRRL 50169 and NRRL 50170. In yet further embodiments, the at least two strains are NRRL 50162 and NRRL 50170.

The phosphate solubilizing microorganisms may be prepared using any suitable method known to the person skilled in the art, such as, solid state or liquid fermentation using a suitable carbon source. These culture methods may be used in the preparation of an inoculum of Penicillium spp. for treating seeds and/or application to an agrononnically acceptable carrier to be applied to soil. The term "inoculum" as used in this specification is intended to mean any form of phosphate solubilizing microorganism, fungus cells, mycelium or spores, bacterial cells or bacterial spores, which is capable of propagating on or in the soil, including on or in the vicinity of plant roots when the conditions of temperature, moisture, etc., are favorable for fungal growth. The phosphate solubilizing microorganism is preferably prepared in the form of a stable spore.

Solid state production of Penicillium spores may be achieved by inoculating a solid medium such as a peat or vermiculite-based substrate, seeds or grains including, but not limited to, corn, soy, potato, oats, wheat, barley, or rice. The sterilized medium (achieved through autoclaving or irradiation) is inoculated with a spore suspension comprising in the range of from 1 x10 ² -1 x10 ⁷ colony forming units per milliliter (cfu/ml) of the appropriate Penicillium spp. and the moisture adjusted to 20 to 50%, depending on the substrate. The inoculated medium is incubated for 2 to 8 weeks at room temperature. The spores may also be produced by liquid fermentation (Cunningham et ai, 1990. Can J Bot. 68:2270-2274). Liquid production may be achieved by cultivating the fungus in any suitable media, such as potato dextrose 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 drying, or fluid bed drying techniques (Friesen, et a/., 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 treatments, such as, but not limited to, chemical seed treatments, carriers (for example, talc, clay, kaolin, silica gel, kaolinite) or polymers (for example, methylcellulose, polyvinylpyrrolidone). Alternatively, a spore suspension of the appropriate Penicillium spp. may be applied to a suitable soil-compatible carrier (for example, peat-based powder or granule) to appropriate final moisture content. The material may be incubated at room temperature, typically for about 1 day to about 8 weeks, prior to use.

The amount of the at least one phosphate solubilizing microorganism varies depending on the type of seed or soil, the type of plant material, 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 experiments for each particular case. Normally, for Penicillium, for example, the application amount falls into the range of from 0.001 to 1 .0 Kg fungal spores and mycelium (fresh weight) per hectare, or 10 ² -10 ⁶ colony forming units (cfu) per seed (when coated seeds are used), or on a granular carrier applying between 1 x10 ⁶ and 1 x10 ¹¹ 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.

Diazotrophs are bacteria and archaea that fix atmospheric nitrogen gas into a more usable form such as ammonia. Examples of diazotrophs include bacteria from the genera Rhizobium spp. (e.g., R. cellulosilyticum, R. daejeonense, R. etli, R. galegae, R. gallicum, R. giardinii, R.

hainanense, R. huautlense, R. indigoferae, R. leguminosarum, R.

loessense, R. lupini, R. lusitanum, R. meliloti, R. mongolense, R.

miluonense, R. sullae, R. tropici, R. undicola, and/or R. yanglingense), Bradyrhizobium spp. (e.g., B. bete, B. canariense, B. elkanii, B.

iriomotense, B. japonicum, B. jicamae, B. liaoningense, B. pachyrhizi, and/or B. yuanmingense), Azorhizobium spp. (e.g., A. caulinodans and/or A. doebereinerae), Sinorhizobium spp. (e.g., S. abri, S. adhaerens, S. americanum, S. aborts, S. fredii, S. indiaense, S. kostiense, S. kummerowiae, S. medicae, S. meliloti, S. mexicanus, S. morelense, S. saheli, S. terangae, and/or S. xinjiangense), Mesorhizobium spp., (M. albiziae, M. amorphae, M. chacoense, M. ciceri, M. huakuii, M. loti, M. mediterraneum, M. pluifarium, M. septentrionale, M. temperatum, and/or M. tianshanense), and combinations thereof. In a particular embodiment, the diazotroph is selected from the group consisting of B. japonicum, R leguminosarum, R meliloti, S. meliloti, and combinations thereof. In another embodiment, the diazotroph is B. japonicum. In another embodiment, the diazotroph is R leguminosarum. In another embodiment, the diazotroph is R meliloti. In another embodiment, the diazotroph is S. meliloti.

Mycorrhizal fungi form symbiotic associations with the roots of a vascular plant, and provide, e.g., absorptive capacity for water and mineral nutrients due to the comparatively large surface 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 embodiment, the endomycorrhizae is a strain of Glomus aggregatum, Glomus brasilianum, Glomus clarum, Glomus deserticola, Glomus etunicatum, Glomus fasciculatum, Glomus intraradices, Glomus monosporum, or Glomus mosseae, Gigaspora margarita, or a combination thereof.

Examples of mycorrhizal fungi include ectomycorrhizae of the phylum Basidiomycota, Ascomycota, and Zygomycota. Other examples include a strain of Laccaria bicolor, Laccaria laccata, Pisolithus tinctorius,

Rhizopogon amylopogon, Rhizopogon fulvigleba, Rhizopogon luteolus, Rhizopogon villosuli, Scleroderma cepa, Scleroderma citrinum, or a combination thereof.

The mycorrhizal fungi include ecroid mycorrhizae, arbutoid

mycorrhizae, or monotropoid mycorrhizae. Arbuscular and

ectomycorrhizae form ericoid mycorrhiza with many plants belonging to the order Ericales, while some Ericales form arbutoid and monotropoid mycorrhizae. In one embodiment, the mycorrhiza may be an ericoid mycorrhiza, preferably of the phylum Ascomycota, such as

Hymenoscyphous ericae or Oidiodendron sp. In another embodiment, the mycorrhiza also may be an arbutoid mycorrhiza, preferably of the phylum 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 mycorrhiza, preferably of the genus Rhizoctonia.

In some embodiments, the agricultural compositions can comprise combinations of any of the above listed components. For example, the agricultural composition can comprise a combination of two different insecticides, a fungicide and any one or more of the above listed bacterial or fungal strains. In other embodiments, the agricultural composition can comprise the substantially pure compounds of Structure A and

chlorantraniliprole, cyantraniliprole or a combination of chlorantraniliprole and cyantraniliprole and one or more of the ingredients in Table 1 of rows 1 , 2 or 3.

TABLE 1

The agricultural composition can be applied by such methods as drenching the growing medium including a propagating material with a solution or dispersion of the agricultural composition, mixing the agricultural composition with growing medium and planting a propagating material in the treated growing medium (e.g., nursery box treatments), or various methods of propagating material treatment whereby the agricultural composition is applied to a propagating material before it is planted in a growing medium.

For growing-medium drenches, the formulation needs to provide the substantially pure Structure A compound, generally after dilution with water, in solution or as particles small enough to remain dispersed in the liquid. Water-dispersible or soluble powders, granules, tablets, emulsifiable concentrates, aqueous suspension concentrates and the like are formulations suitable for aqueous drenches of growing media.

Drenches are most satisfactory for treating growing media that have relatively high porosity, such as light soils or artificial growing medium comprising porous materials such as peat moss, perlite, vermiculite and the like. The drench liquid comprising the substantially pure Structure A compound can also be added to a liquid growing medium (i.e.

hydroponics), which causes the substantially pure Structure A compound to become part of the liquid growing medium. One skilled the art will appreciate that the amount of substantially pure Structure A compound needed in the drench liquid for efficacy (i.e. biologically effective amount) will vary with several factors including, but not limited to, plant species, propagating material type and environmental conditions. The

concentration of substantially pure Structure A compound in the drench liquid is generally between about 10 ^"3 M to 10 ^"12 M of the composition, more typically between about 10 ^"6 M to 10 ^"10 M. One skilled in the art can easily determine the biologically effective concentration necessary for the desired level of efficacy.

For treating a growing medium a substantially pure Structure A compound can also be applied by mixing it as a dry powder or granule formulation with the growing medium. Because this method of application does not require first dispersing or dissolving in water, the dry powder or granule formulations need not be highly dispersible or soluble. While in a nursery box the entire body of growing medium may be treated, in an agricultural field only the soil in the vicinity of the propagating material is typically treated for environmental and cost reasons. To minimize application effort and expense, a formulation comprising the substantially pure Structure A compound is most efficiently applied concurrently with propagating material planting (e.g., seeding). For in-furrow application, the substantially pure Structure A formulation (most conveniently a granule formulation) is applied directly behind the planter shoe. For T-band application, the substantially pure Structure A formulation is applied in a band over the row behind the planter shoe and behind or usually in front of the press wheel. One skilled the art will appreciate that the amount of substantially pure Structure A compound needed in the growing medium locus for efficacy (i.e. biologically effective amount) will vary with several factors including, but not limited to, plant species, propagating material type and environmental conditions. The concentration of substantially pure Structure A compound in the growing medium locus is generally between about 10 ^"3 M to 10 ^"12 M of the composition, more typically between about 10 ^"6 M to 10 ^"10 M. One skilled in the art can easily determine the biologically effective amount necessary for the desired level efficacy.

A propagating material can be directly treated by soaking it in a solution or dispersion of the substantially pure Structure A compound. Although this application method is useful for propagating materials of all types, treatment of large seeds (e.g., having a mean diameter of at least 3 mm) is more effective than treatment of small seeds for providing efficacy. Treatment of propagating materials such as tubers, bulbs, corms, rhizomes and stem and leaf cuttings can also provide effective treatment of the developing plant in addition to the propagating material. The formulations useful for growing-medium drenches are generally also useful for soaking treatments. The soaking medium comprises a nonphytotoxic liquid, generally water-based although it may contain nonphytotoxic amounts of other solvents such as methanol, ethanol, isopropanol, ethylene glycol, propylene glycol, propylene carbonate, benzyl alcohol, dibasic esters, acetone, methyl acetate, ethyl acetate, cyclohexanone, dimethylsulfoxide and /V-methylpyrrolidone, which may be useful for enhancing solubility of the substantially pure Structure A compound and penetration into the propagating material. A surfactant can facilitate wetting of the propagating material and penetration of the substantially pure Structure A compound. One skilled the art will appreciate that the amount of substantially pure Structure A compound needed in the soaking medium for efficacy (i.e. biologically effective amount) will vary with several factors including, but not limited to, plant species, propagating material type and environmental conditions. The concentration of substantially pure Structure A compound in the soaking liquid is generally between about 10 ^"5 M to10 ^"12 M of the composition, more typically between about 10 ^"6 M to 10 ^"10 M. One skilled in the art can easily determine the biologically effective concentration necessary for the desired level of efficacy. The soaking time can vary from one minute to one day or even longer. Indeed, the propagating material can remain in the treatment liquid while it is germinating or sprouting (e.g., sprouting of rice seeds prior to direct seeding). As shoot and root emerge through the testa (seed coat), the shoot and root directly contact the solution comprising the substantially pure Structure A compound. For treatment of sprouting seeds of large-seeded crops such as rice, treatment times of about 8 to 48 hours, e.g., about 24 hours, is typical. Shorter times are most useful for treating small seeds.

A propagating material can also be coated with a coating

composition comprising a biologically effective amount of the substantially pure Structure A compound. The coatings of the disclosure are capable of affecting a slow release of the substantially pure Structure A compound by diffusion into the propagating material and surrounding medium. Coatings include dry dusts or powders adhering to the propagating material by action of an adhesive agent such as methylcellulose or gum arabic.

Coatings can also be prepared from suspension concentrates, water- dispersible powders or emulsions that are suspended in water, sprayed on the propagating material in a tumbling device and then dried.

Substantially pure Structure A compounds that are dissolved in the solvent can be sprayed on the tumbling propagating material and the solvent then evaporated. Such compositions preferably include ingredients promoting adhesion of the coating to the propagating material. The compositions may also contain surfactants promoting wetting of the propagating material. Solvents used must not be phytotoxic to the propagating material; generally water is used, but other volatile solvents with low phytotoxicity such as methanol, ethanol, methyl acetate, ethyl acetate, acetone, etc. may be employed alone or in combination. Volatile solvents are those with a normal boiling point less than about 100 °C. Drying must be conducted in a way not to injure the propagating material or induce premature germination or sprouting.

The thickness of coatings can vary from adhering dusts to thin films to pellet layers about 0.5 to 5 mm thick. Propagating material coatings can comprise more than one adhering layer, only one of which need comprise the substantially pure Structure A compound. Generally pellets are most satisfactory for small seeds, because their ability to provide a biologically effective amount of the substantially pure Structure A compound is not limited by the surface area of the seed, and pelleting small seeds also facilitates seed transfer and planting operations. Because of their larger size and surface area, large seeds and bulbs, tubers, corms and rhizomes and their viable cuttings are generally not pelleted, but instead coated with powders or thin films.

Propagating materials contacted with agricultural compositions comprising substantially pure compounds of Structure A in accordance to this disclosure include seeds. The application of the agricultural composition to seeds can accelerate the rate of germination. Suitable seeds include seeds of wheat, durum wheat, barley, oat, rye, maize (corn), sorghum, rice, wild rice, cotton, flax, sunflower, soybean, garden bean, lima bean, broad bean, garden pea, peanut, alfalfa, beet, garden lettuce, rapeseed, cole crop, turnip, leaf mustard, black mustard, tomato, potato, pepper, eggplant, tobacco, cucumber, muskmelon, watermelon, squash, carrot, zinnia, cosmos, chrysanthemum, sweet scabious, snapdragon, gerbera, babys-breath, statice, blazing star, lisianthus, yarrow, marigold, pansy, impatiens, petunia, geranium and coleus. Of note are seeds of cotton, maize (corn), soybean and rice. Propagating materials contacted with substantially pure compounds of Structure A in accordance to this disclosure also include, for example, rhizomes, tubers, bulbs or corms, or viable divisions thereof. Suitable rhizomes, tubers, bulbs and corms, or viable divisions thereof include those of potato, sweet potato, yam, garden onion, tulip, gladiolus, lily, narcissus, dahlia, iris, crocus, anemone, hyacinth, grape-hyacinth, freesia, ornamental onion, wood-sorrel, squill, cyclamen, glory-of-the-snow, striped squill, calla lily, gloxinia and tuberous begonia. Of note are rhizomes, tubers, bulbs and corms, or viable division thereof of potato, sweet potato, garden onion, tulip, daffodil, crocus and hyacinth. Propagating materials contacted with substantially pure compounds of Structure A can also include stems and leaf cuttings.

In some embodiments, a propagating material is contacted with a coating composition comprising the substantially pure Structure A

compound and a film former or adhesive agent. Coating compositions which comprise a biologically effective amount of the substantially pure compound of Structure A and a film former or adhesive agent, can further comprise an effective amount of at least one of the previously mentioned biologically active compounds or agents. Of note are compositions comprising (in addition to the substantially pure Structure A compound and the film former or adhesive agent) arthropodicides of the group

pyrethroids, such as cypermethrin, cyhalothrin, cyfluthrin and beta- cyfluthrin, esfenvalerate, fenvalerate and tralomethrin; carbamates such as fenothicarb, methomyl, oxamyl and thiodicarb; neonicotinoids such as clothianidin, imidacloprid and thiacloprid; neuronal sodium channel blockers such as indoxacarb; insecticidal macrocyclic lactones such as spinosad, abamectin, avermectin and emamectin; γ-aminobutyric acid (GABA) antagonists such as endosulfan, ethiprole and fipronil; insecticidal ureas such as flufenoxuron and triflumuron; and juvenile hormone mimics such as diofenolan and pyriproxyfen.

Generally, the coating composition may further comprise formulation aids such as a dispersant, a surfactant, a carrier and optionally an antifoam agent and a dye. One skilled the art will appreciate that the amount of substantially pure Structure A compound needed for efficacy (i.e. biologically effective amount) will vary with several factors including, but not limited to, plant species, propagating material type and environmental conditions. The coating needs to not inhibit germination or sprouting of the propagating material.

The film former or adhesive agent component of the propagating material coating is composed preferably of an adhesive polymer that may be natural or synthetic and is without phytotoxic effect on the propagating material to be coated. The film former or sticking agent may be selected from polyvinyl acetates, polyvinyl acetate copolymers, hydrolyzed polyvinyl acetates, polyvinylpyrrolidone-vinyl acetate copolymer, polyvinyl alcohols, polyvinyl alcohol copolymers, polyvinyl methyl ether, polyvinyl methyl ether-maleic anhydride copolymer, waxes, latex polymers, celluloses including ethylcelluloses and methylcelluloses, hydroxymethylcelluloses, hydroxypropylcellulose, hydroxymethylpropylcelluloses, polyvinylpyrrolidones, alginates, dextrins, malto-dextrins, polysaccharides, fats, oils, proteins, karaya gum, jaguar gum, tragacanth gum, polysaccharide gums, mucilage, gum arabics, shellacs, vinylidene chloride polymers and copolymers, soybean-based protein polymers and copolymers,

lignosulfonates, acrylic copolymers, starches, polyvinylacrylates, zeins, gelatin, carboxymethylcellulose, chitosan, polyethylene oxide, acrylimide polymers and copolymers, polyhydroxyethyl acrylate, methylacrylimide monomers, alginate, ethylcellulose, polychloroprene and syrups or mixtures thereof. Preferred film formers and adhesive agents include polymers and copolymers of vinyl acetate, polyvinylpyrrolidone-vinyl acetate copolymer and water-soluble waxes. Particularly preferred are polyvinylpyrrolidone-vinyl acetate copolymers and water-soluble waxes. The above-identified polymers include those known in the art and for example some are identified as AGRIMER ^® VA 6 (Vinylpyrrolidone/vinyl acetate copolymers available from Ashland, Inc., Covington, KY) and LICOWAX ^® KST (an ester of montanic acids with multifunctional alcohols available from Clariant International Ltd., Muttenz, Switzerland). The amount of film former or adhesive agent in the formulation is generally in the range of about 0.001 to 100% of the weight of the propagating material. For large seeds the amount of film former or adhesive agent is typically in the range of about 0.05 to 5% of the seed weight; for small seeds the amount is typically in the range of about 1 to 100%, but can be greater than 100% of seed weight in pelleting. For other propagating materials the amount of film former or adhesive agent is typically in the range of 0.001 to 2% of the propagating material weight.

Materials known as formulation aids may also be used in propagating material treatment coatings are well known to those skilled in the art.

Formulation aids assist in the production or process of propagating material treatment and include, but are not limited, to dispersants, surfactants, carriers, antifoams and dyes. Useful dispersants can include highly water-soluble anionic surfactants like BORRESPERSE™ CA (a spray dried calcium lignosulphonate available from Borregaard

Deutschland GmbH, Karlsruhe, Germany), MORWET ^® D425 (naphthalene sulfonate available from AkzoNobel, Amsterdam, Netherlands) and the like. Useful surfactants can include highly water-soluble nonionic surfactants like PLURONIC ^® F108 (a difunctional block copolymer surfactant available from BASF, Florham Park, NJ), BRIJ ^® 78

(polyethylene glycol octadecyl ether available from Sigma-Aldrich, St. Louis. MO) and the like. Useful carriers can include liquids like water and oils which are water-soluble such as alcohols. Useful carriers can also include fillers like woodflours, clays, activated carbon, diatomaceous earth, fine-grain inorganic solids, calcium carbonate and the like. Clays and inorganic solids which may be used include calcium bentonite, kaolin, china clay, talc, perlite, mica, vermiculite, silicas, quartz powder, montmorillonite and mixtures thereof. Antifoam agnets can include water dispersible liquids comprising polyorganic siloxanes like RHODORSIL ^® 416 (mixture of silicone-polyether block copolymer and free polyether available from Rhodia Inc., Cranbury, NJ). Dyes can include water dispersible liquid colorant compositions like PRO-IZED ^® Colorant Red (liquid seed colorant available from Gustafson LLC, Piano, TX). One skilled in the art will appreciate that this is a non-exhaustive list of formulation aids and that other recognized materials may be used depending on the propagating material to be coated and the substantially pure compound of Structure A used in the coating. Suitable examples of formulation aids include those listed herein and those listed in

McCutcheon's 2001, Volume 2: Functional Materials, published by MC Publishing Company. The amount of formulation aids used may vary, but generally the weight of the components will be in the range of about 0.001 to 10000% of the propagating material weight, with the percentages above 100% being mainly used for pelleting small seed. For nonpelleted seed generally the amount of formulating aids is about 0.01 to 45% of the seed weight and typically about 0.1 to 15% of the seed weight. For propagating materials other than seeds, the amount of formulation aids generally is about 0.001 to 10% of the propagating material weight.

Conventional means of applying seed coatings may be used to carry out the coating. Dusts or powders may be applied by tumbling the propagating material with a formulation comprising the substantially pure Structure A compound and a sticking agent to cause the dust or powder to adhere to the propagating material and not fall off during packaging or transportation. Dusts or powders can also be applied by adding the dust or powder directly to the tumbling bed of propagating materials, followed by spraying a carrier liquid onto the seed and drying. Dusts and powders comprising the substantially pure Structure A compound can also be applied by treating (e.g., dipping) at least a portion of the propagating material with a solvent such as water, optionally comprising a sticking agent, and dipping the treated portion into a supply of the dry dust or powder. This method can be particularly useful for coating stem cuttings. Propagating materials can also be dipped into compositions comprising Structure A formulations of wetted powders, solutions, suspoemulsions, emulfiable concentrates and emulsions in water, and then dried or directly planted in the growing medium. Propagating materials such as bulbs, tubers, corms and rhizomes typically need only a single coating layer to provide a biologically effective amount of the substantially pure Structure A compound.

Propagating materials may also be coated by spraying a suspension concentrate directly into a tumbling bed of propagating materials and then drying the propagating materials. Alternatively, other formulation types like wetted powders, solutions, suspoemulsions, emulsifiable concentrates and emulsions in water may be sprayed on the propagating materials. This process is particularly useful for applying film coatings to seeds. Various coating machines and processes are available to one skilled in the art. Suitable processes include those listed in P. Kosters et al., Seed

Treatment: Progress and Prospects, 1994 BCPC Monograph No. 57 and the references listed therein. Three well-known techniques include the use of drum coaters, fluidized bed techniques and spouted beds.

Propagating materials such as seeds may be presized prior to coating. After coating the propagating materials are dried and then optionally sized by transfer to a sizing machine. These machines are known in the art for example, as a typical machine used when sizing corn (maize) seed in the industry.

For coating seed, the seed and coating material are mixed in any variety of conventional seed coating apparatus. The rate of rolling and coating application depends upon the seed. For large oblong seeds such as those of cotton, a satisfactory seed coating apparatus comprises a rotating type pan with lifting vanes turned at sufficient rpm to maintain a rolling action of the seed, facilitating uniform coverage. For seed coating formulations applied as liquids, the seed coating must be applied over sufficient time to allow drying to minimize clumping of the seed. Using forced air or heated forced air can facilitate an increased rate of application. One skilled in the art will also recognize that this process may be a batch or continuous process. As the name implies, a continuous process allows the seeds to flow continuously throughout the product run. New seeds enter the pan in a steady stream to replace coated seeds exiting the pan.

The seed coating process of the present disclosure is not limited to thin film coating and may also include seed pelleting. The pelleting process typically increases the seed weight from 2 to 100 times and can be used to also improve the shape of the seed for use in mechanical seeders. Pelleting compositions generally contain a solid diluent, which is typically an insoluble particulate material, such as clay, ground limestone, powdered silica, etc., to provide bulk in addition to the film former such as one or more of those previously mentioned. After sufficient layers have been built up, the coat is dried and the pellets graded. A method for producing pellets is described in Agrow, The Seed Treatment Market, Chapter 3, PJB Publications Ltd., 1994.

Seed varieties and seeds with specific transgenic traits may be tested to determine which seed treatment options and application rates may complement such varieties and transgenic traits in order to increase rate of germination, increase rate of seedling emergence, increase rate of radicle growth, increase rate of early growth, increase pest control, increase disease control, increase plant height, increase vigor, increase resistance to abiotic environmental stress, and increase biomass and/or yield. Further, the good root establishment and early emergence that results from the proper use of the substantially pure compound of

Structure A seed treatment may result in more efficient nitrogen use, a better ability to withstand drought and an overall increase in yield potential of a variety or varieties containing a certain trait when combined with a seed treatment comprising the substantially pure compound of Structure A.

In another embodiment, the agricultural composition is a foliar formulation. Such formulations will generally include at least one additional component selected from the group consisting of surfactants, solid diluents and liquid diluents, which serve as a carrier. The formulation ingredients are selected to be consistent with the physical properties of the substantially pure compound of Structure A, mode of application and environmental factors such as soil type, moisture and temperature.

Useful formulations include both liquid and solid compositions. Liquid compositions include solutions (including emulsifiable

concentrates), suspensions, emulsions (including microemulsions and/or suspoemulsions) and the like, which optionally can be thickened into gels. The general types of aqueous liquid compositions are soluble concentrate, suspension concentrate, capsule suspension, concentrated emulsion, microemulsion and suspoemulsion. The general types of nonaqueous liquid compositions are emulsifiable concentrate, microemulsifiable concentrate, dispersible concentrate and oil dispersion. The general types of solid formulations are dusts, powders, granules, pellets, prills, pastilles, tablets, filled films (including seed coatings) and the like, which can be water-dispersible ("wettable") or water-soluble.

Substantially pure compounds of Structure A can be (micro)encapsulated and further formed into a suspension or solid formulation; alternatively the entire formulation can be encapsulated (or "overcoated"). Encapsulation can control or delay release of the substantially pure compound of

Structure A. An emulsifiable granule combines the advantages of both an emulsifiable concentrate formulation and a dry granular formulation.

High-strength compositions are primarily used as intermediates for further formulation.

Sprayable formulations are typically extended in a suitable medium before spraying. Such liquid and solid formulations are formulated to be readily diluted in the spray medium, usually water. Spray volumes can range from about one to several thousand liters per hectare, but more typically are in the range from about ten to several hundred liters per hectare. Sprayable formulations can be tank mixed with water or another suitable medium for foliar treatment by aerial or ground application, or for application to the growing medium of the plant. Liquid and dry

formulations can be metered directly into drip irrigation systems or metered into the furrow during planting. Liquid and solid formulations can be applied onto seeds of crops and other desirable vegetation as seed treatments before planting to protect developing roots and other

subterranean plant parts and/or foliage through systemic uptake. Effective foliar formulations will typically contain from about 10 ^"5 M to 10 ^"12 M of the substantially pure compound of Structure A. In a preferred embodiment, formulations contain from about 10 ^"6 M to 10 ^"10 M of the substantially pure compound of Structure A.

In another embodiment of the disclosure, the composition is applied to soil either prior to or following planting of propagating materials.

Compositions can be applied as a soil drench of a liquid formulation, a granular formulation to the soil, a nursery box treatment or a dip of transplants. In some embodiments, the composition comprising the substantially pure compound of Structure A is applied to the soil in the form of a soil drench liquid formulation. Other methods of contact include application of a compound or the agricultural composition by direct and residual sprays, aerial sprays, gels, seed coatings, microencapsulations, systemic uptake, foggers, fumigants, aerosols, dusts and many others. One embodiment of a method of contact is a dimensionally stable fertilizer granule, stick or tablet comprising the substantially pure compound of Structure A. Effective soil formulations will typically contain from about 10 ^{" 5} M to 10 ^"12 M of the substantially pure compound of Structure A. In a preferred embodiment, formulations contain from about 10 ^"6 M to 10 ^"10 M of the substantially pure compound of Structure A.

The methods of this disclosure is applicable to virtually all plant species, including monocots, dicots and gymnosperms. Seeds that can be treated include, for example, wheat (Triticum aestivum L), durum wheat (Triticum durum Desf.), barley (Hordeum vulgare L), oat (Avena sativa L), rye (Secale cereale L), maize (Zea mays L), sorghum (Sorghum vulgare Pers.), rice (Oryza sativa L), wild rice (Zizania aquatica L), millet

(Eleusine coracana, Panicum miliaceum), cotton (Gossypium barbadense L. and G. hirsutum L), flax (Linum usitatissimum L), sunflower (Helianthus annuus L), soybean (Glycine max Merr.), garden bean (Phaseolus vulgaris L), lima bean (Phaseolus limensis Macf.), broad bean (Vicia faba L), garden pea (Pisum sativum L), peanut (Arachis hypogaea L), alfalfa (Medicago sativa L), beet (Beta vulgaris L), garden lettuce (Lactuca sativa L), rapeseed (Brassica rapa L. and B. napus L), cole crops such as cabbage, cauliflower and broccoli (Brassica oleracea L), turnip (Brassica rapa L), leaf (oriental) mustard (Brassica juncea Coss.), black mustard (Brassica nigra Koch), tomato (Lycopersicon esculentum Mill.), potato (Solanum tuberosum L), pepper (Capsicum frutescens L), eggplant (Solanum melongena L), tobacco (Nicotiana tabacum), cucumber

(Cucumis sativus L), muskmelon (Cucumis melo L), watermelon (Citrullus vulgaris Schrad.), squash (Curcurbita pepo L, C. moschata Duchesne, and C. maxima Duchesne.), carrot (Daucus carota L), zinnia (Zinnia elegans Jacq.), cosmos (e.g., Cosmos bipinnatus Cav.), chrysanthemum (Chrysanthemum spp.), sweet scabious (Scabiosa atropurpurea L), snapdragon (Antirrhinum majus L), gerbera (Gerbera jamesonii Bolus), babys-breath (Gypsophila paniculata L, G. repens L. and G. elegans Bieb.), statice (e.g., Limonium sinuatum Mill., L. sinense Kuntze.), blazing star (e.g., Liatris spicata Willd., L. pycnostachya Michx., L. scariosa Willd.), lisianthus (e.g., Eustoma grandiflorum (Raf.) Shinn), yarrow (e.g., Achillea filipendulina Lam., A. millefolium L), marigold (e.g., Tagetes patula L, T. erecta L), pansy (e.g., Viola cornuta L, V. tricolor L), impatiens (e.g., Impatiens balsamina L.) petunia (Petunia spp.), geranium (Geranium spp.) and coleus (e.g., Solenostemon scutellarioides (L.) Codd).

Gymnosperm seeds that can be treated include, for example, pine (Pinus spp.), fir (Abies, spp.), Hemlock (Tsuga spp.) cypress (Cupressus spp.) and Douglas-fir (Pseudotsuga spp.).

Not only seeds, but also rhizomes, tubers, bulbs or corms, including viable cuttings thereof, can be treated according to the dislcosure from, for example, potato (Solanum tuberosum L), sweet potato (Ipomoea batatas L), yam (Dioscorea cayenensis Lam. and D. rotundata Poir.), garden onion (e.g., Allium cepa L.), tulip (Tulipa spp.), gladiolus (Gladiolus spp.), lily (Lilium spp.), narcissus (Narcissus spp.), dahlia (e.g., Dahlia pinnata Cav.), iris (Iris germanica L. and other species), crocus (Crocus spp.), anemone (Anemone spp.), hyacinth (Hyacinth spp.), grape-hyacinth (Muscari spp.), freesia (e.g., Freesia refracta Klatt., F. armstrongii \N .

Wats), ornamental onion (Allium spp.), wood-sorrel (Oxalis spp.), squill (Scilla peruviana L. and other species), cyclamen (Cyclamen persicum Mill, and other species), glory-of-the-snow (Chionodoxa luciliae Boiss. and other species), striped squill (Puschkinia scilloides Adams), calla lily (Zantedeschia aethiopica Spreng., Z. elliottiana Engler and other species), gloxinia (Sinnigia speciosa Benth. & Hook.) and tuberous begonia

(Begonia tuberhybrida Voss.). Stem cuttings can include those from such plants as sugarcane (Saccharum officinarum L.), carnation (Dianthus caryophyllus L.), florists chrysanthemum (Chrysanthemum mortifolium Ramat.), begonia (Begonia spp.), geranium (Geranium spp.), coleus (e.g., Solenostemon scutellarioides (L.) Codd) and poinsettia (Euphorbia pulcherrima Willd.). Leaf cuttings which can include those from begonia (Begonia spp.), african-violet (e.g., Saintpaulia ionantha Wendl.) and sedum (Sedum spp.).

The above recited cereal, vegetable, ornamental (including flower) and fruit crops are illustrative, and should not be considered limiting in any way. For reasons of economic importance, preferred embodiments can include wheat, rice, maize, barley, sorghum, oats, rye, millet, soybeans, peanuts, beans, rapeseed, canola, sunflower, sugar cane, potatoes, sweet potatoes, cassava, sugar beets, tomatoes, plantains and bananas, and alfalfa.

EXAMPLES

The present disclosure is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt it to various usages and conditions. The meaning of abbreviations is as follows: "Ac" means an acetyl group, "Bz" means a benzoyl group, "t-BDMS" means a tertiary-butyl-dimethyl-silyl group, "Hz" means hertz, "MHz" means megahertz, "ppm" means parts per million, "HOD " means hydrogen deuteriumoxide, "μ " means micron, "g" means gram(s), "mg" means milligrams, "kg" means "kilogram(s), "ml" means milliliter(s), "L" means liter(s), "min" means minutes, "h" means hour, "eq." means equivalents, "mmol" means millimole(s), "mol" means mole(s), "M" means molar, "MW" means molecular weight, "M/e calc" means calculated mass to charge ratio.

General Methods and Materials for Chemical Syntheses Unless specified, all the reagents were purchased from Aldrich Chemical Co (St. Louis, MO). Cis-vaccenic acid and palmitoleic acid were purchased from MP Biochemicals LLC (Solon, OH). Thin layer

chromatography was performed on pre-coated plates of Silica Gel 60 F254 (EM Science) and the spots were visualized with a spray containing 5% sulfuric acid in ethanol, followed by heating. Column chromatography was done on silica gel 60 (230 - 400 mesh, EM Science). ¹ H NMR spectra were recorded at 500 MHz. The hydrogen chemical shifts in organic solvents are expressed relative to deuterated methylenechloride, with a reference chemical shift of 5.36 ppm. For solutions of compounds in deuterium oxide or deuterated methanol, the hydrogen chemical shift values are expressed relative to the HOD signal (4.75 ppm at 296 °K).

The present disclosure is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt it to various usages and conditions.

Example 1

Synthesis of 2-deoxy-1 ,3,4,6-tetra-O-acetyl-2-phthalimido-D- glucopyranose

Product 3

D-Glucosamine hydrochloride (Product 1 , 1 .0 kg) was suspended in methanol (5.0 L) and vigorously stirred. NaOH (184.8 g) was dissolved in minimum deionized water and added to the D-glucosamine/methanol (MeOH) suspension. The suspension was stirred for 15 min and the insoluble material (sodium chloride) was filtered off by vacuum filtration.

To the filtrate, phthalic anhydride (342 g) was added and the solution was stirred until most of the solid dissolved (about 30 min). This was then followed by addition of triethylamine (468 g) and stirring for 10 to 15 min. To the resulting clear solution, another portion of phthalic anhydride (342 g) was added and the mixture was allowed to stir overnight at room temperature. Product began to precipitate out after two hours. The precipitated product was filtered and the residue was washed with minimum ice cold methanol so as to remove the yellow color from the product. The residue was then washed three times with acetonitrile, with enough solvent added to the filter to completely immerse the solid, and dried at room temperature under high vacuum. The weight of the white solid, Product 2, was 954 g. ¹ H-NMR (D ₂ O): 7.74-7.56 (phthalimido hydrogens), 5.42 (H-1 a), 4.94 (H-1 β), 4.17 and 4.01 (H-6), 3.27 (CH ₂ of N-ethyl group), 1 .35 (CH ₃ of N-ethyl group).

The Product 2 from above (1 .01 Kg, made from two batches) was placed in a 10 L 3-neck round bottom flask set up with an overhead electric stirrer, an N ₂ inlet and an addition funnel. Acetic anhydride (3 L) and N,N-dimethylaminopyridine (1 .0 g) were added to the flask and stirred vigorously. Pyridine (2.8 L) was added slowly and the reaction mixture was stirred for 2 days at room temperature. The reaction mixture was quenched with ice-water (4 L) and the product was extracted with methylene chloride (CH2CI2). The organic layer was repeatedly washed with aqueous hydrochloric acid solution, and then with saturated sodium bicarbonate solution. The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated to dryness. The product was recrystallized from hot ethanol. Weight of the recrystallized Product 3 was 701 g. ¹ H-NMR (CD ₂ CI ₂ ) δ: 7.91 -7.80 (phthalimido hydrogens), 6.62 (H-1 ), 5.59 (H-3), 5.21 (H-4), 4.47 (H-2), 4.36 and 4.16 (H-6), 4.06 (H-5), 2.12, 2.06, 2.02, 1 .88 (acetyl methyl groups). Thus the above NMR chemical shift data verified the structure of Product 3, 2-deoxy-1 ,3,4,6- tetra-O-acetyl-2-phthalimido-D-glucopyranose.

Example 2

Synthesis of Monomer A

Preparation of intermediate Product 4:

Product 3 Product 4

Product 3 (464 g) was dissolved in toluene and the solvent was evaporated. This was repeated and the remaining solid was placed on a high vacuum line overnight.

The dried solid was dissolved in minimum CH2CI2 (ca. 600 ml), and stirred well. To this, 4-methylbenzenethiol (181 g, 1 .45 mol, 1 .5 eq.) was added followed by the dropwise addition of boron trifluoride diethyl etherate (BF3-etherate; 165 g, 1 .16 mol, 1 .2 equivalent, over 180 min). The reaction mixture was stirred overnight. White crystals formed in the morning when stirring was stopped. The crystals were filtered, giving Product 4A. The filtrate was diluted with CH2CI2, washed sequentially with saturated NaHCO3 solution, water, then bicarbonate solution, and dried giving Product 4B. Products 4A and 4B were extensively washed with anhydrous methanol and dried under vacuum. Since the NMR spectrums of 4A and 4B products were identical, these two were combined (Product 4, 426.3 g).

¹ H-NMR (CD2CI2) δ: 7.96-7.80 (phthalimido hydrogens), 7.36 & 7.13 (S-aromatic hydrogens), 5.78 (H-3), 5.69 (H-1 ), 5.13 (H-4), 4.33 (H-2), 4.30 & 4.12 (H-6), 3.93 (H-5), 2.36 (S-Ph-Me group), 2.13, 2.04, 1 .85 (methyls of acetyl groups). Thus the NMR spectrum verified the structure of Product 4, as shown above. Preparation of intermediate Product 5

Product 4 Product 5

Product 4 (350 g) was suspended in nearly 4 L of dry methanol. To this, 35 ml of 0.5 M sodium methoxide solution was added and the solution immediately turned basic. The suspension was left stirring at room temperature overnight. The solid deposited was filtered and washed with dichloromethane, giving pure Product 5 (232 g). The filtrate was neutralized with sulfonic acid resin and concentrated to dryness. The dry solid was washed with CH ₂ CI ₂ and dried, giving impure Product 5 (43.8 g). ¹ H-NMR (CD ₃ OD) of pure 5 δ: 7.87-7.76 (phthalimido hydrogens), 7.22 & 6.99 (S- aromatic hydrogens), 5.46 (H-1 ), 4.18 (H-2), 4.03 (H-3), 3.89 & 3.70 (H-6), 3.39 (H-5), 3.37 (H-4), 2.22 (S-Ph-Me group). Thus the NMR spectrum verified the structure of Product 5, as shown above.

Preparation of intermediate Product 6:

Product 5 (295 g; 638 mmol) was suspended in dry toluene (1 L) and evaporated under vacuum. This procedure was repeated once more to ensure the removal of methanol contaminant that is detrimental to the reaction. 265 grams total product was recovered. The residue after toluene evaporation was suspended in CH2CI2 (3 L) in a 3-neck flask fitted with an overhead stirrer and the suspension was stirred under dry nitrogen atmosphere. The flask was cooled in an ice bath and the following reagents were added: 126g pyridine, 500mg N,N-dimethylaminopyridine; and 171 g benzoyl chloride (slowly added by means of an addition funnel in drops over 60 min). The reaction mixture was milky white, but began to clear when all of the benzoyl chloride was added. The reaction was allowed to stir for 18 h at room temperature. The reaction was diluted with CH2CI2 and was washed with water (2x), 1 M aqueous HCI (2x), then saturated NaHCO ₃ and dried with MgSO .

The crude product was recrystallized in 8 liters of hot EtOH, crystals were filtered, and washed in EtOH giving Crop 6A (225 g). The filtrate was concentrated to dryness giving Crop 6B (131 g). A second recrystallization of Crop 6A was done to give pure Product 6 (172g). The residue (40 g) from the filtrate of the second recrystallization had product 6 of purity greater than 95%, as determined by NMR. Crop 6B was not further processed as NMR analysis showed that it had a significant amount of undesired products and was therefore recycled back to Product 5.

¹ H-NMR (CD2CI2) δ: 8.14, 7.88, 7.69, 7.57, 7.41 (benzoate hydrogens), 7.80-7.72 (phthalimido hydrogens), 7.34 & 7.00 (S-aromatic hydrogens), 5.93 (H-3), 5.79 (H-1 ), 4.77 & 3.99 (H-6), 4.47 (H-2), 4.03- 3.99 (H-5), 3.91 (H-4), 3.25 (OH), 2.31 (S-Ph-Me group). Thus the NMR spectrum verified the structure of Product 6, as shown above. Preparation of Monomer A

Product 6 (171 .9 g; 275.6 mmol) was dissolved in minimum methylene chloride (350 ml_) containing collidine (41 .7 g; 344.5 mmol; 1 .25 eq.). t- BDMS-Triflate (80. Og; 303.1 mmol; 1 .1 eq.) was added drop-wise by addition funnel over 50 minutes. The reaction mixture was allowed to stir overnight. The reaction mixture was diluted with methylene chloride and washed sequentially with ice-cold water, 0.5 M aqueous HCL (ice cold), then aqueous saturated NaHCO3. It was then dried with MgSO ₄ , filtered and concentrated to give Monomer A as a white solid (207 g). The product was dissolved in dry toluene and concentrated to dryness before use in a glycosylation reaction. The 207 g of Monomer A product recovered was essentially equal to the theoretical yield, calculated to be 203.4 g.

¹ H-NMR (CD ₂ CI ₂ ) δ: 8.16 - 7.41 (benzoate hydrogens, phthalimido hydrogens), 7.30 & 6.95 (S-aromatic hydrogens), 5.97 (H-3), 5.82 (H-1 ), 4.89 & 4.49 (H-6), 4.40 (H-2), 4.14 (H-4), 4.01 (H-5), 2.30 (S-Ph-Me group), 0.80 (t-butyl group on silicon), 0.09& -0.16 (methyl groups of silicon). Thus the NMR spectrum verified the structure of Monomer A, as shown above. Example 3

Synthesis of Monomer B

Preparation of intermediate Product 7:

Product 3 Product 7

To ensure that the starting glycoside was free of EtOH traces, Product 3 (60.0 g; 126 mmol) was dissolved in toluene and evaporated. It was then dissolved in anhydrous CH ₂ CI ₂ (500 ml) containing MeOH (6.5 g;

202 mmol; 1 .6 eq.). Tin tetrachloride (SnCI ₄ ; 18.4 g; 70.5 mmol; 0.56 eq.) was diluted with CH2CI2 (25 ml) and added dropwise. The reaction mixture was poured over ice water and shaken well. This was repeated once more and then the organic layer was washed twice with aqueous saturated NaHCO3, dried with MgSO^ filtered, and concentrated. The crude product was recrystallized from hot EtOH, giving crystals of product 7(43.1 g). The 49.8 g crude yield of Product 7 was 88% of the theoretical yield, calculated to be 56.6 g, while the recrystallized Product 7 yield of 43.1 g was 76%.

¹ H-NMR (CD ₂ CI ₂ )5: 7.86-7.74 (phthalimido hydrogens), 5.78 (H-3), 5.31 (H-1 ), 5.18 (H-4), 4.31 (H-2), 4.34 & 4.20 (H-6), 3.88 (H-5), 2.20, 2.03, 1 .86 (methyls of acetyl groups). Thus the NMR spectrum verified the structure of Product 7, as shown above. Preparation of intermediate Product 8:

Product 7 Product 8

Product 7 (141 .0 g; 314 mmol) was suspended in MeOH (1000 ml), and NaOMe (0.5 M, 10 ml) was added. The methyl glycoside Product 7 did not readily dissolve in MeOH. The solution was tested to ensure basicity. The reaction was stirred overnight. The solution became clear. Examination of the reaction mixture by TLC (EtOAc-hexane-EtOH;

10:20:1 ) indicated the disappearance of the starting material and the formation of a polar product (near the origin). The solution was neutralized with sulfonic acid resin, filtered, and concentrated to dryness. Weight of the residue, called Product 8, was 105.3 g, which probably includes some MeOH.

The crude yield of 105.3 g of Product 8 was essentially equal to the theoretical yield, calculated to be 101 .3 g. ¹ H-NMR (CD ₃ OD) δ: 7.85-7.80 (phthalimido hydrogens), 5.07 (H-1 ), 4.21 (H-2), 3.94 (H-3), 3.92 & 3.74 (H-6), 3.40 (H-5), 3.40 (OCH ₃ ), 3.38 (H-4). Thus the NMR spectrum verified the structure of Product 8, as shown above. Preparation of Monomer B

Product 8 Monomer B

Product 8 (crude; 105.3), after being evaporated with toluene-DMF, was suspended in CH ₂ CI ₂ (500 ml). Pyridine (61 .8 g; 782 mmol; 2.5 eq.) was added first, followed by the drop-wise addition of benzoyl chloride (88 g; 626 mmol; 2.0 eq.) to the mixture. The reaction mixture was allowed to stir at room temperature for 24 h. It was then diluted with CH ₂ CI ₂ and washed sequentially with H ₂ O,1 M HCI (2X), then aqueous saturated sodium bicarbonate solution, dried with MgSO^ filtered, and concentrated.

The product was purified by chromatography on silica gel, using 3:8 EtOAc-hexane as the eluant. The weight of the purified product was 1 16.1 g. The product was about 90% pure as determined by NMR. A portion (21 .1 g) of this product was crystallized from diethylether-hexane to obtain pure crystalline material (13.8 g) of Monomer B.

¹ H-NMR (CD ₂ CI ₂ ) δ: 8.15, 7.92, 7.67, 7.56, 7.42 (benzoate hydrogens), 7.83-7.74 (phthalimido hydrogens), 5.93 (H-3), 5.40 (H-1 ), 4.82 & 4.72 (H- 6), 4.43 (H-2), 4.03-3.92 (H-5, H-4), 3.50 (OCH ₃ ), 3.33 (OH). Thus the NMR spectrum verified the structure of monomer B, as shown above. Example 4

Synthesis of Derivatized Glucosamine Disaccha de Structural Characterization of Oligoglucosamine Derivatives:

The structures of the coupled products described below were confirmed by proton NMR and mass spectrometry as follows. The chemical shifts of hydrogens H-3 and H-1 of the phthalimido glucosamine unit appeared in proton NMR spectrum at chemical shifts between 5 and 6.5 ppm. The hydrogen H-3 appeared as a doublet of a doublet with a coupling constant of about 8-10 Hz. By counting the number of these hydrogen signals, the length of the oligoglucosamine can easily be determined, for the

disacchahde to the pentasaccharide. For oligoglucosamine derivatives of 6 and above, the signals for these hydrogens started to overlap. However, a sufficient number of these signals could be identified to confirm the structure. A similar observation was seen for the anomeric hydrogens, which appeared as a doublet with a coupling constant of about 8-8.5 Hz, thereby confirming the β-glycosidic configuration. Furthermore, the chemical shift of H-4 in the terminal glucosamine unit appeared around 3.5 ppm, when the corresponding carbon carried a hydroxyl group. This was shifted to 3.7 ppm upon glycosylation at this site. Thus, H-4 could be used as a reporter group for establishing the success of the glycosylation reaction. Further proof of structure was obtained by MALDI and

electrospray mass spectral data of the product, which are indicated for each compound.

Synthesis of Dinner Product 9

Monomer A Monomer B Product 9

Monomer A (80.6 g, 109.3 mmol, 1 .2 eq.) and Monomer B (48.4 g, 91 .1 mmol), both previously evaporated with toluene once, were dissolved in CH ₂ CI ₂ (150 ml_) in a 3-necked, 500 ml flask. 4A Molecular sieve was added (5 g). The mixture was cooled to -60° C under nitrogen atmosphere with vigorous stirring. After 10 min, N-lodosuccinimide (NIS; 44.3 g; 196.7 mmol; 2.2 eq.) was added as a dry powder, followed by the dropwise addition of a solution of triflic acid (TfOH; 13.7 g, 91 .1 mmol, 1 .0 eq.) and methyltriflate (14.9 g, 54.8 mmol, 1 .0 eq.) in CH ₂ CI ₂ . The reaction mixture was left at -55° C for an additional 4 hr. An additional 100 ml of the triflic acid/methyltriflate solution was added to the reaction mixture dropwise to reduce of the viscosity. The reaction mixture was filtered cold over a celite pad into a filter flask containing 1 :1 saturated sodium thiosulfate-sodium bicarbonate solution that was stirred thoroughly during filtration. The flask and residue on the filter were rinsed with CH ₂ CI ₂ and the combined filtrate was worked up as follows.

The filtrate was poured into a separatory funnel. The contents were thoroughly mixed, the aqueous solution separated, and the organic layer washed one more time with saturated aqueous sodium thiosulfate solution, followed by water, and aqueous saturated sodium bicarbonate solution. The solution was then dried with magnesium sulfate, filtered and concentrated. Weight of the crude product was 1 1 1 .1 g. Analytically pure sample was prepared by subjecting the crude product to separation by silica gel chromatography, using ethyl acetate-hexane (3:8) as eluant. 1 H-NMR (CD ₂ CI ₂ ) δ: 8.17 - 7.19 (phthalimido and benzoate hydrogens), 6.1 1 and 5.76 (2 x H-3), 5.74 and 5.31 (2 x H-1 ), 4.36 and 4.32 (2 x H-2), 4.32 and 3.93 (2 x H-4), 3.90 and 3.53 (2 x H-5), 4.65, 4.38, 4.12, and 3.63 (4 x H-6), 3.38 (OCH ₃ ), 0.68 (t-butyl), -0.12, -0.40 (2 x CH ₃ ). Mass spec: M. wt. Calc. 1 144.37; Obs. M+Na = 1 167.5. Thus the NMR spectrum verified the structure of Product 9, as shown above. The crude product as such was used in the next step, where complete removal of the tBDMS was accomplished.

Example 5

Removal of the Silicon Group from Disaccharide Product 9 for Chain

Extension

Preparation of intermediate Product 10:

Product 9 Product 10

Product 9 (1 1 1 .1 g) was dissolved in THF (350 ml). To this solution, a 1 M solution of acetic acid (1 10 ml) and a 1 M solution of n- tetrabutylammonium fluoride in THF (1 10 ml) were added and the reaction mixture was stirred at room temperature for 3 days. Completion of the reaction was ascertained by TLC using EtOAC-HexEtOH (4:8:1 ) as a solvent, which indicated that the reaction was complete. The solvent of the reaction was evaporated on high vacuum (without heat) and the residue was dissolved in CH2CI2, washed sequentially with water, 1 M aqueous

HCI, 10% sodium thiosulfate aqueous solution, and finally, with saturated aqueous NaHCO ₃ . The solution was then dried with MgSO ₄ , filtered and concentrated. The resulting solid was treated with diethyl ether which resulted in a gluey material. The supernatant was filtered and the gluey material was repeatedly washed with diethyl ether. To the filtrate, hexane was added to precipitate any ether soluble product and this was filtered (Fraction B, 5.9 g). The final filtrate from ether-hexane was concentrated to dryness (Fraction C).

The NMR spectrum indicated that Fraction B product had about 5% silicon impurity (peak around 0 ppm) along with the major desired disaccharide. Fraction A was contaminated about 10% with tBDMS impurities and a tetrabutylammonium derivative. Therefore, Fraction A was resuspended in 600 ml of ether, mixed for about 10 minutes, filtered and the process was repeated once more (weight of the solid recovered was 77.3 g). This solid was purified once more by dissolving the product in EtOAc and precipitating the product with the aid of hexane (weight of the product recovered was 71 .7 g). The filtrates were combined, hexane was added to precipitate the remaining product and additional 10.8 g of the product was recovered. ¹ H-NMR (CD ₂ CI ₂ ) δ: 8.12 - 7.14 (phthalimido and benzoate hydrogens), 6.14 and 5.73 (2 x H-3), 5.72 and 5.34 (2 x H- 1 ), 4.37 and 4.34 (2 x H-2), 4.10 and 3.69 (2 x H-4), 3.97 and 3.44 (2 x H- 5), 4.66, 4.18, 4.12- 4.06 (4 x H-6), 3.38 (OCH ₃ ), 3.35 (OH). Mass spec: M. wt. Calc. 1030.98 ; Obs. M+Na = 1053.1 . Thus the NMR spectrum verified the structure of Product 10, as shown above. Example 6

Synthesis of Trimer Product 11

Synthesis of trimer Product 11

Monomer A Product 10 Product 11

Monomer A (88.6 g; 120 mmol; 1 .5 eq.) and Product 10 (82.5 G; 80.0 mmol) were dissolved in CH2CI2 (100 ml) in a flask. Molecular sieve

(4A, 5.0 g) was added. The flask was placed in a -55 °C water bath and stirred for 15 min. NIS (48.6 g; 216 mmol) was added as a powder to the cold solution, while maintaining vigorous stirring. A solution of methyl triflate (13.1 g; 80 mmol; 1 .0 eq.) and TfOH (12 g; 80 mmol; 1 .9 eq.), both dissolved together in CH ₂ CI ₂ (5 ml), was added to the cold solution in drops by means of an addition funnel over 60 min. After 6 h at -60°C to - 50°C, the reaction mixture was poured over saturated NaHCOs and saturated sodium thiosulfate aqueous solution (1 :1 , 400 ml) contained in an Erlenmeyer flask and thoroughly stirred. Additional CH ₂ CI ₂ (200 ml) was added and the contents were thoroughly mixed for 10 min, the aqueous solution separated, and the organic layer washed with 0.6% aqueous bleach solution, deionized water, and aqueous saturated CH ₂ CI ₂ solution. The solution was then dried with MgS04, filtered and concentrated. To remove the excess monomer impurity from the trisaccharide, the crude product was suspended in diethyl ether (600 ml), the solid thoroughly mixed and the supernatant filtered. This process was repeated three times and the residue finally dissolved in CH ₂ CI ₂ , then concentrated to dryness giving 93.5 g of product 1 1 . About 40% volume of hexane was added to the filtrate and the precipitated material filtered, redissolved in CH ₂ CI ₂ and concentrated to dryness under vacuum to obtain an additional amount of Product 11 (26.0 g). ¹ H-NMR (CD ₂ CI ₂ ) δ (only select hydrogen chemical shifts are reported): 8.13 - 7.12 (phthalimido and benzoate hydrogens), 6.03, 5.88, and 5.62 (3 x H-3), 5.64, 5.48, and 5.29 (3 x H-1 ), 3.77 (H-4 of the terminal glucosamine unit), 3.90 (H-5 of the terminal glucosamine unit), 4.63 (H-6 of the terminal glucosamine unit), 3.35 (OCHs), 0.64 (t-butyl), -0.18, -0.33 (2 x CH ₃ of the silicon unit). Mass spec: Exact MW calc. 1643.49; Obs. M+Na = 1666.3. Thus the NMR spectrum verified the structure of Product 11 , as shown above.

Example 7

Removal of the Silicon Group from Trisaccharide Product 11

Preparation of intermediate Product 12:

Product 11 Product 12

Product 1 1 was dissolved in minimum THF (500 ml). To this solution, 1 M solution of acetic acid (150 ml) and a 1 M solution of n- tetrabutylammonium fluoride in THF (150 ml) were added and the reaction mixture was stirred at room temperature for 3 days. The reaction mixture was evaporated to dryness, the residue redissolved in CH ₂ CI ₂ , washed sequentially with deionized water, 1 M HCI, 1 % aqueous bleach solution (to remove the dark brown color), and saturated CH ₂ CI ₂ solution, then concentrated to dryness.

In order to remove the nonpolar silicon and other impurities, the solid was dissolved in minimum ETOAc. Hexane was added in drops (the final solvent ratio EtOAc-hexane was 17:14). This resulted in a gluey material. The liquid was filtered and the gluey material redissolved in EtOAc (200 ml) and precipitated with hexane (100 ml) as described above. Finally, diethyl ether was added to solidify the gluey material and the solid was filtered. The solid was redissolved in CH ₂ CI ₂ and concentrated to dryness giving 81 .4 g of Product 12.

The filtrate EtOAc-Hexane-ether was concentrated to dryness. The residue was suspended in diethyl ether, shaken well and filtered. This process was repeated twice. Finally, the precipitate was dissolved in CH ₂ CI ₂ and concentrated to dryness to obtain additional product 12 (16.5 g). ¹ H-NMR (CD ₂ CI ₂ ) δ (only select hydrogen chemical shifts are reported): 8.08 - 7.16 (phthalimido and benzoate hydrogens), 6.03, 5.92, and 5.59 (3 x H-3), 5.67, 5.48, and 5.29 (3 x H-1 ), 3.56 (H-4 of the terminal glucosamine unit), 3.91 (H-5 of the terminal glucosamine unit), 4.63 (H-6 of the terminal glucosamine unit), 3.35 (OCH ₃ ), 3.01 (OH), 0.64. Mass spec: Exact m. wt. Calc. 1529.41 ; Obs. M+Na = 1553.4. Thus the NMR spectrum verified the structure of product 12, as shown above.

Example 8 Synthesis of Diglucosamine Product 13

Product 10 Step 1 = NaOMe/MeOH

MR = Merrifield Resin Step 2 = MR-CH2-NH(CH ₂ )2NH ₂ /

n-Butanol

Product 13

Step 1 :_The derivatized disaccharide Product 10 (14.1 g, 13.7 mmol) was dissolved in anhydrous methanol (600 ml). Sodium methoxide solution (0.5 M, 7.5 ml) was added and the solution was stirred at room temperature for 24 h. Examination by TLC (EtOAc-hexane-EtOH =

20:20:1 and EtOAc-hexane-EtOH = 5:5:1 ) showed the disappearance of the starting material. The reaction was neutralized with sulfonic acid resin, filtered and concentrated to dryness. NMR of the product indicated incomplete de-O-benzoylation. The product was redissolved in anhydrous MeOH (600 ml), 0.5 M sodium methoxide solution (7 ml) was added and the solution was refluxed for 3 days. It was then neutralized with sulfonic acid resin, filtered and concentrated to dryness. Step 2: The product from Step 1 was dissolved in 250 ml of n-butanol. Polystyrene-ethylenediamine resin (26.0 g) was added and the slurry was heated to 105° C with stirring for 24 h. It was then filtered, concentrated to dryness, and the resulting material was redissolved in water and washed with CH ₂ CI ₂ . The aqueous layer was concentrated to dryness.

Examination of the product by proton NMR (presence of signals between 7-8 ppm) showed incomplete phthalimide removal. The product was redissolved in n-butanol (100 ml) containing 10 g of freshly prepared MR- ethylenediamine resin and heated to 100° C for 16 h, filtered over a celite pad and concentrated to dryness. Weight of the product, designated Product 13, was 4.0 g. ¹ H-NMR (D ₂ O) δ: 4.48 and 4.33 (2 x H-1 ), 3.98, 3.95, 3.83, 3.76 (4 x H-6), 3.67, 3.54, 3.43, 3.40 (2 x H-3, 2 x H-4), 3.60 (OCH ₃ ), 2.70 and 2.66 (2 x H-2). Mass spectral data (electrospray): m/e calc. 354.16 (100%); Obs. M+1 = 355.1 . Thus the NMR and MALDI spectra verified the structure of Product 13, as shown above.

Example 9

Synthesis of T glucosamine Product 14

M ₂ )2NH ₂ /

Product 14

The trisaccharide Product 12 (6.1 g) was suspended in MeOH (300 ml), then NaOMe (0.5 M, 7 ml) was added and stirred at room temperature for 2 days. The mixture was then heated to 65° C for 24 h. Examination of the reaction mixture by proton NMR indicated that all benzoate groups had been removed. A white precipitate appeared in the reaction flask. The flask was cooled in an ice bath for 15 min and filtered. The filtrate was neutralized with acidic resin, and concentrated to dryness (Fraction B). The residue in the filter was washed with hot DMF and concentrated to dryness (Fraction A).

Fractions A and B were separately suspended in n-butanol (200 ml), treated with MR-ethylenediamine resin (20 g) and heated to 100° C for 24 h. The hot solution from Fraction A was filtered over a celite pad and washed with 1 :1 methanol-water. The combined filtrate was concentrated to dryness (Fraction C, 853 mg). Proton NMR of the product indicated it to be the desired triglucosamine Product 14. ¹ H-NMR (D ₂ O) δ: 4.54, 4.52, and 4.37 (3 x H-1 ), 4.01 , 3.98, 3.87, 3.80 (6 x H-6), 3.63 (OCHs), 2.78, 2.74, and 2.70 (3 x H-2). Mass spectral data (ESI): m/e calc. 515.23 (100%); Obs. M+Na = 516.2.

Similarly, the reaction mixture from Fraction B was filtered, washed and concentrated to dryness. The yellow solid was dissolved in water and the aqueous layer was extracted twice with CH ₂ CI ₂ to remove the byproduct methyl benzoate. Finally, the aqueous layer was concentrated to dryness, the residue redissolved in water, and lyophilized (Fraction E). NMR analysis of the Fraction E product indicated incomplete phthalimide removal. The solid was suspended in n-butanol (100 ml) and heated to 104° C. MR-Ethylenediamine resin (9 g) was added and the reaction was continued for two days. The hot reaction mixture was filtered over a Celite pad, washed with 1 :1 MeOH:water (30 ml), and then with water (2 x 15 ml). The combined filtrate was concentrated to dryness. The residue was dissolved in water and lyophilized (Fraction F). The NMR spectrum of Fraction F showed that though the major product was the desired triglucosamine Product 14, it was contaminated with some incompletely deprotected trisaccharide.

Germination Assay for Examples 10, 11 and 13

All materials, with the exception of the seeds (corn or soybean) were sterilized before use. An aqueous solution of the test compounds (25 ml_, 10 ^"7 M in Dl-water) was prepared for each set of five repeat experiments. A fungicide solution was added to each of the aqueous solutions of the test compounds. Five Petri dishes and 100 seeds were used to test one composition. The seeds were inspected for uniformity and lack of cracks in the seed prior to use. A piece of filter paper was used to cover the inner side of each Petri dish to allow uniform distribution of testing solution. Twenty seeds were placed on the filter paper area of one Petri dish. Each corn seed of examples 10 and 1 1 was placed on the filter paper with the flat side of the corn seed facing upwards. The seeds were placed on the filter paper so that the seeds were not touching each other. 5 ml_ of the test compound solution was carefully poured in the Petri dish. Control experiments were set up the same way with 20 seeds and 5 ml_ of deionized water per dish without any active species. The lid was placed on the Petri dish and was sealed with Para-film. Five dishes with repeat experiments were stacked. Each stack of dishes was wrapped twice with aluminum foil to prevent the seeds receiving any light and the stacks were germinated in dark at the ambient laboratory conditions. The stacks were inspected after 24, 36, 48 and 60 hours. The number of germinated seeds was counted and the percent of germination on each dish was calculated. Radicle emergence was used as the germination indicator. The dishes were placed unwrapped at room temperature for one day and the number of germinated seeds was counted to assure seeds were able to germinate and difference in germination yields were not caused by poor seed quality. Statistical analyses were performed by calculating the standard deviation of the five repetitions for each

experiment. The data was deemed acceptable when the standard deviation was less than 10 percent. Experimental data for these above tests wherein the standard deviation was greater than 10 percent was not used.

Cold Stress Germination Assay for Example 12

A seed coating composition was prepared comprising fungicide, insecticide, film forming binder, colorant, Product 14 (3 x 10 ^"7 M) and water. A control coating was also prepared using the identical ingredients, but excluding Product 14. Corn seeds were coated with the seed coating composition using spray treatment method. Five petri dishes and 100 seeds were used to test each seed coating composition. Each corn seed was inspected for uniformity and lack of cracks in the seeds prior to use. A piece of filter paper was used to cover the inner side of each petri dish to allow uniform distribution of water. Twenty corn seeds were placed on the filter paper area of one petri dish with the flat side of the corn seed facing upwards. The seeds were placed one the filter paper so that the corn seeds were not touching each other. 5 ml_ of water was added to each petri dish. The dishes were sealed, stacked and placed in an incubator set to 10°C. The dishes were examined twice per day at the times indicated in Table 3. During the observation period, one stack was removed from the incubator and observed for germination. The dishes were then rotated within the stack and placed back into the incubator before removing another treatment stack.

Statistical analyses were performed by calculating the standard deviation of the five repetitions for each experiment. The data was deemed acceptable when the standard deviation was less than 10%. Standard deviations are indicated in brackets next to their respective treatment averages in the Tables included in Examples 10 to 13. Results are statistically significant when the standard deviations of the treatment averages do not overlap.

Example 10

Effect of Product 13 on Corn Seed Germination at Room

Temperature

Table 1 shows the percent germination of corn seeds treated with diglucosamine Product 13 vs. control treatment maintained at room temperature at selected time points.

TABLE 1

Corn seeds treated with aqueous solutions of Product 13 exhibited statistically significant increases in percent germination at 48 and 60 hours following treatment initiation compared to the control treatment (Table 1 ). Results were deemed statistically significant when the standard deviations (indicated in brackets) of the treatment averages do not overlap.

Example 1 1

Effect of Product 14 on Corn Seed Germination at Room

Temperature

Table 2 shows the percent germination of corn seeds treated with triglucosamine Product 14 vs. control treatment maintained at room temperature at selected time points.

TABLE 2

Corn seeds treated with aqueous solutions of Product 14 exhibited statistically significant increases in percent germination at 48 and 60 hours compared the control treatment (Table 2). Results were deemed statistically significant when the standard deviations (indicated in brackets) of the treatment averages do not overlap.

Example 12

Effect of Product 14 on Corn Seed Germination under Cold Stress Table 3 shows the percent germination of corn seeds treated with triglucosamine Product 14 vs. control treatment maintained under cold stress (10°C) at selected time points. TABLE 3

Corn seeds coated with Product 14 and subjected to cold stress (10°C) conditions exhibited statistically significant increases in percent germination at 132, 144, 156 168 and 180 hours compared the control treatment (Table 3). Results were deemed statistically significant when the standard deviations (indicated in brackets) of the treatment averages do not overlap.

Table 4 shows the percent increase in percent germination of corn seed treated with triglucosamine Product 14 vs. control treatment and maintained under cold stress (10°C) at selected time points.

TABLE 4

Example 13

Effect of Product 13 and Product 14 on Soybean Seed Germination at

Room Temperature

Percent germination of soybean seeds treated with diglucosamine Product 13 triglucosamine or triglucosamine Product 14 vs. control treatment at room temperature at selected time points.

TABLE 5

Soybean seeds treated with aqueous solutions of Product 13 and Product 14 exhibited statistically significant increases in percent

germination at 12 and 24 hours following treatment initiation compared to the control treatment (Table 5). Results were deemed statistically significant when the standard deviations (indicated in brackets) of the treatment averages do not overlap.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.