Compositions comprising a mixture of lipo-chitooligosaccharide(s) and plant biostimulant(s), and uses thereof for increasing plant growth and crop yields

Abstract

Described herein are methods and compositions comprising a combination of lipo- chitooligosaccharide(s) (LCOs) and plant biostimulant(s) for increasing plant growth, and particularly crop yield. Synergistic benefits are obtained when combining the lipo- chitooligosaccharide(s) with the plant biostimulant(s).

Description

COMPOSITIONS COMPRISING A MIXTURE OF LIPO-CHITOOLIGOSACCHARIDE(S) AND PLANT BIOSTIMULANT(S), AND USES THEREOF FOR INCREASING PLANT GROWTH AND CROP YIELDS CROSS REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to provisional patent application No. US 63 / 514,716 filed on July 20, 2023, the content of which is incorporated herein in its entirety. FIELD OF THE INVENTION

[0002] The invention relates to the field of agriculture, and more particularly to synergistic effects associated with a combination of lipo-chitooligosaccharide(s) and plant biostimulant(s) in increasing plant growth, and particularly crop yield. BACKGROUND OF THE INVENTION

[0003] With climate changes and the growth of the global population, agriculture is now, more than ever, under strong pressure to feed the planet. The agricultural industry is thus permanently searching for ways to improve production and yield, particularly in conditions that are becoming warmer, drier or wetter, more variable and less stable. Pest control is also a major concern.

[0004] Under nitrogen-limiting conditions, Gram-negative soil bacteria commonly named as rhizobia can establish root symbiosis with certain legumes and fix atmospheric nitrogen. These bacterial include genera of Rhizobium, Bradyrhizobium, Sinorhizobium, mesorhizobium, Phylorhizobium, and Azorhizobium. Rhizobia and form specialized organs called nodules on the roots, and sometimes stems, of legumes. Nodule formation is a highly specialized process that is modulated by signal molecules including plant-to- bacteria signal molecules and bacteria-to-plant signal molecules.

[0005] Lipo-chitooligosaccharides (LCOs) are examples of bacteria-to-plant signal molecules. Lipo-chitooligosaccharides (LCOs) are known to affect several host plant physiological processes, including inducing root hair deformation, inducing ontogeny ofcompete nodule structures, inducing cortical cell division and inducing the expression of host nodulin genes essential for infection thread formation LCOs have also been shown to activate defense-related enzymes. Patent US 7,250,068 teaches increasing photosynthesis in plants by exposing the plants to lipo-chitooligosaccharides. Patent publication US 2021 / 0235704 discloses combinations of lipo-chitooligosaccharides for enhancing plant growth. Patent US 10,973,230 teaches the combined used of lipo- chitooligosaccharides and chitinous compounds for enhancing plant growth and yield. Patents US 8,992,653 and US 11,560,340 disclose seed treatment methods comprising contacting seeds with LCO. Patent publication US 2018 / 0235219 teaches compositions comprising lipo-chitooligosaccharides and microorganisms for enhancing plant growth. Although there is more an more interest about using LCOs, there is still a need for compositions comprising such molecules for increasing yield in commercial agriculture and large-scale farming.

[0006] Bacillus thuringiensis MS 20 produces Bacillin 20, a peptide that has been tested in the field for biostimulant efficacy against infections from Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato (see International PCT application PCT / IB2024 / 052728, filed on March 21, 2024). Likewise, patent publication US 2003 / 0228679 discloses bacteria of the species Bacillus subtilis (i.e., NEB4 and NEB5) and of the species Bacillus thurigiensis (i.e., NEB17) which can promote plant growth. Particularly, B. thuringiensis NEB17 produces a polypeptide named Thuricin-17, a polypeptide which has been suggested of having a positive impact on plant growth, at least under certain conditions (see international PCT patent publication WO 2008 / 138129; Marion P. et al., Agronomy for sustainable development 35 (2015): 749-757; Gray, E. J., and Donald L. Smith, Soil biology and biochemistry 37, no.3 (2005): 395-412; Smith D. et al., Frontiers in plant science 6 (2015): 722; Lee K. D. et al., Planta 229 (2009): 747-755; Jung, W.-J. et al., Journal of Plant Biology 51 (2008): 145-149; Subramanian S. et al., PLoS One 11, no.8 (2016): e0160660; Subramanian S. et al., Frontiers in plant science 7 (2016): 1314).

[0007] Nevertheless, plant-microbe interaction is an area that is largely unexplored and unexploited for the improvement and optimization of plant production. As such, prior to the present invention, no one had ever combined lipo-chitooligosaccharides (LCOs)and a plant biostimulant such as Bacillin 20 in a single mixture, nor tested the effect of such combination on plants, let alone agricultural production or yield.

[0008] Accordingly, there is also a need for compositions and methods allowing to modulate and / or take advantage of plant-to-bacteria and bacteria-to-plant communications and molecular signaling in agriculture.

[0009] There is also a need for compositions and methods allowing to increase agricultural production in commercial and large-scale agriculture.

[0010] There is particularly a need for compositions combining both lipo- chitooligosaccharides (LCOs) and a plant biostimulant such as Bacillin 20 in a single mixture, the combination providing more benefits on plant growth, including crop yield, than each individual components used individually.

[0011] There is also a need for methods in which compositions capable of increasing agricultural production or crop yield are contacted with the plants (e.g., roots or seed).

[0012] There is particularly a need for seed coating compositions that can provide benefits such as improving or enhancing growth parameters such as crop yields.

[0013] The present invention addresses these needs and other needs as it will be apparent from the review of the disclosure and description of the features of the invention hereinafter. BRIEF SUMMARY OF THE INVENTION

[0014] According to one aspect, the invention relates to a composition for increasing plant growth comprising a combined mixture of at least one lipo-chitooligosaccharides (LCO) and one or more plant biostimulant.

[0015] According to another aspect, the invention relates to a composition comprising at least one lipo-chitooligosaccharide (LCO) and one or more plant biostimulant, wherein said at least one LCO and said one or more plant biostimulant are present in amounts effective to synergistically enhance one or more growth parameters of a plant when said composition is applied to said plant.

[0016] According to another aspect, the invention relates to a treated seed comprising a plant seed that is at least partially coated with a seed treatment composition, wherein said seed treatment composition comprises at least one lipo-chitooligosaccharide and one or more one or more plant biostimulant.

[0017] According to another aspect, the invention relates to a method for enhancing plant growth, comprising applying at least one lipo-chitooligosaccharide (LCO) and one or more plant biostimulant to said plant in amounts effective to synergistically enhance one or more growth parameters of said plant.

[0018] According to another aspect, the invention relates to a method for enhancing plant growth, comprising applying at least one lipo-chitooligosaccharide (LCO) and one or more plant biostimulant to a plant seed in amounts effective to synergistically enhance one or more growth parameters of the plant that grows from said seed.

[0019] According to another aspect, the invention relates to a method for increasing crop yield, comprising contacting plants with at least one lipo-chitooligosaccharide (LCO) and one or more plant biostimulant, wherein said contacting synergistically enhance crop yield of plants contacted with both compounds, as compared to contacting plants with only the at least one lipo-chitooligosaccharide (LCO) or only the one or more plant biostimulant.

[0020] According to another aspect, the invention relates to a method of increasing corn yield, said method comprising applying at least one lipo-chitooligosaccharide (LCO) and one or more plant biostimulant to corn seed in amounts effective to synergistically increase the yield of corn grown from said corn seed, and wherein said at least one lipo- chitooligosaccharide and said one or more plant biostimulant are applied to said corn seed prior to planting.

[0021] According to another aspect, the invention relates to a kit for stimulating plant growth, comprising: (i) a first container comprising a concentrated solution of at least one LCO; and (ii) a second container comprising a concentrated solution of one or more plant biostimulant.

[0022] According to another aspect, the invention relates to a kit for stimulating plant growth, comprising: (i) a first container comprising a mixture of at least one LCO and one or more plant biostimulant; and (ii) a second container comprising at least one of a herbicide, an insecticide, a fungicide, a bactericide, and a nutrient.

[0023] According to another aspect, the invention relates to the use of at least one LCO and one or more plant biostimulant for stimulating plant growth, wherein said at least one LCO and one or more plant biostimulant are applied on a plant simultaneously or concurrently.

[0024] Additional aspects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments which are exemplary and should not be interpreted as limiting the scope of the invention. DETAILED DESCRIPTION OF EMBODIMENTS

[0025] The following description provides embodiments by which the invention may be practised. It will be understood that other embodiments may be made without departing from the scope of the invention disclosed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. General overview

[0026] Lipo-chitooligosaccharides (LCOs) are known to promote plant growth by triggering the formation of root nodules which are utilized by Rhizobium bacteria for nitrogen fixation in leguminous plants.

[0027] Plant biostimulants are natural and environmentally friendly substances with positive effects on plant growth. Plant biostimulants can enhance plant metabolism and physiology when applied in small doses.

[0028] Despite the above, plant-microbe interactions has largely been unexplored and unexploited for the improvement and optimization of plant production, relief of plant stress, and disease resistance.

[0029] The present invention proposes to modulate and / or take advantage of plant- to-bacteria and bacteria-to-plant communications and molecular signaling for agriculture.

[0030] More specifically, therefore, the present invention concerns the demonstration that contacting lipo-chitooligosaccharide(s) (LCOs) with plant biostimulant(s) significantly increases plant growth, and particularly crop yield.

[0031] Applicant has found that synergistic effects can be obtained by combining lipo- chitooligosaccharide(s) (LCOs) with plant biostimulant(s). In a particular set of experiments, a composition of the present invention comprises a mixture of at least one LCO and one or more plant biostimulant (e.g., Bacillin 20) synergistically increased yield of corn.

[0032] Prior to the present invention, no one had ever combined lipo- chitooligosaccharides (LCOs) and a peptidic plant biostimulant such as Bacillin 20 in a single mixture, nor tested the effect of such combination on plants, let alone agricultural production or yield. The surprising benefits associated with such combination thus open the way to numerous applications in the field of agriculture. Lipo-chitooligosaccharides (LCOs)

[0033] As used herein, the term “lipo-chitooligosaccharides” or "LCO" broadly refers to a Nod factors which is under the control of at least one nodulation gene (nod gene) common to rhizobia. LCO therefore relates to a bacteria-to-plant signal molecule which induces the formation of nodules in legumes and enables the symbiotic bacteria to colonize same. Broadly, LCOs are lipo-chitooligosaccharide signal molecules, acting as phytohormones, comprising an oligosaccharide moiety having a fatty acid condensed at one of its end. LCOs from B. japonicum have been characterized in U.S. Pat. Nos. 5,175,149 and 5,321,011, which are incorporated herein by reference. Additional LCOs, including chemical structures thereof, have been described also in the following USpatents and US patent publications, which are incorporated herein by reference: US 7,250,068; US 8,992,653; US 10,973,230; US 11,560,340; US 2021 / 0235704 and US 2018 / 0235219. LCOs are also commercially available from Novozymes (Milwaukee, WI, USA) under the brand names Ratchet™, Torque™ IF, and B360™. In embodiments, the present invention encompasses the uses of one or more of these known LCOs.

[0034] The present invention encompasses compositions comprising various LCOs and it is not limited the LCOs exemplified hereinafter. Indeed, while the present examples hereinafter refer to LCOs obtained from B. japonicum, it is envisioned that other LCOs may also be effective as that of the LCOs exemplified herein. For instance, effective LCOs can be synthetic or naturally occurring. LCOs may be isolated and / or purified or purchased commercially (see above). LCOs may be isolated and / or purified from a species of rhizobia which is capable of entering into a nitrogen fixation relationship with a plant including but not limited to, Bradyrhizobium spp., Rhizobium spp., Sinorhizobium spp. Azorhizobium spp. and Thizobium spp., such as Bradyrhizobium japonicum, Rhizobium leguminosarum, S. melilot, Thizobium meliloti and Allorhizobium spp. In one particular embodiment, the LCO is obtained from Bradyrhizobium japonicum strain 532C (see Soulemanov et al., Microbial Research, Vol. 157, 1, 2022, p. 25-28). In one particular embodiment the chemical name of the LCO, based on its structure, is Nod Bj-V (C18:1, MeFuc). It is within the skills of those in the art to identify and test LCOs displaying beneficial properties on crops (preferably synergistic properties), when combined with one or more plant biostimulant (such as Bacillin 20) as disclosed and claimed herein. Plant biostimulants and Bacilin 20

[0035] As used herein, the term "plant biostimulant" or “biostimulant" refers to a biological agent or combination of agents, which application to a plant enhances one or more metabolic and / or physiological processes of the plant or part thereof (e.g., stress mitigation, respiration, photosynthesis, nucleic acid uptake, ion uptake and / or nutrient delivery). In embodiments, the plant biostimulant is a peptide produced by a bacterium, for instance Bacillus thuringiensis or Bacillus subtilis, including but not limited to Bacillus thuringiensis MS 20, Bacillus thurigiensis NEB17, Bacillus subtilis NEB4 and Bacillus subtilis NEB5.

[0036] In embodiments, the plant biostimulant consists of Bacillin 20, a peptide produced the bacterium Bacillus thuringiensis strain 20 (BtMS20). BtMS20 was deposited at the International Depositary Authority of Canada (IDAC) on November 3rd, 2020 under Accession No.301020-01. In embodiments, the Bacillin 20 peptide comprises the amino acid sequence DWTCWSCLVCAACSVELLNLVTAATGASTAS (SEQ ID NO:1).

[0037] In other embodiments, the plant biostimulant consists of Thuricin 17, a peptide produced by Bacillus thuringiensis strain NEB17 (the strain was deposited at the International Depositary Authority of Canada, Winnipeg, Manitoba, Canada, on March 27, 2003 under Accession No. 301020-01. In embodiments, the Thuricin 17 peptide comprises the amino acid sequence DWTCWSCLVCAACSVELLNLVTAATGASTAS (SEQ ID NO:1).

[0038] In other embodiments, the plant biostimulant consists of a peptide produced by Bacillus subtilis strain NEB4 or a peptide produced by Bacillus subtilis strain NEB5. The B. subtilis strain NEB4 was deposited at the International Depositary Authority of Canada, Winnipeg, Manitoba, Canada, in 2003 under Accession No. AF406704. The B. subtilis strain NEB5 was deposited at the International Depositary Authority of Canada, Winnipeg, Manitoba, Canada, in 2003 under Accession No. AF406705.

[0039] The plant biostimulant may comprises an isolated peptide comprising SEQ ID NO: 1 as defined hereinabove, as well as any fragments of SEQ ID NO: 1, or any isolated protein, fusion protein or peptide comprising SEQ ID NO: 1, and having substantially similar biological activity as the peptide of SEQ ID NO: 1, particularly in displaying plant biostimulant activity and / or in improving crop yield in combination with LCO(s) as described herein.

[0040] In embodiments, the plant biostimulant may comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of SEQ ID NO:1. Sequence identity may be determined using any suitable software, for instance by using the BLAST™ algorithms (e.g., BLASTp), available through the internet at the National Center for Biotechnology Information (NCBI).

[0041] The peptide may comprise a fragment of SEQ ID NO:1, the fragment comprising at least 25, 26, 27, 27, 28, or 29 consecutive amino acids of SEQ ID NO:1. In accordance with the present invention the fragment should have substantially identical plant biostimulant activity and / or be able to improve crop yield in combination with LCO(s), in a substantially identical manner to than peptide of SEQ ID NO:1 as described herein.

[0042] Plant biostimulants useful in practicing the methods of the invention, including Bacilin 20, Thuricin 17, peptide(s) from B. subtilis strain NEB4 or B. subtilis strain NEB5 and / or related peptides may be obtained in a number of ways. For example, any polypeptide of interest may be screened either sequentially in either order, or simultaneously, for a desired biological activity, including but not limited to plant biostimulant activity and / or improving crop yield in combination with LCO(s) as described herein.

[0043] In one embodiment plant biostimulant polypeptides are obtained from a bacterial species that express the polypeptides. For instance, suitable bacterial strain may be cultured under conditions sufficient for expression of the polypeptide and the polypeptide recovered from the culture medium. The polypeptide may be purified e.g., by chromatography (e.g., high-performance liquid chromatography), gel electrophoresis, filtration, dialysis, precipitation, centrifugation, etc. or combinations thereof. It may also be possible to obtain the biostimulant peptides of interest by chemical synthesis and / or by overexpression in a bacterial system.

[0044] In embodiments, Bacilin 20 is produced by the bacterium BtMS20 and it is obtainable therefrom (e.g., purified). According to one particular approach, the bacterium BtMS20 is cultured in King’s B liquid medium at 28 ±2 °C on an orbital shaker for at least 96 hours, the entire culture is extracted with of n-butanol (shaking for 30 min keeping overnight at room temperature and the separated butanol phase is collected and evaporated under vacuum at 50 °C using a rotary evaporator. The dried extract is resuspended in 20% acetonitrile and loaded on PrepSep C18™ cartridge (Fisher Scientific, cat# 03-251-292) and fractionated using 20, 30, 35, 40, 60, 70 and 100% acetonitrile (in water, v / v). These fractions are collected, and aliquots are used for HPLC analyses and Bacilin 20 quantifications in fractions.

[0045] Accordingly, the present invention also encompasses bacterial extracts, such as extracts from BtMS20, comprising one or more Bacilin 20 polypeptides as defined herein. The present invention further encompasses compositions comprising peptides (e.g., Bacilin 20 and / or fragment or peptides deriving therefrom as defined herein), proteins and bacterial strain (e.g., BtMS20) as defined herein.

[0046] The present invention further encompasses bacterial extracts, such as extracts from Bacillus thurigiensis NEB17 comprising one or more Thuricin 17 polypeptides as defined herein, or extracts from B. subtilis strain NEB4 or from B. subtilis strain NEB5, compositions comprising such peptides (e.g., isolated peptides and / or fragment deriving therefrom), and proteins and bacterial strains (e.g., NEB17, NEB4 and NEB5) as defined herein. Uses and benefits of synergistic compositions

[0047] the present invention may be applied to the plant either before, during or after planting and may be applied to, for example, plant leaves, stems, roots, or seeds.

[0048] A particular aspect of the invention concerns compositions combining both, at least one lipo-chitooligosaccharide (LCO) with one or more biostimulant. Applicant has found that synergistic effects on plant growth (e.g., crop yield) can be obtained when combining such compounds.

[0049] Accordingly, the present invention encompasses compositions comprising at least one lipo-chitooligosaccharide (LCO) and at least one biostimulant in amounts effective to synergistically enhance one or more growth parameters of a plant when said composition is applied to the plant, e.g., the seed from which said plant is grown.

[0050] In embodiments, the term “amounts effective to synergistically enhance one or more growth parameters of the plant” refers to a quantity which is sufficient to result in enhancement of the growth of the plant as compared to growth of a control-treated (or untreated) crop plant. In particular embodiments, enhancement of growth is displayed as an increased crop yield. Examples of growth parameters are provided hereinafter.

[0051] As used herein, “synergistically enhance”, “synergistic enhancement” or “synergistic combination” and grammatical variants thereof) refers to a combined effect that is more than a simple additive effect. In the context of the present invention, it means that the combined use of at least one lipo-chitooligosaccharide (LCO) and one or more plant biostimulant provides benefit(s) to the plant that is(are) greater than the sum of benefit(s) for the at least one lipo-chitooligosaccharide (LCO) and the one or more plant biostimulant when used separately. For instance, the Examples hereinafter demonstrate that corn seed treatment with a mixture comprising LCO + Bacillin 20 provided an increase in corn yield that was greater than simple a coating of LCO alone or a coating of Bacillin 20 alone, thereby suggesting a synergistic effect when using both compounds in combination. In preferred embodiments, both compounds are applied to the plant(s) concurrently or contemporaneously, for instance by contacting the plant(s) with a mixture comprising both compounds. It is also conceivable to obtains synergistic effects if the compounds are applied to or contacted with the plant(s) (e.g., seed) in sequential order (e.g., one before or after the other).

[0052] The yield-promoting activity of the compositions of the invention may be observable over a broad range of concentrations. In embodiments, the LCO(s) is(are) present in a concentrated liquid mixture at a concentration between about 10-7M to about 10-9M and it is used as a diluted liquid solution (e.g., a dilutive factor of 10X, 50X, 100X, 500X, 1000X or more) to be applied on plants at a concentration of about 10-7M to about 10-12M, preferably about 10-9M to about 10-12M, and more preferably about 10-8M to about 10-10M.

[0053] In embodiments, a dose of about 8500 mg of LCO is used to treat seeds for over 40000 ha of corn (i.e., about 25 kg of corn seeds / ha). In embodiments, a dose of about 8500 mg of LCO is used to treat seeds for over 20,000 ha of soybean (i.e., about 70-100 kg of soybean seeds / ha). In embodiments, a dose of about 6000 mg to about 22 000 mg of LCO is used to treat about 750,000 kg to about 2,500,000 kg of corn seeds.

[0054] In embodiments, the plant biostimulant is present in a concentrated liquid mixture at a concentration between about 10-6M to about 10-10M and it is used in a diluted liquid solution (e.g., a dilutive factor of 10X, 50X, 100X, 500X, 1000X or more) and appliedon plants at a concentration of about 10-7M to about 10-13M, preferably about 10-8M to about 10-12M, and more preferably about 10-9M to about 10-11M.

[0055] In embodiments, a dose of about 200 mg of Bacilin 20 (in an isolated form or as part of a bacterial extract) is used to treat seeds for over 40,000 ha of corn (i.e., about 25 kg of corn seeds / ha). In embodiments, a dose of about 200 mg of Bacilin 20 is used to treat seeds for over 20,000 ha of soybean (i.e., about 70-100 kg of soybean seeds / ha). In embodiments, a dose of about 150 mg to about 470 mg of Bacilin 20 is used to treat about 750,000 kg to about 2,500,000 kg of corn seeds.

[0056] Compositions of the invention may contain dilutents, adjuvants, excipients, carriers, wetting agents, surfactants, anti-freezing agents, etc. known in the art as suitable for inclusion in plant growth and / or crop yield promoting compositions. In another aspect, the invention provides a composition comprising a LCO and a plant biostimulant as described herein, and a carrier or diluent. The compositions may be in, for example, solid (such as powdered) or liquid form. In embodiments the composition is sterilized for greater stability and the components remain stable and active for more than 2 years.

[0057] As used herein, the term "carrier" means an agronomically acceptable carrier, a oil compatible carrier, a seed-compatible carrier and / or a foliar-compatible carrier. An "agronomically acceptable carrier" means any material which can be used to deliver the actives (e.g., LCO(s) + biostimulant(s), etc.), to a plant, plant part or plant growing locus (e.g., foliage, seed, soil). As used herein, the term "soil compatible carrier" means any material which can be added to a soil without causing / having substantial adverse effect on plant growth, soil structure, soil drainage, or the like. As used herein, the term "seed-compatible carrier" means any material which can be added to a seed without causing / having an adverse effect on the seed, the plant that grows from the seed, seed germination, or the like. As used herein, the term "foliar-compatible carrier" means any material which can be added to a plant or plant part without causing / having an adverse effect on the plant, plant part, plant growth, plant health, or the like.Uses and methods of increasing plant growth and plant production

[0058] Anotherenhancing plant growth and / or for increasing crop yield comprising by using a synergistic combination of at least one LCO and one or more biostimulant (e.g., Bacillin 20).

[0059] As used herein, the term "plant" can refer to a whole plant, any part thereof, or a cell or tissue culture derived from a plant. Thus, the term "plant" may refer to any of whole plants, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, plant cells and seeds. The term "plant" includes all naturally occurring and non-naturally occurring plant populations, including, agricultural, horticultural and silvicultural plants. Thus, the term "plant" encompasses plants obtained by conventional plant breeding and optimization methods (e.g., marked-assisted selection) and plants obtained by genetic engineering (e.g., transgenic), including cultivars protectable and not protectable by plant breeders' rights.

[0060] The composition of the present invention is preferably applied in an amount effective for increasing plant growth. In embodiments, the composition of the present invention is applied in an effective amount. Terms like "effective amount," "effective concentration," and "effective dosage" (and grammatical variants thereof) refer to an amount, concentration or dosage that is sufficient to cause a desired effect (e.g., enhanced plant growth). As used herein, the terms "enhanced growth", "enhanced plant growth", and the like refer to an improvement in one or more characteristics or parameters of plant growth and / or plant development as compared to one or more control plants (e.g., a plant germinated from an untreated seed or an untreated plant). Exemplary plant growth / development characteristics or parameters include, but are not limited to, biomass, height, leaf length, leaf mass, leaf number, leaf surface area, leaf volume, nutrient uptake, root area, root diameter, root length, root mass, root nodulation, root number, root surface area, root volume, nitrogen fixation, seed germination, seedling emergence, shoot diameter, shoot length, shoot mass, shoot number, shoot surface area, shoot volume, plant spread, plant survival rate, and plant or crop yield.

[0061] As used herein, the terms “increasing crop yield” or “improving yield” and the like refer to an improvement in one or more characteristics of plant yield or production, as compared to one or more control plants (e.g., a plant germinated from an untreated seed or an untreated plant). Exemplary plant yield / production characteristics include, but are not limited to, increased biomass, increased fruit number increased bolls, increased seed number or size, or a combination thereof as measured by bushels per acre, increased root number, increased root mass, increased root volume, increased leaf area, increased plant stand, increased plant vigor, improved disease resistance, reduced or inhibited pathogen infestation, faster seedling emergence (e.g., enhanced emergence), faster germination, (e.g., enhanced germination), or combinations thereof) when compared to an untreated plant or untreated plant part. In embodiments, the increase of crop yield associated with using the composition of the invention is more than 10, 20, 30, 40, 50, 60, or 70%, compared to a crop from control plants (e.g., UTC).

[0062] In embodiments, the at least one LCO is applied to plant seeds at a rate of about 0.8 ng per kg to about 8000 ng per kg, or about 8 ng per kg to about 800 ng per kg, or about 80 ng per kg to about 400 ng per kg. In embodiments, the biostimulant is applied to plant seeds at a rate of about 0.1 ng per kg to about 15000 ng per kg, or about 1 ng per kg to about 1500 ng per kg, or about 10 ng per kg to about 150 ng per kg.

[0063] In embodiments, the composition comprising both the LCO and biostimulant can be applied in an amount that ranges between about 0.1 to about 10 liter / A, or between about 0.5 to about 5 liter / A, or between about 1 to about 3 liter / A

[0064] The absolute value of the amount / concentration / dosage that is sufficient to cause desired effect(s) may be affected by factors such as size (e.g., area, total acreage, etc.) of land being treated, the type and condition of soils, the species, variety and / or condition of the plant, the environment (e.g., whether the plant is in a pot, a greenhouse, a field), etc. Those skilled in the art will understand how to select an effective amount / concentration / dosage using routine dose-response experiments.

[0065] A composition in accordance with the present invention may be applied by any suitable means or method including, but not limited to, seed coating, application to seedsprior to and / or during planting, root inoculation, rood dipping, soil drenching, soil application (e.g., injection), foliar spray (e.g., high- or low-pressure spraying), etc. The composition may be applied either in a solid form (e.g., as a free-flowing powder) or liquid form (such as in an aqueous carrier). The composition may also be applied to a delivery vehicle, wherein the delivery vehicle serves as a means of transporting the LCO and biostimulant to the soil, plant, seed, field, etc. For example, the compounds or composition can be applied to a delivery vehicle (e.g., a particle, a polymer, or a substrate) to be used in filtration systems for the treatment of irrigation water. In some embodiments, the compounds or composition is to be applied to a polymer as a wetting agent and / or gel that releases water as needed.

[0066] Seed coating and root dipping prior to planting are two preferred techniques. Seed coating, root irrigation, root dipping, soil drenching, foliar applications such as spray treatments of leaves, etc. are well-known in the art and the method of administration of a composition of the present invention to the seeds, root, leaves, etc. can be adapted by a skilled artisan to meet particular needs. For instance, it may be envisioned to deliver the composition of the invention to seeds in slow-release formulations, such as beads or gels. The skilled person can readily determine suitable application regimes for the composition.

[0067] The invention also concerns plant seeds pre-treated with a composition of the invention, e.g., the composition is applied to the seeds in advance of planting (e.g., a seed coating that has occurred 1 week, 2 weeks, 4 weeks, 2 months, 3 months, 4 months, or 6 months, or 1 year or more prior to planting). Accordingly, in another aspect the invention relates to plant seeds treated and / or coated with a composition as defined herein. In embodiments, the material coated on dry seeds is effective for at least 1 year or at least 2 years.

[0068] The invention also related to foliar applications with a composition of the invention. As used herein, the term "foliage" refers to those portions of a plant that normally grow above the ground, including, but not limited to, leaves, stalks, stems, flowers, fruiting bodies, and fruits. As used herein, the terms "foliar application", "foliarly applied" and grammatical variations thereof, refer to the application of one or more active ingredients to the foliage of a plant (e.g., to the leaves of the plant). Application may beeffected by any suitable means, including, but not limited to, spraying the plant with a composition comprising the active ingredient(s). In some embodiments, the active ingredient(s) is / are applied to the leaves, stems and / or stalk of the plant and not to the flowers, fruiting bodies or fruits of the plant.

[0069] The compositions of the invention can be applied at a particular time, or one or more times, depending on parameters including, but not limited to, soil types, soil conditions, environmental conditions, time of planting, plant susceptibility, etc.

[0070] In one particular embodiment, the method for increasing crop yield comprises contacting plants with at least one lipo-chitooligosaccharide (LCO) and one or more plant biostimulant.

[0071] In one particular embodiment, the method for enhancing plant growth comprises applying at least one lipo-chitooligosaccharide (LCO) and one or more plant biostimulant to a plant seed in amounts effective to synergistically enhance one or more growth parameters of the plant that grows from said seed.

[0072] When treating seeds, the at least one lipo-chitooligosaccharide (LCO) and the one or more plant biostimulant can be applied by a variety of techniques including, but not limited to, high- or low-pressure spraying, coating, immersion, and injection. Once treated, seeds can be planted in natural or artificial soil and cultivated using conventional procedures to produce plants. After plants have propagated from seeds treated with the compounds or composition, the plants may further be treated with one or more applications of the disclosed composition or one or both individual compounds.

[0073] A composition in accordance with the present invention may additionally or alternatively be applied to the growing environment of the plant or seed rather than directly to the plant or seed. By “growing environment” is meant the area sufficiently proximal to the plant or seed (such as to the soil adjacent to the plant or seed) that the composition and / or individual components can affect have a desired beneficial effect on the plant. If the composition is applied to the soil, it may be applied before, during or after planting.

[0074] Compositions in accordance with the present invention may also find additional useful preventive and / or curative applications for various agricultural crops (e.g., cereals), fruits (e.g., small fruits, citrus), vegetables, grass, turf, ornamental plants, etc.

[0075] While the present invention has been demonstrated in the Examples using corn, the invention should not be so limited. Indeed, it will be clear to a person skilled in the art to which the present invention pertains that other types of plants may respond similarly to the combined LCO+biostimulant application, by displaying an increase in crop yield thereof.

[0076] Therefore, the compositions and methods of the invention are not limited to use with any particular plant or plant-type. In embodiments, the invention is used for enhancing growth of one or more of monocot, dicot, C3 plants and C4 plants. Examples of plants with which the compositions and methods of the invention may be practiced include, without limitation: cereals (e.g., wheat, rye, maize, corn, sorghum, oat, barley, bulgur, rice, millet, and canola), rice (black rice, brown rice, red rice), tree nuts (e.g., almonds, cashews, pecans, pistachios, etc.) ground nuts (e.g., peanuts), fruits (e.g., tomato), vegetables (e.g., potato), oil seed crops (e.g. canola, soybeans, cotton), forage crops (e.g., alfalfa and clover), plants of lesser agricultural importance (e.g., lupine, sainfoin, trefoil, and even some small tree species), horticultural tree species (e.g., peach, apple, plum, pear, mango), forestry tree species (e.g., spruce, pine, fir, maple, oak, poplar), flowers, cannabis, grass, turf, and ornamental plants. These may be grown in the fields or in greenhouses.

[0077] As used herein, the term "agriculturally beneficial agent" refers to any agent (e.g., chemical or biological agent) or combination of agents the application of which causes and / or provides a beneficial and / or useful effect in agriculture (e.g., enhanced plant growth). Such agents include, but not limited to, agriculturally beneficial microorganisms (e.g., rhizobacteria), biostimulants, nutrients, chemical fertilizers, flavonoids, pesticides (e.g., acaricides, fungicides, gastropodicides, herbicides, insecticides, nematicides, rodenticides, and virucides), and plant signal molecules such as lipo-chitooligosaccharides, chitooligosaccharides, chitinous compounds, flavonoids, jasmonic acid or derivatives thereof, linoleic acid or derivatives thereof, linolenic acid orderivatives thereof, karrikins, etc. In embodiments, the methods of the invention further comprise applying at least one of a herbicide, an insecticide, a fungicide, a bactericide, and a nutrient.

[0078] As used herein, the terms "agriculturally beneficial microorganisms," "agriculturally beneficial microbe," "beneficial microorganism," and "beneficial microbe" refer to a microorganism having at least one agriculturally beneficial property (e.g., the ability to produce an agriculturally beneficial agent, such as a plant signaling molecule described herein; to enhance nutrient and / or water uptake; to promote and / or enhance nitrogen fixation; to enhance growth; to enhance seed germination; to enhance seedling emergence; to increase seed number or size; to break the dormancy or quiescence of a plant; etc.).

[0079] The compositions of the invention may be used, and the methods of the invention practiced, wherever plants are grown. Accordingly, the present invention encompasses uses of the compositions and methods as defined herein in a variety of plant environments such as fields, greenhouse facilities, vertical farms, urban greening systems, hydroponic systems and laboratories. The compositions may be used with plants that are grown at temperatures above 30°C, at which temperatures nitrogen fixing rhizobacteria are generally most active, or also at low temperatures, such as at an average daily root zone temperature below 28, 26, 24, 22, 20, 18, 16, 14, 12, or 10°C.

[0080] Examples of potentially useful herbicides include, but are not limited to, Roundup™, atrazine, 2,4-D (Weed-B-Gon™), dicamba (Banvel™), imazethapyr (Pursuit™), metolachlor (Dual™), S-metolachlor (Dual II Magnum™), pendimenthalin (Prowl™), clethodim (Select™), triclopyr (Garlon™), clopyralid (Stinger™), Fluro Star™, flufosinate (Liberty™), halosulfuron (Permit™), isoxaben (Gallery™), sethoxydim (Poast™), bentazon (Basagran™), pyraflufen-ethyl (Epic™), fomesafen (Reflex™), trifluralin (Treflan™), mesotrione (Callisto™), flumioxazin (Valor), dicamba (Banvel™, Clarity™), and imazapyr (Arsenal™).

[0081] Examples of potentially useful insecticides include, but are not limited to, pyrethoids, pyrethins, neonicotinoids, organophosphates, carbamates, spinosad,imidacloprid, fipronil, malathion, permethrin, cypermethrin, and chlorpyrifos. Examples of potentially useful bactericide include, but are not limited to, copper sulfate, zinc sulfate, peracetic acid, quaternary ammonium compounds, streptomycin, kasugamycin, tetracycline hydrochloride, phyton 27™, aureobasidium pullulans, Actigard™, and Agri- strep™. Examples of potentially useful nutrients include, but are not limited to, nitrogen, phosphorus, potassium, sulphur, magnesium, calcium and other plant micronutrients.

[0082] Examples of potentially useful fungicides include, but are not limited to, copper fungicide that includes copper sulfate, copper sulfate pentahydrate, copper hydroxide, copper oxychloride sulfate, cuprous oxide, copper octanoate, and mixtures thereof. Existing commercial examples include, but are not limited to, Kocide 3000TM, Kocide 2000™, Cuprofix Ultra 40 Disperss™, Nordox 75 WG™, Champ WG™, Cueva™, Badge SC™, Basic Copper 53™, Bonide™ copper fungicide, Camelot O™, Nu-Cop 3L™, C-O- C-S WDG™, Previsto™, Badge X2™, Badge MAXX™, Cuproxat™ flowable copper fungicide, tribasic copper sulfate, Nordox 30 WG™, Copper-Count-N™ fungicide, Cuprablau Z 35 WP™ and Captain Jack’s™ liquid copper fungicide.

[0083] The compositions and methods of the invention may provide numerous benefits. In embodiments, using the composition of the provide one or more of the following benefits: − mitigating negative effects and stress induced by application of herbicide(s) and pesticide(s) to plants and crops (e.g., by at least 2%, 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25%, 30% or more); − increasing crop yields (e.g., by at least 2%, 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25%, 30% or more); − increasing plant growth (e.g., by at least 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25% or more); − increasing leaf area, greenness, number and / or biomass (e.g., by at least 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25% or more); − stimulating the plant root system (e.g., by at least 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25% or more); − stimulating bacterial production of phytohormone(s); − stimulating bacterial production of siderophore(s);− increasing induced resistance to plant pathogens (e.g., by at least 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25% or more); − stimulating production of antibiotics; − enhancing early seedling, germination and / or growth (e.g., by at least 5%, 10%, 15%, 20%, 25% or more); − increasing nodule number (e.g., by at least 5%, 10%, 15%, 20%, 25% or more); − increasing plant dry weight (e.g., by at least 5%, 10%, 15%, 20%, 25% or more); − increasing root irrigation nodule number (e.g., by at least 5%, 10%, 15%, 20%, 25% or more); − increasing concentration of root irrigation in shoot tissue (e.g., by at least 5%, 10%, 15%, 20%, 25% or more); − increasing photosynthetic rate (e.g., by at least 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25% or more); − increasing root and shoot length (e.g., by at least 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25% or more); − increasing in energy and anti-oxidant biosynthetic pathways (e.g., by at least 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25% or more); − up-regulating defense related genes (e.g., by at least 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25% or more); − mitigating salt-related stress; − providing bacteriocin capabilities; − activating carbon energy and metabolisms pathway proteins; − providing positive metagenomic effects by increasing number of root microbes which enhances crop growth (e.g., by at least 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25% or more); − enhancing root-hair production and nutrient uptake (e.g., by at least 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25% or more); − protecting crops from abiotic stress; − improving plant resistance to disease(s) (e.g., caused by viruses, fungi, and / or bacteria);− reducing the need for synthetic fertilizers and pesticides yields (e.g., by at least 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25% or more); − promotion of symbiotic relationship between bacteria, inoculants and host crop; − increasing number of root microbes (e.g., by at least 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25% or more); − acting as a signaling compound (e.g., hormone-like activity); − increasing farm revenues; − improving global food security; and − cost efficient and economical to apply.

[0084] In embodiments, benefit(s) is(are) observed or determined by comparing plant(s) contacted or not with the two compounds or with the composition of the invention. In embodiments, benefit(s) is(are) observed when comparing plant(s) contacted with the composition versus plant(s) contacted with a reference treatment (e.g., UTC or compounds individually). In embodiments, seeds are treated prior to planting and the reference treatments consist of a LCO and a plant biostimulant (e.g., Bacillin 20), both applied individually as a seed coating. In embodiments, the benefit(s) is(are) observed or determined about 3, 5, 7, 14, 21, 28, 35, 42, 60 days, or about 3 months, or about 4 months after contacting the plant with the two compounds or with the composition of the invention. Packages / Kits

[0085] The composition in accordance with the invention may be provided in the form of a package or kit containing the individual components (i.e., LCO(s) and plant biostimulant(s) separately) or a composition comprising both components, and optionally additional elements such as instructions for uses in promoting plant growth.

[0086] In one embodiment, the kit comprises separate containers such as bottles, e.g., a bottle comprising the LCO(s) and another bottle comprising the plant biostimulant(s) (e.g., Bacillin 20), and optional instructions for mixing both product prior use. In embodiments, the LCO(s) and biostimulant(s) are provided in a concentrated format (either as a mixture or as individual components) for dilution prior use (e.g., 10x, 50x, 100x, 500x, 1000x, 10000x concentrate).

[0087] In another embodiment, the kit comprises a first container having a composition comprising both LCO(s) and the plant biostimulant(s) in a concentrated or diluted form, and a second container comprising at least one agriculturally beneficial ingredient as defined above.

[0088] A concentrate of LCO(s), a concentrate of the plant biostimulant(s) or a concentrated mixture thereof may, as necessary, be formulated with suitable agricultural excipients such as agronomically acceptable carriers, polymers, wetting agents, surfactants, anti-freezing agents, etc.

[0089] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered within the scope of this invention and covered by the claims appended hereto. The invention is further illustrated by the following example, which should not be construed as further or specifically limiting. EXAMPLE

[0090] Example 1: Combination of lipo-chitooligosaccharides (LCOs) and plant biostimulant Bacillin 20 increases agricultural production

[0091] Field trials were conducted in Arkansas over a period of 3 months (June 2022 to Sept.2022) to evaluate the effectiveness and activity of a composition according to the present invention. Four (4) different seed treatments were compared: mixture comprising both lipo-chitooligosaccharides (LCOs) and Bacillin 20; each individual component individually and untreated check (UTC) for reference (control). Bacillin 20

[0092] The Bacillin 20 used for the field trials consisted of a Bacillin 20 aqueous stock solution. Bacillin 20 was purified from Bacillus thuringiensis MS20 strain. The process used to isolate Bacillin 20 involved the following steps: 1) microbial culture production; 2) centrifugation and membrane filtration; 3) butanol extraction of organic metabolites; 4) evaporation of butanol and concentration of extract; 5) fractionation of the organicextract using acetonitrile solvent; 6) purification of Bacillin 20 using HPLC; and 7) mass spectrometry analysis of Bacillin 20 to confirm identity.

[0093] During step 1), Bacillus thuringiensis MS20 was cultured in Kings B medium incubated in a shaker incubator at 125 rpm and at 30 °C for at least 96 h. At step 2), bacterial mass was removed by centrifugation and membrane filtration to obtain a cell- free supernatant. The butanol extraction of step 3) was carried out by mixing and vigorous shaking the microbial cell free supernatant with 1-butanol and phase partitioning of 1- butanol. At step 4) the butanol was evaporated using a rotary evaporator and the organic extract was suspended in 20% acetonitrile. Next, at step 5), the organic extract was fractionated using solid phase extraction (SPE) cartridges to remove undesirable and "junk" compounds, retaining only active fractions of Bacillin 20 for further purification. At step 6) Bacillin 20 was purified conducting high performance liquid chromatography (HPLC) using a gradient of acetonitrile / water to a target of 85-95% purity of Bacillin 20. At step 7), mass spectrometry analysis was done on a purified sample of Bacillin 20 to confirm identity of the peptide using molecular weight.

[0094] The purified Bacillin 20 (i.e., about 85 to about 95% purity) was diluted in phosphate buffered saline to obtain a stock solution comprising a 10-9M concentration of Bacillin 20. The stock solution of Bacillin 20 was diluted 100-fold in water to a final ready- to-use diluted 10-11M working solution which was used for seed treatment. Lipo-chitooligosaccharides (LCOs)

[0095] The lipo-chitooligosaccharides (LCOs) were obtained from Bradyrhizobium japonicum 532C strain in accordance with the procedure described by Soulemanov A, Prithiviraj B, Carlson RW, Jeyaretnam B, Smith DL.2002. Isolation and characterization of the major nod factor of Bradyrhizobium japonicum strain 532C. Microbiological Research.157: 25-8.

[0096] Culturing: Bradyrhizobium japonicum strain 532C bacterium (Liphatech Inc., Milwaukee, MI, USA) was cultured in yeast extract mannitol (YEM) broth at 28±2 °C and shaken at 150 rpm. At the end of the exponential growth phase (day 5–7), genistein(Sigma Chemical Co, MO, USA) was added to a final concentration of 5 μM from a stock solution (1 mM) made with 100% methanol.

[0097] Incubation and evaporation: The culture was incubated for 48 h and at the end of the incubation, 12 L of the culture was extracted with 0.4 V of HPLC grade n-butanol. The organic fraction was collected and evaporated in a rotary evaporator at 50 °C under vacuum. The resultant brown viscous material was re-dissolved in 18% acetonitrile.

[0098] Elution: 20 mL of this material was loaded on a ExtractClean™ SPE C-18 column and eluted five times with 50 mL of 25% acetonitrile. All eluents were collected and the presence or the absence of nod factor was tested by HPLC. The next elutions were done with 5 mL of 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56% and 58% acetonitrile. All eluents were collected, and the presence of nod factor was tested by HPLC. Usually, the eluents with acetonitrile concentration from 48% to 52% contained the pure nod factor.

[0099] Chromatography: The HPLC instrument was fitted with Waters™ 1525 binary pumps, a Waters™ 2487 absorbance detector set at 214 nm and a Waters 717 Plus Autosampler™. C18 reverse phase column (0.46 x 25 cm, 5 µ) (Vydac, CA, USA; catalogue # 218TP54, Mandel) was used. A gradient from 18:82 acetonitrile:water to 40:60 acetonitrile:water during 20 min was programmed to develop the chromatogram. [000100] Determination of Concentration: The Nod factor peak was identified by comparing with the retention time of the standard (Nod Bj-V (C18:1, MeFuc)) of strain USDA110. The concentration of the Nod factor was calculated by the comparison of the area of the peak of the standard, at a known concentration. [000101] Freeze Drying: Eluted fractions containing high concentrations of pure Nod factor were freeze dried and used for mass spectrometry and biological activity tests. [000102] Mass spectrometry: Molecular weight identification was done by LC-MS analysis. The results confirmed that the major Nod factor (Nod Bj-V, C18:1, MeFuc) was present in the samples.[000103] Stock Solution: The purified LCOs (i.e., about 85% to about 95% purity) was diluted in phosphate buffered saline to obtain a stock solution comprising a 10-6M concentration of LCOs. The stock solution of LCOs was diluted 100-fold in water to a final ready-to-use diluted 10-8M working solution which was used for seed treatment. Bacillin 20 + LCO [000104] The Bacillin 20 + LCO used for the field trials consisted of a combination of the peptide Bacillin 20 and the LCO. Small quantities of the stock solutions of Bacillin 20 and LCO were mixed and diluted 100x to prepare a working solution comprising both components which was later used for seed treatment. Seed treatment [000105] Briefly, Zea Mays L corn seeds (variety P1718VYHR) were treated prior to planting with working solutions comprising: LCO alone; Bacillin 20 alone; a mixture of both LCO + Bacillin 20; or just water (control or UTC). The seed treatment comprised soaking the corn seeds in the different solutions followed by air drying before planting. [000106] The seeds from the different treatments were planted in adjacent plots of 10 ft wide x 40 feet long for an individual plot area of 400 square feet. Each seed treatment was repeated in six different plots for a total area of 2400 square feet per treatment. [000107] For Experiment A, planting (bedding method) was on June 6, 2022 and emergence was observed about 6 days later (June 12, 2022). For Experiment B, planting (bedding method) was a day later (June 7, 2022) and emergence was also after 6 days (June 3, 2022). Corn was harvested 3 months later for both experiments (September 29, 2022) using a Wintersteiger Quantum™ plot. During culture, all corn plants were subjected to same maintenance conditions, including herbicides (Roundup PowerMax™, Me-too- lachlor™ and Permit™), fertiliser (urea) and irrigation. [000108] Harvested corn from each treatment was weighed for each of the six replicate and an average yield was calculated for each treatment.[000109] As shown in Table 1 and Table 2 below, combining LCO + Bacillin 20 provided synergistic results in terms of corn yield, for both experiments. [000110] Table 1: Corn Response to LCO & Bacillin 20 subsequent to seed coating (Experiment A) Treatments Yield % yield increase (Bushel per acre) compared to UTC UTC 121 0 LCO 121 0.00% Bacillin 20 125 3.31% LCO + Bacillin 20 128 5.79% [000111] Table 2: Corn Response to LCO & Bacillin 20 subsequent to seed coating (Experiment B) Treatments Yield % yield increase (Bushel per acre) compared to UTC UTC 117 0 LCO 121 3.42% Bacillin 20 123 5.13% LCO + Bacillin 20 132 12.82% [000112] The data of Table 1 and Table 2 clearly demonstrate a positive effect on corn yield of corn seed treated with either LCO or Bacillin 20 when compared to untreaded seeds (UTC). More interestingly treatment with a mixture comprising both LCO + Bacillin 20 was even more beneficial and even synergistic, since the combination provided a yield increased greater than the simple addition of LCO alone and Bacillin 20 alone. [000113] These results thus confirm that combining at least one lipo- chitooligosaccharide (LCO) with one or more plant biostimulant in accordance with the present invention provides substantial benefits in the fields of agriculture, particularly for improving crop yields.[000114] Example 2: Combination of lipo-chitooligosaccharides (LCOs) and plant biostimulant Bacillin 20 increases soybean yield [000115] Field trials were conducted in Brazil to evaluate the effectiveness of Bacillin 20 alone, LCO alone, and a mixture of Bacillin 20 and LCO, as growth-promoting inoculants, applied via seed treatment, on growth and yield variables, in soybean crop. [000116] Primavera do Leste, MT: this trial took place at the Ceres Consultoria Agronômica on Rodovia MT 130, km 30, at an altitude of 589 meters. The climate there was tropical with distinct wet and dry seasons, an average annual temperature of 23.7 °C, and annual precipitation of 1,868 mm. The soil was classified as Red Latosol with a sandy clay loam texture. [000117] Barreiras, BA: the trial occurred at the Experimental Station of Multicrop Research and Development on Rodovia BA 460, km 9, at an altitude of 725 meters. The climate was tropical, with an average annual temperature of 23.3 °C and precipitation of 1,273 mm, making it the driest among the study locations. The soil was also a Red Latosol but with a sandy texture. [000118] Campo Alegre de Goiás, GO: This was hosted in the Central-West region, at an altitude of 868 meters. This area had a tropical climate with dry winters, an average annual temperature of 21.2 °C, and rainfall of 1,525 mm. The soil was a dystrophic red Latosol with a clayey texture. [000119] Gurupi, TO: this trial was held at the Multcrop experimental station. The climate there was tropical with dry winters, featuring a good distribution of rainfall, an average annual temperature of 26.1 °C, and annual precipitation of 1,776 mm. The soil was classified as a Dystrophic Red-Yellow Latosol with a clay loam texture. [000120] Treatments and Management: The trials involved four treatments: a control without any treatment, application of the test product Bacillin 20 (referred to as ABIO), application of the LCO from Bradyrhizobium japonicum strain 532C (see above) and a combination of Bacillin 20 and LCO. All treatments were applied to seeds via a single seed application prior to sowing. After the application, seeds were dried before planting.Fertilization and crop maintenance were conducted according to regional recommendations and needs. [000121] Assessments: The impact of the treatments on soybean plants was evaluated through various growth and production parameters. These included germination tests, Emergency Speed Index (ESI), crop stand, plant vigor, plant height, fresh and dry mass of both the aerial parts and roots, number of pods per plant, number of seeds per pod, thousand-grain weight, and overall yield. Germination tests were performed in accredited laboratories immediately after seed treatment. The ESI was calculated using the Maguire (1962) formula. Plant vigor was assessed visually at the V4 stage, while other parameters were measured at appropriate growth stages. [000122] Experimental Design: The experimental design was a randomized complete block design (RCBD) with four treatments and six replications. Each plot measured 54 m² and was spaced 0.5 meters apart to avoid contamination between treatments. The plant population was set at 300,000 seeds per hectare. [000123] Dosage for the treatments in the trials was as follows: 1. Control: Only water was applied to the seeds. 2. Bacillin 20 (referred to as ABIO): The test product Bacillin 20 (ABIO) was seed applied at a dose of 0.50 mL / kg of seeds. 3. LCO: The LCO was also seed applied at a dose of 0.50 mL / kg of seeds. 4. Bacillin 20 (ABIO) + LCO: A combination of Bacillin 20 (ABIO) and LCO was seed applied, with each product dosed at 0.50 mL / kg of seeds, resulting in a total application volume of 1.00 mL / kg of seeds. [000124] The application method involved coating the products onto the seeds using at the rate of 10 mL / kg of seeds, which included both water and the products. After treating the seeds with the treatments the seeds were dried and prepared for sowing. [000125] The trials assessed various growth and production parameters of the soybean plants. These included: • Germination: The percentage of seeds that successfully germinated, determined through laboratory tests.• Emergency Speed Index (ESI): A measure of the speed at which seeds emerged, calculated using the Maguire (1962) formula. • Crop Stand: The number of plants that emerged per linear meter, counted at the V4 stage. • Plant Vigor: Visual assessment of plant health and robustness, rated on a scale from 1 to 9. • Plant Height: The height of the plants measured at the R1 growth stage. • Fresh Mass of the Aerial Part (FMAP): The weight of the above-ground parts of the plants immediately after cutting. • Dry Mass of the Aerial Part (DMAP): The weight of the above-ground parts after drying in an oven at 60°C. • Fresh Root Mass (FRM): The weight of the roots immediately after cutting. • Dry Root Mass (DRM): The weight of the roots after drying in an oven at 60°C. • Number of Pods per Plant (NPP): The average number of pods produced by each plant. • Number of Grains per Pod (NGP): The average number of grains contained in each pod. • Thousand-Grain Weight: The weight of one thousand grains, adjusted to 13% moisture content. • Yield: The total weight of grains harvested per hectare, also adjusted to 13% moisture content. [000126] These assessments provided a comprehensive evaluation of the impact of the different treatments on the growth and yield of the soybean crops. [000127] Results. Results are presented in Tables 3 to 5 hereinafter, where “ABIO” represents Bacillin 20 and where “LCO” represents LCO from Bradyrhizobium japonicum strain 532C as described above. [000128] The following results can be observed from the data presented in Table 3 (important numbers are highlighted): A) ABIO+LCO 3.3% increase in stands (Primavera do Leste / MT);B) ABIO+LCO 3.2% increase in emergence (Barreiras / BA); C) ABIO+LCO 3% increase in stand (Barreiras / BA); D) ABIO+LCO 70% increase in vigor (Barreiras / BA); E) ABIO+LCO 17.3% increase in emergence (Campo Alegre de Goiàs / GO); F) ABIO+LCO 2.1% increase in stand (Campo Alegre de Goiàs / GO); and G) ABIO+LCO 4.3% increase in stand (Campo Alegre de Goiàs / GO) [000129] Table 3: Variables in the initial growth of soybean plants in different locations.[000130] The following results can be observed from the data presented in Table 4 (important numbers are highlighted): A) ABIO+LCO 40.9% increase in height (Barreiras / BA) B) ABIO+LCO 40.9% increase in Fresh Mass Aerial Part (Barreiras / BA) C) ABIO+LCO 63.3% increase in Dry Mass Aerial Part (Barreiras / BA) D) ABIO+LCO 160% increase in Fresh Root Mass (Barreiras / BA)E) ABIO+LCO 80% increase in Dry Root Mass (Barreiras / BA) F) ABIO+LCO 7.6% increase in height (Campo Alegre de Goiàs / GO) G) ABIO+LCO 4.1% increase in Fresh Mass Aerial Part (Campo Alegre de Goiàs / GO) H) ABIO+LCO 6.8% increase in Dry Mass Aerial Part (Campo Alegre de Goiàs / GO) I) ABIO+LCO 6.7% increase in Fresh Root Mass (Campo Alegre de Goiàs / GO) J) ABIO+LCO 16.7% increase in Dry Root Mass (Campo Alegre de Goiàs / GO) K) ABIO+LCO 7.9% increase in height (Gurupi / TO) L) ABIO+LCO 9.1% increase in Dry Mass Aerial Part (Gurupi / TO) M) ABIO+LCO 12.5% increase in Fresh Root Mass (Gurupi / TO) N) ABIO+LCO 23% increase in Dry Root Mass (Gurupi / TO) . [000131] Table 4: Soybean vegetative growth variables in different locations[000132] The following results can be observed from the data presented in Table 5 (important numbers are highlighted): A) ABIO+LCO 0.8% increase in Yield (Primavera do Leste / MT)B) ABIO+LCO 1.1% increase in Number of Pods per Plant (Barreiras / BA) C) ABIO+LCO 1.5% increase in Yield (Barreiras / BA) D) ABIO+LCO 2.6% increase in Number of Pods per Plant (Campo Alegre de Goiàs / GO) E) ABIO+LCO 4.2% increase in Number of Grains per Pod (Campo Alegre de Goiàs / GO) F) ABIO+LCO 2.1% increase in 1000 grain weight(g) (Campo Alegre de Goiàs / GO) G) ABIO+LCO 5.6% increase in Yield (Campo Alegre de Goiàs / GO) H) ABIO+LCO 2.9% increase in Number of Pods per Plant (Gurupi / TO) I) ABIO+LCO 3.7% increase in Number of Grains per Pod (Gurupi / TO) J) ABIO+LCO 5.5% increase in Yield (Gurupi / TO) [000133] Table 5: Components of soybean productivity in different locations.[000134] Conclusions: 1. ABIO and “ABIO+LCO” provided a statistically significant increase in Vigor in Barreiras / BA and Emergency Speed Index (ESI) in Campo Alegre de Goiás / GO in relation to the Control; 2. In Barreiras / BA and Gurupi / TO, the inoculant treatments (ABIO+LCO) performed statistically better than the control in the soybean vegetative growth variables: specifically, in Barreiras / BA with the variables Fresh and dry mass of the aerial part (FMAP and DMAP) and Fresh and dry root mass (FRM and DRM), and in Gurupi / TO with Plant height and Fresh and dry root mass (FRM and DRM). 3. All locations had numerical increases in relation to the control, highlighting ABIO+LCO with relative average increases of 3.3%. [000135] Overall, these results confirm that ABIO alone (i.e. Bacillin 20), and ABIO in combination with at least one lipo-chitooligosaccharides (LCO) increase growth and production parameters of soybean plants. Accordingly, combining LCO(s) with one or more plant biostimulant, in accordance with the present invention, is commercially beneficial in the fields of agriculture, particularly for soybean. [000136] Example 3: Combination of lipo-chitooligosaccharides (LCOs) and plant biostimulant Bacillin 20 increases corn crop development and yield [000137] Field trials were conducted in Brazil to evaluate the effectiveness of Bacillin 20 alone, LCO alone, and a mixture of Bacillin 20 and LCO, as growth-promoting compounds applied via seed treatment on corn crop development and yield. The trials were conducted at two locations: Primavera do Leste / MT and Gurupi / TO. [000138] Treatments and Management: The treatments included: 1) a control with only water applied at a spray volume of 10 mL / kg seed, 2) ABIO at 0.82 mL / kg seeds, applied with water at 10 mL / kg seed, 3) LCO at 0.82 mL / kg seeds, and 4) a combination of ABIO and LCO, each at 0.82 mL / kg seeds. The experimental design consisted of randomized blocks with four treatments and six replications. A minimum of 60,000 seeds per hectare were used, with three seeds per linear meter, resulting in a minimum plot size of 54.0 m²(12 rows, 9 meters long, spaced 0.5 meters apart), and 0.5-meter borders between plots. Each plot contained 324 seeds. [000139] Measured Parameters: Parameters measured included a germination test post-inoculation and pre-seeding, emergency speed index (ESI) from sowing to the end of emergence over 6 meters linear per plot, and crop stand at the V6 growth stage over 10 meters linear per plot. Plant vigor was visually assessed at the V6 and V10 growth stages, graded from 1 to 10. Plant height, fresh and dry mass of the aerial part, and fresh and dry root mass were measured at the V10 growth stage with five plants per plot. At harvest, average cob length and diameter, number of grains per cob, number of rows of grains per cob, number of grains per row, 1000 grain weight, and yield (all corrected to 13% moisture) were assessed. Application and planting took place between January and March 2024. [000140] Results: Most of the best results were observed using the combination of ABIO and LCO, summarized below: 1. Dry Root Mass Increase (Primavera do Leste / MT) a. Best performing treatment: LCO+ABIO b. Percentage Increase: 25.3% 2. Dry Aerial Mass Increase (Primavera do Leste / MT) a. Best performing treatment: LCO+ABIO b. Percentage Increase: 23.7% 3. Fresh Aerial Mass Increase (Primavera do Leste / MT) a. Best performing treatment: LCO+ABIO b. Percentage Increase: 6.9% 4. Fresh Root Mass Increase (Primavera do Leste / MT) a. Best performing treatment: LCO+ABIO b. Percentage Increase: 5.3% 5. Yield Increase (Primavera do Leste / MT) a. Best performing treatment: LCO+ABIO b. Percentage Increase: 2.0% 6. Average Cob Length Increase (Primavera do Leste / MT) a. Best performing treatment: LCO+ABIOb. Percentage Increase: 8.1% 7. Fresh Aerial Mass Increase (Gurupi / TO) a. Best performing treatment: LCO+ABIO b. Percentage Increase: 6.4% 8. Dry Aerial Mass Increase (Gurupi / TO) a. Best performing treatment: LCO+ABIO b. Percentage Increase: 4.1% [000141] In addition, it is worth noting that the 2023 / 2024 corn season in Brazil presented high precipitation which affected the Primarevera do Leste location. The months of February, March and April were the rainiest months relative to the entire historical average in the trial location. Despite these exceptionally difficult conditions, the “ABIO+LCO” treatment presented higher relative increases in aerial and root mass, which may have positively influenced the higher corn yield, which presented around 139 kg / ha more than the Control. [000142] Conclusions: Altogether these results confirm that combining at least one lipo- chitooligosaccharide (LCO) with one or more plant biostimulant such as ABIO favorably increase growth and production parameters of corn. Furthermore, the results confirm that the combination ABIO+LCO is effective even under difficult climate conditions such as heavy rain. Accordingly, combining LCO(s) with one or more plant biostimulant, in accordance with the present invention, is commercially beneficial in the fields of agriculture, particularly for corn, and under various climate conditions. * * * [000143] Headings are included herein for reference and to aid in locating certain sections. These headings are not intended to limit the scope of the concepts described therein, and these concepts may have applicability in other sections throughout the entire specification. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.[000144] The singular forms “a”, “an” and “the” include corresponding plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes one or more compounds and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein. [000145] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, concentrations, properties, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present specification and attached claims are approximations that may vary depending upon the properties sought to be obtained. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the embodiments are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors resulting from variations in experiments, testing measurements, statistical analyses, and such. [000146] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the present invention and scope of the appended claims.

Claims

AMENDED CLAIMS received by the International Bureau on 23 December 2024 (23.12.2024)1. A composition for increasing plant growth, comprising a combined mixture of at least one lipo-chitooligosaccharide (LCO) and one or more plant biostimulant, wherein said one or more plant biostimulant comprises at least one of Thuricin 17 and Bacillin 20, and wherein said composition displays synergistic activity in increasing said plant growth compared to said at least one lipo-chitooligosaccharide (LCO) and said one or more plant biostimulant individually.

2. The composition of claim 1 , wherein said at least one LCO is obtained from a rhizobia selected from the group consisting of Rhizobium, Bradyrhizobium, Sinorhizobium, mesorhizobium, Phylorhizobium, and Azorhizobium.

3. The composition of claim 1 or 2, wherein said at least one LCO is obtained from Bradyrhizobium japonicum.

4. The composition of any one of claims 1 to 3, wherein said composition is a concentrate comprising said at least one LCO at a concentration of about 10'7M to about 10-9M.

5. The composition of any one of claims 1 to 4, wherein said composition is a ready-to-use composition comprising said at least one LCO at a concentration of about 10-7M to about 10-12M.

6. The composition of any one of claims 1 to 5, wherein said one or more plant biostimulant consists of Bacillin 20.

7. The composition of any one of claims 1 to 6, wherein said composition is a concentrate comprising said one or more plant biostimulant at a concentration of about 10-6M to about 10-10M, or about 10-7M to about 10-13M.

8. The composition of any one of claims 1 to 7, wherein an increase in plant growth is displayed by an increased in crop yield.

9. The composition of any one of claims 1 to 8, wherein said plant is selected from the group consisting of cereals, rice, tree nuts, ground nuts, fruits, vegetables, oil seed crops, forage crops,horticultural tree species, forestry tree species, flowers, cannabis, grass, turf, and ornamental plants.

10. The composition of any one of claims 1 to 9, wherein said composition consists of a composition formulated for seed coatings.

11. The composition of any one of claims 1 to 9, wherein said composition consists of a composition formulated for an application to the roots.

12. The composition of any one of claims 1 to 9, wherein said composition consists of a composition formulated for foliar application.

13. The composition of any one of claims 1 to 12, wherein said composition provides for at least of the following benefits:- mitigating negative effects and stress induced by application of herbicide(s) and pesticide(s) to plants and crops;- increasing crop yields;- increasing plant growth;- increasing leaf area, greenness, number and / or biomass;- stimulating the plant root system;- stimulating bacterial production of phytohormone(s);- stimulating bacterial production of siderophore(s);- increasing induced resistance to plant pathogens;- stimulating production of antibiotics;- enhancing early seedling, germination and / or growth;- increasing nodule number;- increasing plant dry weight;- increasing root irrigation nodule number;- increasing concentration of root irrigation in shoot tissue;- increasing photosynthetic rate;- increasing root and shoot length;- increasing in energy and anti-oxidant biosynthetic pathways;- up-regulating defense related genes;- mitigating salt-related stress;providing bacteriocin capabilities; activating carbon energy and metabolisms pathway proteins; providing positive metagenomic effects by increasing number of root microbes which enhances crop growth; enhancing root-hair production and nutrient uptake; protecting crops from abiotic stress; improving plant resistance to disease(s); reducing the need for synthetic fertilizers and pesticides yields; promotion of symbiotic relationship between bacteria, inoculants and host crop; increasing number of root microbes; acting as a signaling compound; increasing farm revenues; improving global food security; and cost efficient and economical to apply.

14. The composition of claim 13, wherein said benefit is determined by comparing a crop contacted or not with said composition.

15. The composition of any one of claims 1 to 14, wherein said composition provides higher yield per acre.

16. A composition comprising at least one lipo-chitooligosaccharide (LCO) and one or more plant biostimulant, wherein said one or more biostimulant comprises at least one of Thuricin 17 and Bacillin 20, and wherein said at least one LCO and said one or more plant biostimulant are present in amounts effective to synergistically enhance one or more growth parameters of a plant when said composition is applied to said plant.

17. The composition of claim 16, wherein said at least one lipo-chitooligosaccharide (LCO) and one or more plant biostimulant together synergistically enhance crop yield of plants contacted with said composition.

18. The composition of claim 16 or 17, wherein said composition when applied to seeds is effective to synergistically enhance crop yield of plants grown from said seeds.

19. The composition of any one of claims 16 to 18, as further defined in any one of claims 3 to 15.

20. A treated seed comprising a plant seed that is at least partially coated with a seed treatment composition, wherein said seed treatment composition comprises a composition as defined in any one of claims 1 to 17.

21. The treated seed of claim 20, wherein a plant grown from said treated seed exhibits increased plant growth as compared to a control plant grown from an untreated seed.

22. The treated seed of claim 20 or 21 , wherein said treated seed has been packaged and stored for at least one month prior to planting.

23. A method for enhancing plant growth, comprising applying at least one lipo- chitooligosaccharide (LCO) and one or more plant biostimulant to said plant in amounts effective to synergistically enhance one or more growth parameters of said plant, and wherein said one or more biostimulant comprises at least one of Thuricin 17 and Bacillin 20.

24. The method of claim 23, wherein enhancement of said one or more growth parameters comprises increased crop yield.

25. The method of claim 23 or 24, wherein said at one least one LCO is applied to said plant at a concentration of about 10-7M to about 10-12M, and wherein said one or more plant biostimulant is applied a concentration of about 10-7M to about 10-13M.

26. The method of claim 25, wherein said at one least one LCO is applied to said plant at a concentration of about 10-8M to about 10-10M, and wherein said one or more plant biostimulant is applied a concentration of about 10-9M to about 10-11M.

27. The method of any one of claims 23 to 26, wherein said applying comprises seed coating.

28. The method of any one of claims 23 to 26, wherein said applying comprises at least one of root dipping, root irrigation and soil drenching29. The method of any one of claims 23 to 26, wherein said applying comprises foliar application.

30. The method of any one of claims 23 to 29, wherein said method further comprises applying to said plant at least one of the following: a herbicide, an insecticide, a fungicide, a bactericide, and a nutrient.

31. The method of any one of claims 23 to 30, wherein said method provides for at least one of the benefits listed in claim 13.

32. The method of claim 31 , wherein said at least one benefit is determined by comparing a plant contacted or not with said composition.

33. The method of any one of claims 23 to 32, wherein said method provides higher yield per acre.

34. The method of any one of claims 23 to 33, wherein said plant is selected from the group consisting of cereals, rice, tree nuts, ground nuts, fruits, vegetables, oil seed crops, forage crops, horticultural tree species, forestry tree species, flowers, cannabis, grass, turf, and ornamental plants.

35. The method of any one of claims 23 to 34, wherein said at least one LCO is obtained from a rhizobia selected from the group consisting of Rhizobium, Bradyrhizobium, Sinorhizobium, mesorhizobium, Phylorhizobium, and Azorhizobium.

36. The method of any one of claims 23 to 35, wherein said at least one LCO is obtained from Bradyrhizobium japonicum.

37. A method for enhancing plant growth, comprising applying at least one lipo- chitooligosaccharide (LCO) and one or more plant biostimulant to a plant seed in amounts effective to synergistically enhance one or more growth parameters of the plant that grows from said seed, wherein said one or more biostimulant comprises at least one of Thuricin 17 and Bacillin 20.

38. The method of claim 37, further comprising one or more of the features as defined in any one of claims 24 to 36.

39. A method for increasing crop yield, comprising contacting plants with at least one lipo- chitooligosaccharide (LCO) and one or more plant biostimulant, wherein said one or more biostimulant comprises at least one of Thuricin 17 and Bacillin 20, and wherein said contactingsynergistically enhance crop yield of plants contacted with both compounds, as compared to contacting plants with only the at least one lipo-chitooligosaccharide (LCO) or only the one or more plant biostimulant.

40. The method of claim 39, further comprising one or more of the features as defined in any one of claims 24 to 36.

41. A method of increasing corn yield, said method comprising applying at least one lipo- chitooligosaccharide (LCO) and one or more plant biostimulant to corn seed in amounts effective to synergistically increase the yield of corn grown from said corn seed, wherein said one or more biostimulant comprises at least one of Thuricin 17 and Bacillin 20, and wherein said at least one lipo-chitooligosaccharide and said one or more plant biostimulant are applied to said corn seed prior to planting.

42. The method of claim 41 , further comprising one or more of the features defined in any of claims 24 to 36.

43. A kit for stimulating plant growth, comprising: (i) a first container comprising a concentrated solution of at least one LCO; and (ii) a second container comprising a concentrated solution of one or more plant biostimulant, wherein said one or more biostimulant comprises at least one of Thuricin 17 and Bacillin 20.

44. The kit of claim 43, further comprising: (iii) a third container comprising at least one of a herbicide, an insecticide, a fungicide, a bactericide, and a nutrient.

45. A kit for stimulating plant growth, comprising: (i) a first container comprising a mixture of at least one LCO and one or more plant biostimulant, wherein said one or more biostimulant comprises at least one of Thuricin 17 and Bacillin 20; and (ii) a second container comprising at least one of a herbicide, an insecticide, a fungicide, a bactericide, and a nutrient.

46. The kit of any one of claims 43 to 45, wherein said at least one LCO is obtained from a rhizobia selected from the group consisting of Rhizobium, Bradyrhizobium, Sinorhizobium, mesorhizobium, Phylorhizobium, and Azorhizobium.

47. The kit of any one of claims 43 to 46, wherein said at least one LCO is obtained from Bradyrhizobium japonicum.

48. The kit of any one of claims 43 to 46, wherein said at least one LCO is at a concentration of about 10-7M to about 10-9M.

49. The kit of any one of claims 43 to 48, wherein said one or more plant biostimulant is at a concentration of about 10-6M to about 10-10M.

50. The kit of any one of claims 43 to 49, for use in seed coatings applications, application to the roots or for foliar application.

51. Use of at least one LCO and one or more plant biostimulant for stimulating plant growth, wherein said one or more plant biostimulant comprises at least one of Thuricin 17 and Bacillin 20, wherein said at least one LCO and one or more plant biostimulant are applied on a plant simultaneously or concurrently, and wherein said at least one LCO and said one or more plant biostimulant are used in amounts effective to synergistically enhance one or more growth parameters of a plant.

52. The use of claim 51 , comprising applying to said plant a composition as defined in any one of claims 1 to 15.

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