Peptide having plant biostimulant activity, bacterial strain producing same and uses thereof for promoting plant growth and crop yields

Abstract

Described herein are polypeptides having plant biostimulant activity, bacterial strains producing these polypeptides and uses thereof for promoting plant growth. In embodiments the polypeptide comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In embodiments the polypeptides are sactipeptides comprising one to four Cys-to-alpha carbon bond(s). The peptides are particularly useful for plant biostimulation. Also described are bacterial extracts coprising the peptides, nucleic acid molecules encoding these peptides and bacterial strains producing the same.

Description

PEPTIDE HAVING PLANT BIOSTIMULANT ACTIVITY, BACTERIAL STRAIN PRODUCING SAME AND USES THEREOF FOR PROMOTING PLANT GROWTH AND CROP YIELDS CROSS REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to patent application US 63 / 673,252 filed on July 19, 2024, the content of which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION

[0002] The invention relates to the field of plant growth, and more particularly to plant biostimulants derived from bacteria. BACKGROUND OF THE INVENTION

[0003] Biostimulants are natural and environmentally friendly substances with positive effects on plant growth. Biostimulants can enhance plant metabolism and physiology when applied in small doses. The most common categories of biostimulants are seaweed extracts, peptides and amino acids, humic substances and beneficial microorganisms (e.g., fungi and bacteria). For instance, bacteriocins are proteins produced by prokaryotes that are bactericidal and / or bacteristatic against organisms related to the producer strain. Examples of beneficial microorganisms plant growth promoting rhizobacteria (PGPR) are characterized for their ability to increase plant growth. They include bacteria found in soil near plant roots, on the surface of plant root systems, in spaces between cells of the root epidermis and cortex or inside specialized cells of root nodules.

[0004] Plant-microbe interactions have a huge impact on both agriculture and horticulture but are largely unexplored and unexploited for the improvement and optimization of plant production, relief of plant stress, and disease resistance. There are a limited examples of patent documents in this field. For instance, US patent publication No. 2003 / 0228679 discloses plant growth bacteria of the species Bacillus subtilis (i.e., NEB4) and of the species Bacillus thurigiensis (i.e., NEB17). International PCT patent publication WO 2008 / 138129 describes a purified polypeptide named Thuricin 17 that is a bacteriocin produced by Bacillus thurigiensis NEB17, that peptide having plant growth and / or disease resistance promoting activity. Discovery and purification of Thuricin 17 wasfirst published by Gray et al. in 2006 and it was found to have a molecular weight of 3162 Da (Gray et al., Journal of Applied Microbiology, Volume 100, Issue 3, 1 March 2006, Pages 545–554). A related bacteriocin named “Thuricin H” produced also by Bacillus thuringiensis (strain SF361) was later characterized in 2009 and 2011. Thuricin H appears to have same amino acid sequence than Thuricin 17, but a different molecular weight of 3139.51 Da due posttranslational modifications (Lee et al., FEMS Microbiol Lett 299 (2009) 205-213; Sit et al., Angew. Chem. Int. Ed. (2011), 50, 8718-8721). Patent US 9,333,227 describes genetically engineered microbial cells that can produce bacteriocins. US patents No. 9,868,675 and No. 10,053,392 describe a consortium of bacteria strains for enhancing the availability of soil phosphorous and other macronutrients to plants.

[0005] Accordingly, there is a need for natural and environmentally friendly biostimulating substances with positive effects on plant growth.

[0006] Particularly, there is a need for peptides having bactericidal and bacteriostatic activity, for isolated bacteria producing such peptides and for methods using such peptides and isolated bacteria for plant biostimulation, including promoting plant growth.

[0007] 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

[0008] According to one aspect, the invention relates to purified polypeptides and purified sactipeptides. In preferred embodiments, the polypeptides and the sactipeptides have plant biostimulant activity.

[0009] According to one particular aspect, the invention relates to a purified polypeptide selected from the group consisting of: (a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3;(b) a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; (c) a polypeptide which is a fragment of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, the fragment comprising at least 25, 26, 27, 27, 28, or 29 consecutive amino acids of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; wherein said purified peptide is a sactipeptide comprising one, two, three or four Cys-to-alpha carbon bond(s); wherein said purified polypeptide has plant biostimulant activity.

[0010] According to one particular aspect, the invention relates to a purified polypeptide, wherein said purified peptide consists of a sactipeptide comprising one, two, three or four Cys-to-alpha carbon bond(s), wherein said peptide is encoded by a nucleotide sequence comprising SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or by a nucleotide sequence comprising SEQ ID NO:4, and wherein said purified polypeptide has plant biostimulant activity.

[0011] According to one particular aspect, the invention relates to an isolated sactipeptide represented by Formula 1:Formula 1 wherein the lines represent Sulfur to Alpha-Carbon Thioether Linkage; and wherein the sactipeptide comprises a LC / MS mass of 3137.36 Daltons (C134H212N34O45S4). The peptides preferably displays plant biostimulant activity.

[0012] According to another aspect, the invention relates to bacterial extracts and compositions for plant biostimulation comprising polypeptides sactipeptides as defined herein.

[0013] According to another aspect, the invention relates to a method for plant biostimulation, comprising applying to the plant, polypeptide(s), sactipeptide(s), bacterial extract(s) and / or composition(s) as defined herein. In embodiments, application of the polypeptide(s), sactipeptide(s), bacterial extract(s) and / or composition(s) provides at least one of the following benefits to the plant, compared to a plant not contacted with said polypeptide(s), sactipeptide(s), bacterial extract(s) and / or composition(s): an increase of nodule number; an increase of plant dry weight or yield; an increase of root irrigation nodule number; an increase of root irrigation concentration; an increase of leaf photosynthetic rate; an increase of leaf greenness; an increase of photosynthetic rate; an increase of plant growth; an increase of production of plant stress-response proteins, an increase of seed germination; an increase of expression of plant defense-related enzymes; an increase of nitrogen fixation; and an increase of leaf area.

[0014] According to another aspect, the invention relates to an isolated polynucleotide encoding a polypeptide and / or a sactipeptide as defined herein, or the complement thereto. According to another related aspect, the invention relates to a vector, a host cell comprising the isolated polynucleotide defined herein.

[0015] According to another aspect, the invention relates to an isolated bacterium comprising a polynucleotide as defined herein and / or encoding a polypeptide as defined herein and / or a sactipeptide as defined herein.

[0016] 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. BRIEF DESCRIPTION OF THE FIGURES

[0017] In order for the invention to be readily understood, embodiments of the invention are illustrated by way of example in the accompanying figures.

[0018] Figures 1A and 1B shows possible open reading frames for encoding Peptide CXC-1 from the nucleotide sequence of SEQ ID NO:4.

[0019] Figure 2 is a 3D model of the amino acid sequence of Peptide CXC-1.

[0020] Figure 3 is a 3D model similar to Figure 2, higlithing the four Cys-to-alpha carbon bonds / linkage in Peptide CXC-1.

[0021] Further details of the invention and its advantages will be apparent from the detailed description included below. DETAILED DESCRIPTION OF EMBODIMENTS

[0022] In the following description of the embodiments, references to the accompanying figures are illustrations of an example by which the invention may be practiced. It will be understood that other embodiments may be made without departing from the scope of the invention disclosed. General overview

[0023] The invention provides a peptide having plant biostimulant activity, namely “Peptide CXC-1” and also related peptides having similar biological activity. The invention further provides a Bacillus thurigiensis strain producing Peptide CXC-1. The invention also provides methods for promoting plant growth, increasing production of plant stress- response proteins and / or inducing expression of plant defense-related enzymes by using these peptides and strains. Peptide CXC-1 and other peptides

[0024] One main aspect of the present invention concerns purified or isolated peptides having plant biostimulant activity.

[0025] As used herein, the term “polypeptide” or “peptide” encompasses any chain of naturally or non-naturally occurring amino acids (either D- or L-amino acids), regardless of length (e.g., at least 5, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 100 or more amino acids) or post-translational modification (e.g., Cys-to-alpha carbon bonds, glycosylation, phosphorylation, acylation, lipidation, thiolation, oxidation, etc.) or the presence of one or more non-amino acyl groups (for example, sugar, lipid, etc.) covalently linked to thepeptide, and includes, for example, natural proteins, synthetic or recombinant polypeptides and peptides, hybrid molecules, peptoids, peptidomimetics, etc. In embodiment, polypeptides in accordance with the present comprises SEQ ID NO: 1, a fragment thereof or having ≥ 75% identity with SEQ ID NO: 1.

[0026] By “isolated” or “purified” is meant that the polypeptide is substantially separated or isolated from the components such as other polypeptides, proteins, or lipids, carbohydrates, etc. that accompany the polypeptide in its natural environment. Thus, for example, a polypeptide that is chemically synthesised or produced by recombinant technology will generally be substantially free from its naturally associated components and be considered to be purified. Typically, the purified polypeptide will constitute at least 60%, 70%, 75%, 80%, 90%, 95%, 98% or 99% by weight, of the total material in a sample (i.e., a sample of the purified polypeptide will contain less than 40%, 30%, 20%, 10%, 5%, 2% or 1% by weight of components such as other polypeptides, proteins, lipids, carbohydrates, etc. that accompany the polypeptide in its natural environment).

[0027] As used herein, the terms “plant biostimulant activity” or “plant biostimulation” include, but is not limited to, promoting plant growth, increasing production of plant stress-response proteins, inducing expression of plant defense-related enzymes, improving nodulation (e.g., increased number of nodules), improving nitrogen fixation (e.g., increased nitrogen concentration as measured by mg / g dry weight of plant material), increasing leaf area, increasing seed germination, increasing leaf greenness (e.g., as measured by SPAD), increasing photosynthesis (µmol cm-2s-1), increasing accumulated dry-weight of the plant and / or increasing yield. Any increase or improvement can be measured in a treated plant (e.g., a plant treated with a polypeptide according to the invention) relative to an untreated control plant.

[0028] As used herein, the term “purified or isolated peptides having plant biostimulant activity” or similar expression(s) refers to an isolated peptide having any one of SEQ ID NO: 1 to 3 as defined hereinafter, as well as any fragments or SEQ ID NO: 1 to 3, or any isolated protein, fusion protein or peptide comprising any of SEQ ID NO: 1 to 3, and having substantially similar biological activity as the peptide of SEQ ID NO: 1,particularly in displaying bactericidal and / or bacteriostatic effects against Bacillus strains other than Bacillus thurigiensis, and / or in having plant biostimulant activity.

[0029] Sactipeptides have been described in the art (e.g., Hudson et al, J. Am. Chem. Soc. May 22, 2019; 141(20): 8228–8238). In embodiments, the peptide of the invention consists of a sactipeptide comprising one, two, three or four Cys-to-alpha carbon bond(s), e.g. sulphur-to-alpha carbon bonds. In embodiments, the peptide of the invention comprises one, two, three or four of the following linkages: Cys4-Ser28; Cys7-Thr25; Cys10-Thr22; and Cys13-Asn19. In preferred embodiments, the peptide of the invention comprises all four linkages.

[0030] In one embodiment, the peptide comprises the amino acid sequence DWTCWSCLVCAACSVELLNLVTAATGASTAS (SEQ ID NO:1).

[0031] In one embodiment, the peptide comprises the amino acid sequence: DWTCWSCLVCAACSVEX17X18X19X20X21X22X23ATGASTX30X31(SEQ ID NO: 2), wherein: ^ X17is a hydrophobic amino acid, or any one of L, A, V, I, M, F, Y, or W; ^ X18is a hydrophobic amino acid, or any one of L, A, V, I, M, F, Y, or W; ^ X19is a polar amino acid, or any one of N, S, T, or Q; ^ X20is a hydrophobic amino acid, or any one of L, A, V, I, M, F, Y, or W; ^ X21is a hydrophobic amino acid, or any one of V, L, A I, M, F, Y, or W; ^ X22is a polar amino acid, or any one of T, N, S, or Q; ^ X23is a hydrophobic amino acid, or any one of A, L, V, I, M, F, Y, or W; ^ X30is a hydrophobic amino acid, or any one of A, L, V, I, M, F, Y, or W; ^ X31is a polar amino acid, or any one of S,T, N, or Q;

[0032] In one embodiment, the peptide comprises the amino acid sequence: X1X2TCWSCLVCAACSVEX17X18X19X20VTAATGASTAS (SEQ ID NO: 3), wherein: ^ X1is a negatively charged amino acid, or any one of D, or E ^ X2is a hydrophobic amino acid, or any one of W, A, V, I, L, M, F, or Y; ^ X17is a hydrophobic amino acid, or any one of L, A, V, I, M, F, Y, or W; ^ X18is a hydrophobic amino acid, or any one of L, A, V, I, M, F, Y, or W;^ X19is a polar amino acid, or any one of N S, T, or Q; ^ X20is a hydrophobic amino acid, or any one of L, A, V, I, M, F, Y, or W;

[0033] In embodiments, the peptide comprises 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, SEQ ID NO:2, or SEQ ID NO:3. 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). In embodiments, substitutions in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 are conservative amino acid substitutions, i.e., a substitution that can be made without substantial loss of the relevant function of the original peptide. In embodiments the change or substitution of the amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing. Preferably, a peptide with a substitution has plant biostimulant activity, more preferably it has a plant biostimulant activity substantially identical to the peptide of SEQ ID NO:1.

[0034] In embodiments, the peptide comprises a fragment of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, the fragment comprising at least 25, 26, 27, 27, 28, or 29 consecutive amino acids of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, respectively. Preferably, the fragment has plant biostimulant activity, more preferably it has a plant biostimulant activity substantially identical to the peptide of SEQ ID NO:1.

[0035] In embodiments, the peptide is different (i.e., distinct) from the polypeptide “Thuricin 17” (Mw = 3162 Da) produced by Bacillus thurigiensis NEB17 (see international PCT publication WO 2008 / 138129). In embodiments, the peptide is also different from “Thuricin H” produced by Bacillus thurigiensis SF361 (Mw = 3139.51 Da) (see Lee et al., FEMS Microbiol Lett 299 (2009) 205-213; Sit et al., Angew. Chem. Int. Ed. (2011), 50, 8718-8721).

[0036] In one embodiment, the peptide has a theoretical molecular mass of 3145.477 Daltons (i.e. molecular weight based on amino acid sequence C134H212N34O45S4).

[0037] In one embodiment, the peptide is characterized as having a monoisotopic mass of 3137.3603 Daltons (C134H212N34O45S4) [M+3H 1046.793 amu].

[0038] The present invention encompasses peptides comprising post-translational modifications including, but not limited to, Cys-to-alpha carbon bonds and other known post-translation like glycosylation (e.g., Asparagine (N), Arginine (R), Threonine (T), Serine (S)) acylation (e.g., Lysine (L)), acetylation (e.g., Lysine (L)), phosphorylation (e.g., Arginine (R), Tyrosine (Y), Threonine (T), Serine (S), Aspartate (D), Histidine (H), Cysteine (C)), lipidation (e.g., Cysteine (C)), thiolation (e.g., Cysteine (C)), oxidation (e.g., Methionine (M)) and pupylation (e.g., Lysine (L)).

[0039] In preferred embodiments, the purified polypeptide in accordance with the present invention consists of Peptide CXC-1. As used herein, the term “Peptide CXC-1” refers to a sactipeptide characterized by a monoisotopic mass of 3137.3603 Daltons (C134H212N34O45S4) [M+3H 1046.793 amu]. In embodiments, Peptide CXC-1 consists of a sactipeptide comprising Formula 1:Formula 1 wherein the lines represent Cys-to-alpha carbon bonds for the following amino acids: Cys4-Ser28; Cys7-Thr25; Cys10-Thr22; and Cys13-Asn19.

[0040] In embodiments, the peptide comprises a 3D structure as depicted in Figure 2 and / or Figure 3.

[0041] Based on its amino acid sequence, Peptide CXC-1 appears to be a class IId bacteriocin. Peptide CXC-1 is expected to display both bactericidal and bacteriostatic effects against other Bacillus strains (i.e., other than Bacillus thurigiensis).

[0042] Peptide CXC-1 has a low molecular weight (e.g., below 3200 Da), it is expected to be stable across a pH range of 1.0–9.25, it is expected to be highly heat resistant and to be inactivated by treatment with proteolytic enzymes. Methods of production and Biological activities

[0043] Polypeptides that are useful in practicing the methods of the invention, including Peptide CXC-1, can 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 for bactericidal and / or bacteriostatic effects and / or for plant biostimulant activity.

[0044] In embodiments polypeptides in accordance to the invention are obtained from a bacterial species that express the polypeptides. For instance, suitable bacterial strains may be cultured under conditions sufficient for expression of the polypeptide(s) and the polypeptide(s) may be recovered from the culture medium. The polypeptide(s) may be purified e.g., by chromatography (e.g., high-performance liquid chromatography), gel electrophoresis, filtration, dialysis, precipitation, centrifugation, etc. or combinations thereof.

[0045] Accordingly, the invention encompasses methods for producing a polypeptide of interest as defined herein, the methods comprising: (i) culturing a suitable host cell comprising a polynucleotide encoding the polypeptide (e.g., bacterial strain CXC-1, see hereinafter) under conditions sufficient for expression of the polypeptide encoded by said polynucleotide; and (ii) recovering said polypeptide. The host cell can be transformed or transfected with nucleic acid encoding the polypeptide. The host cell may be a prokaryotic host cell, for example a bacterial cell, or a eukaryotic cell, such as a yeast, plant, or animal cell.

[0046] It may also be possible to obtain peptide(s) of interest by chemical synthesis and / or by overexpression in a bacterial system. For the synthesis, it may be necessary to provide key enzymes that may be only found in the original bacteria and that may be necessary for the proper folding of the peptide(s). One of this(these) enzyme(s) may be alanine dehydrogenase which may be involved in formation of Cys-to-alpha carbon bonds.

[0047] In embodiments, Peptide CXC-1 is produced by the bacterium CXC-1 and it is obtainable therefrom (e.g., purified). According to one particular approach, bacterium CXC-1 is cultured in King’s B liquid medium at 28 ±2 °C on an orbital shaker for at least 72 hours, the entire culture is extracted with 40% n-butanol (shaken for 30 min and allowed to stand overnight at room temperature in a separatory funnel. The butanol phase is collected and evaporated at 45 °C using a rotary evaporator. The dried extract is resuspended in 20% acetonitrile and loaded on PrepSep C18™ cartridge (Fisher Scientific, cat#11-131-12) and fractionated using 35% (acetonitrile:water, v / v) , 43% and 100% acetonitrile. These fractions are collected and aliquots are used for HPLC analyses and Peptide CXC-1 quantification is determined in various fractions.

[0048] The present invention also encompasses bacterial extracts, such as extracts from bacterium CXC-1, comprising one or more the polypeptide of the invention, particularly Peptide CXC-1 as defined herein and / or related conformers.

[0049] In embodiments the polypeptide of the invention (and / or bacterial extracts comprising same) possesses one or more of the following properties: ^ increasing plant growth (e.g., by at least 2%, 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 antibiosis and biocontrol of plant pathogens ^ stimulating production of antibiotics; ^ enhancing early seedling growth of soybean and corn (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 leaf photosynthetic rates when applied on leaves (e.g., by at least 3%, 4%, 5%, 6%, 7%, 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 crop yields (e.g., by at least 2%, 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25% or more); ^ protecting crops from abiotic stress; ^ improving plant resistance to disease(s); ^ 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); ^ act as a signaling compound (e.g., hormone-like activity); ^ increasing farm revenues (e.g., by at least 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25% or more); ^ improving global food security; and ^ it is cost efficient and economical to apply.

[0050] Methods for testing compounds for bactericidal, bacteriostatic and / or properties are known in the art. For example, a zone of inhibition assay such as an agar disc diffusion assay may be used to test polypeptide(s) of interest or bactericidal or bacteriostatic activity against various indicator strains.

[0051] Likewise, assessment of the plant biostimulant activity of polypeptide(s) in accordance with the present invention may be accomplished by known methods. For instance, for assessing plant growth promoting activity, a polypeptide of interest may be applied by leaf spray or root irrigation to test plants, such as soybean plants. Plants may then be grown under controlled environment conditions (growth chamber or greenhouse) for e.g., about 40 days. At harvest, data may be collected concerning e.g., plant height, leaf greenness, leaf area, nodule number, nodule dry weight, shoot and dry root weight or length, nitrogen content and photosynthesis and compared to controls.

[0052] Assessment of plant disease resistance promoting activity of polypeptide(s) of interest may also be accomplished by known methods, such as by detecting or measuring a reduction in pathogen infestation of a plant, or indirectly by detecting or measuring increased production of one or more secondary metabolites that function to improve the resistance of a plant to pathogen attack. Exemplary secondary metabolites include lignification-related enzymes such as phenylalanine ammonia lyase (PAL), and tyrosine ammonia lyase (TAL), antioxidative enzymes such as peroxidase (POD), catalase (CAT), and superoxidase dismutase (SOD), and total phenolic compounds. Various methods for detecting or measuring increases in enzyme activity levels in plants (e.g., PAL, TAL, POD, CAD and SOD) are known in the art and exemplary techniques are described herein in the exemplification section. Similarly, techniques for determining concentrations or levels of total phenolic compounds are known and exemplary methods are described in the examples herein. Nucleic acids, vectors and transgenic plants

[0053] Another aspect of the invention relates to isolated polynucleotides encoding a polypeptide as defined herrein, or the complement of such polynucleotides.

[0054] As used herein, the term "polynucleotide" refers to as a "nucleic acid" or "nucleic acid molecule" of any length, and it encompasses ribonucleotides, deoxyribonucleotides, or modified forms of either type of nucleotide. The term polynucleotide includes single and double stranded forms of DNA or RNA. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. The polynucleotides of the invention include full-length genes and cDNA molecules as well as a combination of fragments thereof.

[0055] The polynucleotides of the invention are preferably "isolated" polynucleotides by which it is meant that they are not present in their naturally occurring form associated with the 5' and / or 3' sequences with which they are normally found. The polynucleotides are separated from at least one or both of the 5' or 3' sequences with which they are normally associated. For example, a nucleic acid molecule of the invention, inserted into a vector or linked to a foreign promoter, is in "isolated" form.

[0056] In embodiments, the polynucleotide according to the present invention encodes a polypeptide that has plant biostimulant activity and that comprises the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or it comprises a polypeptide having a certain minimum level of identity to these sequences and / or it is a fragment thereof, as defined previously in paragraphs (a), (b) and (c) in paragraph

[0009] .

[0057] In one embodiment, the isolated nucleic acid molecule comprises sequence SEQ ID NO: 4 as defined below, or the complement thereto, or consecutive nucleotides (e.g. at least 300, 400, 500, 550 or 600 nucleotides) of sequence SEQ ID NO: 4 positioned on any of first, second or third reading frames encoding for the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 (e.g., Figure 1 showing two different possible open reading frames for Peptide CXC-1). In embodiments, the peptide is encoded by open reading frame 1, as illustrated in Figure 1. In embodiments, the peptide is encoded by open reading frame 3, as illustrated in Figure 1.

[0058] In embodiments ,the isolated nucleic acid molecule encodes a peptide having a predicted molecular weight of 4054.85 Da (with leader peptide) and a mature peptide having a predicted molecular weight of 3144.77 Da. 1 cagaaaaggg ggtaggtcaa atggaaacac cagtagtaca accaagggac tggacttgtt 61 ggagttgttt agtatgtgca gcatgttctg tggaattatt aaatttagtt actgcggcaa 121 caggggctag tactgcaagc taatatcctt tcagaagttt tttggttaaa agcactaaga 181 gaatgagcac ttttggtcaa attagacttt ctaggagaaa aataaataat caaaaaggag 241 atgttctaaa tggaaacacc agtagtacaa ccaagggatt ggacttgttg gagttgctta 301 gtatgtgcag catgttctgt ggaattatta aatttagtta ctgcggcaac aggggctagt 361 actgcaagct aatatccttt tagaagtttt ttggttaaag gcactaagag aatgagcact 421 tttggtcaaa tgaattttct agaagaaaaa taaataacca aaaaggagat gttctaaatg 481 gaaacaccag tagtacaacc aagggattgg acttgttgga gttgcttagt atgtgcagca 541 tgttctgtag agttattaaa tctagttact gcggcaaccg gagctagtac tgcaagttaa 601 aatttaaaaa tgtgagagtg tttat [SEQ ID NO: 4]

[0059] As depicted in Figure 1, two open reading frames are possible for the nucleotide sequence of SEQ ID NO: 4. Both reading frames encode the amino acid sequence of SEQ ID NO: 13:METPVVQPRDWTCWSCLVCAACSVELLNLVTAATGASTAS [SEQ ID NO: 13].

[0060] As depicted also in Figures 1A and 1B, Reading Frame 1 may produce two copies of the peptide of SEQ ID NO: 1, whereas Reading Frame 3 may produce 1 copy of the peptide. Acordingly, activation of the gene for SEQ ID NO: 4 may theotically yield to simultaneous production of up to three (3) copies of the peptide of SEQ ID NO: 1.

[0061] As depicted also in Figures 1A and 1B, the nucleic acids encoding the amino acids of SEQ ID NO: 1 per se, are represented by SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 defined below: gattggactt gttggagttg cttagtatgt gcagcatgtt ctgtggaatt attaaattta gttactgcgg caacaggggc tagtactgca agc [SEQ ID NO: 5] gattggactt gttggagttg cttagtatgt gcagcatgtt ctgtagagtt attaaatcta gttactgcgg caaccggagc tagtactgca agt [SEQ ID NO: 6] gactggactt gttggagttg tttagtatgt gcagcatgtt ctgtggaatt attaaattta gttactgcgg caacaggggc tagtactgca agc [SEQ ID NO: 7]

[0062] The invention also encompasses isolated nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule that comprises any of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or to the complement thereto.

[0063] The invention also encompasses fragments of the isolated nucleic molecules defined hereinabove, this fragment having lengths of at least about 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 100. Such fragments may be useful, for instance, as probes in hybridization reactions to identify new polypeptides related to Peptide CXC-1 that have bacteriocin activity and / or plant biostimulant activity, and / or as PCR primers for amplifying such sequences. The invention also relates to vectors, such as plasmid vectors, viral vectors, expression vectors, etc. comprising any of the polynucleotides of the invention. The term "vector" refers to a nucleic acid molecule, which is capable oftransporting another nucleic acid to which it has been linked. Preferred vectors are those capable of autonomous replication and / or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors" or “recombinant expression vectors”. Recombinant expression vectors of invention can be constructed by standard techniques known to one of ordinary skill in the art.

[0064] A recombinant expression vector comprising a nucleic acid sequence of the present invention may be introduced into a host cell, which may include a living cell capable of expressing the protein coding region from the defined recombinant expression vector. The living cell may include both a cultured cell and a cell within a living organism, preferably a plant. Accordingly, the invention also relates to host cells containing the recombinant expression vectors of the invention. The host cell may be a prokaryotic host cell, for example a bacterial cell, or a eukaryotic cell, such as a yeast, plant, or animal cell. Vectors can be constructed in accordance to desired features (e.g., to contain one or more transcriptional regulatory sequences or elements such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals, etc.) and can be introduced into cells via conventional transformation or transfection techniques.

[0065] In another aspect, the invention relates to a transgenic plant comprising a polynucleotide as described above. The plant may express the polypeptide encoded by the polynucleotide, obviating the need to apply the polypeptide to the plant in order to obtain the benefits of the plant growth and / or disease resistance promoting activities of the polypeptide. Exemplary plants which could be genetically modified using a polynucleotide in accordance with the invention include, without limitation, legumes, such as soybean, peanut, pulses (e.g., pea and lentil), bean, forage crops (e.g., alfalfa and clover), plants of lesser agricultural importance (e.g., lupine, sainfoin, trefoil, and even some small tree species); tomato; corn; horticultural tree species (e.g., peach, apple, plum, pear, mango), forestry tree species (e.g., spruce, pine, fir, maple, oak, poplar), and small grain cereals or canola. Transgenic plants stably transformed with a nucleic acid molecule encoding a polypeptide of the invention sequence may be prepared by any of a variety of methods know in the art such as, without limitation Agrobacterium-mediatedtransformation, particle bombardment or electroporation. Alternatively, viral vectors may be used to transform plant cells. Bacillus thuringiensis strain CXC-1

[0066] Peptide CXC-1 is produced by a bacterium Bacillus thuringiensis CXC-1 (referred herein as “bacterial strain CXC-1”). Bacterial strain CXC-1 was deposited at the International Depositary Authority of Canada (IDAC) on July 3rd, 2024 and was assigned Accession No.030724-01.

[0067] The biostimulant Peptide CXC-1 is produced by the bacterial strain CXC-1. As such, the bacterial strain CXC-1 is expected to have plant biostimulant activity and that strain could thus find numerous applications for promoting plant growth, increasing production of plant stress-response proteins and / or inducing expression of plant defense- related enzymes by using these peptides and strains.

[0068] Accordingly, the present invention also encompasses compositions comprising bacterial strain CXC-1 and uses thereof for plant biostimulation.

[0069] In embodiments, the bacterial strain CXC-1 is cultured in King’s B broth / agar plates (King et al., The Journal of laboratory and clinical medicine 44.2 (1954): 301-307). In embodiments, the strain CXC-1 is grown by inoculating 1000 ml of the medium in a 2000 ml conical flask with 1 mL of a CXC-1 subculture. The flask is incubated in a rotary- shaker (Forma Scientific Inc, Model 4580™) set to 28 ℃ ± 2 ℃, and 120 rpm. The bacterial culture is allowed to grow for 4-5 days and is then ready to isolate Peptide CXC-1. Commercial applications and uses

[0070] The invention further relates to methods for promoting plant growth, for increasing production of plant stress-response proteins and / or for inducing expression of plant defense-related enzymes by using any of the peptides and strains described herein.

[0071] Polypeptides and microbial may be applied either before, during or after planting and may be applied to, for example, plant leaves, stems, roots, or seeds. As usedherein and in the claims, the term “plant” includes without limitation whole plants, plant parts, organs, leaves, stems and roots. The polypeptide 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 polypeptide can affect a growth or disease resistance promoting effect on the plant. If the polypeptide is applied to the soil, it may be applied before, during or after planting.

[0072] The polypeptide(s) may be applied by any suitable means, either in solid (e.g., as a free-flowing powder) or liquid form (such as in an aqueous carrier). Leaf spray, seed treatment and root drenching are the preferred techniques. The polypeptide(s) may also be applied to various portions of the plant or seed in slow-release formulations, such as beads or gels. The skilled person can readily determine suitable application regimes for the polypeptide. In one embodiment, the polypeptide is applied in an aqueous carrier at a concentration of about 10-9, 10-10, 10-11or 10-nM, equivalent to a total of 15.8, 1.58 and 0.158 ng pot-1(where each pot contained ten plants), respectively. In one embodiment, the polypeptide(s) is applied in an aqueous carrier at a concentration of about 10-10, 10-11, 10-12or 10-nM, equivalent to a total of 1.58, 0.158, 0.0158 ng plant-1, respectively.

[0073] In practicing the methods of the invention, the polypeptide(s) may be used alone, in the form of a plant growth and / or disease resistance promoting composition and / or in combination (i.e., concurrently, before or after) with other compounds or organisms including but not limited to, organic and chemical fertilizers, flavonoids, rhizobacteria, plant growth promoting rhizobacteria such as bacterial strain CXC-1 and BtNEB17. Accordingly, the present invention encompasses compositions comprising peptides (e.g., Peptide CXC-1 and / or fragment or peptides deriving therefrom as defined herein), with or without proteins and bacterial strains (e.g. strain CXC-1) as defined herein.

[0074] Such compositions may contain diluents, adjuvants, excipients, carriers, etc. suitable for inclusion in a plant growth and / or disease resistance promoting compositions as are known in the art. In another aspect, the invention provides a composition comprising a purified polypeptide as described above, and a carrier or diluent. The compositions may be in, for example, solid (such as powdered) or liquid form. In embodiments thecomposition is sterilized for greater stability and the polypeptides remain stable and active for more than 2 years.

[0075] The plant growth and / or disease resistance promoting composition may be provided in the form of a kit containing the composition and e.g., instructions for use of the composition for promoting plant growth.

[0076] The composition may take the form of plant seeds pre-treated with composition of the invention, e.g., the composition is applied to the seeds in advance of 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 or at least 2 years.

[0077] Elicitors of plant pathogen defence mechanisms may be used in conjunction with the methods and compositions of the invention. Suitable elicitors may include oligosaccharides, such as oligoglucans, oligochitins, oligochitosans and oligogalacturonides. Plant growth and / or disease resistance promoting compositions in accordance with the invention may contain such elicitors, or be packaged together with the elicitor. Such elicitors may be applied to plants, seeds, or the growing environment of the plant together with or separately from the polypeptide possessing plant growth and / or disease resistance promoting activity.

[0078] Plants planted, germinated or grown in the presence of the polypeptides and / or bacterial strain of the invention may exhibit an increase in plant growth, such as an increase in one or more of nodulation, nitrogen fixation, height, increased seedling emergence, leaf area, seed germination, leaf greenness, photosynthesis, or shoot, root, or total dry weight, relative to a plant that has not been treated with the plant growth and / or disease resistance promoting composition.

[0079] Similarly, plants planted, germinated or grown in the presence of the polypeptides and / or bacterial strain of the invention may exhibit one or more characteristics of improved disease resistance, such as, for example, reduced or inhibited pathogen infestation, increased activity of a lignification-related enzyme such as Phenyl ammonia lyase (PAL) or Tyrosine ammonia lyase (TAL), or an anti-oxidative enzyme suchas Peroxidase, Superoxide dismutase (SOD), Guaiacol peroxidase (POD), Ascorbate peroxidase (APX), Polyphenol oxidase (PPO). It may also increase the concentration of phenolics.

[0080] Increases of enzyme activity of more than 10, 20, 30, 40, 50, 60, or 70% may be obtained by the methods of the invention. Increases in concentration of total phenolics of more than 1, 5, 10, 15 or 20% may be obtained by the methods of the invention.

[0081] The compositions of the invention may be used, and the methods of the invention may be practiced, wherever plants are grown, such as in greenhouse, field, or laboratory conditions. 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.

[0082] The methods of the invention are not limited to use with any particular plant or plant-type. Exemplary plants with which the methods of the invention may be practiced include, without limitation: legumes, such as soybean, peanut, pulses (e.g., pea and lentil), bean, forage crops (e.g., alfalfa and clover), plants of lesser agricultural importance (e.g., lupine, sainfoin, trefoil, and even some small tree species); tomato; corn; horticultural tree species (e.g., peach, apple, plum, pear, mango), forestry tree species (e.g., spruce, pine, fir, maple, oak, poplar), and small grain cereals such as wheat, barley, oat, rice, turf grass, and canola, as well as fruits, vegetables, ornamentals, cannabis, and other greenhouse grown crops or field grown crops.

[0083] In emboddiments, application of Peptide CXC-1 on either plant leaves or roots, increases nodule numbers, shoot nitrogen concentration and total amount of nitrogen fixation. When applied as a leaf spray, Peptide CXC-1 is expected to increase leaf photosynthetic rates, leaf greenness and leaf area. When applied to roots, Peptide CXC-1 is expected to increase photosynthetic rates, leaf greenness, leaf area and plant dry matter accumulation.

[0084] In embodiments, Peptide CXC-1 is used a liquid solution at a concentration between about 5x10-9M to about 5x10-11M.

[0085] In embodiments, a dose of about 200 mg of Peptide CXC-1 (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 Peptide CXC-1 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 250 mg of Peptide CXC-1 is used to treat about 750,000 kg to about 2,500,000 kg of seeds.

[0086] Additional bacterial strains and peptides

[0087] The present invention envisions the use of additional strains of bacteria for obtaining peptides having biostimulant activity in accordance with the present invention. Indeed, knowledge of the peptide and nucleotide sequences of Peptide CXC-1 permits the identification of Peptide CXC-1 homologs in other bacterial strains. Tables A, B, C, D and E below provide the results of NCBI’s BLAST nucleotides and NCBI’s BLAST protein searches using default parameters, these tables listing additional bacteria producing peptides having strong homology with Peptide CXC-1. Accordingly, the present invention encompasses the uses of these bacteria, their nucleic acid sequences and the polypeptides encoded therefrom as a plant biostimulant in accordance with the uses and commercial applications described herein.

[0088] Table A: Selected BLASTn results for Peptide CXC-1 gene sequence 1 Gene 1 Sequence [SEQ ID NO: 8]: atggaaacaccagtagtacaaccaagggattggacttgttggagttgcttagtatgtgcagcatgttctgtggaattattaaatt tagttactgcggcaacaggggctagtactgcaagctaa S.# DESCRIPTION Query Percent Accession # Coverage Identity (%) (%) 1 Bacillus thuringiensis strain 100.00 100.00 CP050184.1 HER1410 plasmid pLUSID1, complete sequence 2 Bacillus thuringiensis strain 100.00 100.00 CP040783.1 HM-311 plasmid p1, complete sequence 3 Bacillus thuringiensis strain 100.00 100.00 CP032609.1 QZL38 plasmid p.2, complete sequenceBacillus cereus strain HBL-AI 100.00 100.00 CP023246.1 plasmid unnamed1, complete sequence Bacillus cereus C1L plasmid 100.00 100.00 CP022446.1 pC1L1, complete sequence Bacillus thuringiensis strain 100.00 100.00 CP014284.1 Bt185 plasmid pBT1850294, complete sequence Bacillus thuringiensis strain 100.00 100.00 CP011350.1 YC-10 plasmid pYC1, complete sequence Bacillus thuringiensis serovar 100.00 100.00 CP009998.1 kurstaki strain HD 1 plasmid unnamed1, complete sequence Bacillus thuringiensis serovar 100.00 100.00 CP010090.1 galleriae strain HD-29 plasmid pBMB426, complete sequence Bacillus thuringiensis serovar 100.00 100.00 CP007616.1 kurstaki str. YBT-1520 plasmid pBMB400, complete sequence Bacillus thuringiensis serovar 100.00 100.00 CP004860.1 kurstaki str. YBT-1520 plasmid pBMB422, complete sequence Bacillus thuringiensis strain 100.00 100.00 CP083157.1 ABTS-1857 plasmid pCH_181-a, complete sequence Bacillus thuringiensis strain 100.00 100.00 CP083145.1 GC-91 plasmid pCH_186-a, complete sequence Bacillus thuringiensis strain 100.00 100.00 CP083117.1 SA11 plasmid pCH_164-a, complete sequence Bacillus thuringiensis strain 100.00 100.00 CP083102.1 ABTS-351 plasmid pCH_183- a, complete sequence Bacillus thuringiensis strain 100.00 100.00 CP083086.1 B401 plasmid pP05_2-a, complete sequence Bacillus thuringiensis strain 100.00 100.00 CP043233.1 Bt-GS57 plasmid unnamed2, complete sequence18 Bacillus thuringiensis strain 100.00 100.00 FJ977580.1 SF361 bacteriocin, thurincin H gene cluster, complete sequence 19 Bacillus thuringiensis 100.00 100.00 FJ159242.1 thuricin17 (tucA1), thuricin17 (tucA2), and thuricin17 (tucA3) genes, complete cds 20 Bacillus cereus Q1 plasmid 100.00 99.19 CP000228.1 pBc239, complete sequence 21 Bacillus thuringiensis serovar 100.00 98.37 CP004877.1 kurstaki str. HD-1 plasmid pBMB431, complete sequence

[0089] Table B: Selected BLASTn results for Peptide CXC-1 gene sequence 2 Gene 2 Sequence [SEQ ID NO: 9]: atggaaacaccagtagtacaaccaagggattggacttgttggagttgcttagtatgtgcagcatgttctgtagagttattaaat ctagttactgcggcaaccggagctagtactgcaagttaa S.# Query Percent Description Coverage Identity Accession # (%) (%) 1 Bacillus thuringiensis strain 100.00 HER1410 plasmid pLUSID1, 100.00 CP050184.1 complete sequence 2 Bacillus thuringiensis strain 100.00 HM-311 plasmid p1, complete 100.00 CP040783.1 sequence 3 Bacillus thuringiensis strain 100.00 QZL38 plasmid p.2, complete 100.00 CP032609.1 sequence 4 Bacillus cereus strain HBL-AI 100.00 plasmid unnamed1, complete 100.00 CP023246.1 sequence 5 Bacillus cereus C1L plasmid 100.00 100.00 CP022446.1 pC1L1, complete sequence 6 Bacillus thuringiensis strain 100.00 Bt185 plasmid pBT1850294, 100.00 CP014284.1 complete sequence 7 Bacillus thuringiensis strain 100.00 YC-10 plasmid pYC1, 100.00 CP011350.1 complete sequenceBacillus thuringiensis serovar 100.00 kurstaki strain HD 1 plasmid unnamed1, complete 100.00 CP009998.1 sequence Bacillus thuringiensis serovar 100.00 galleriae strain HD-29 plasmid 100.00 CP010090.1 pBMB426, complete sequence Bacillus thuringiensis serovar 100.00 kurstaki str. YBT-1520 plasmid 100.00 CP007616.1 pBMB400, complete sequence Bacillus thuringiensis serovar 100.00 kurstaki str. HD-1 plasmid 100.00 CP004877.1 pBMB431, complete sequence Bacillus thuringiensis serovar 100.00 kurstaki str. YBT-1520 plasmid 100.00 CP004860.1 pBMB422, complete sequence Bacillus thuringiensis strain 100.00 ABTS-1857 plasmid 100.00 CP083157.1 pCH_181-a, complete sequence Bacillus thuringiensis strain 100.00 GC-91 plasmid pCH_186-a, 100.00 CP083145.1 complete sequence Bacillus thuringiensis strain 100.00 SA11 plasmid pCH_164-a, 100.00 CP083117.1 complete sequence Bacillus thuringiensis strain 100.00 ABTS-351 plasmid pCH_183- 100.00 CP083102.1 a, complete sequence Bacillus thuringiensis strain 100.00 B401 plasmid pP05_2-a, 100.00 CP083086.1 complete sequence Bacillus thuringiensis strain Bt- 100.00 GS57 plasmid unnamed2, 100.00 CP043233.1 complete sequence Bacillus thuringiensis 100.00 thuricin17 (tucA1), thuricin17 100.00 FJ159242.1 (tucA2), and thuricin17 (tucA3) genes, complete cds Bacillus thuringiensis strain 100.00 FJ977580.1 SF361 bacteriocin, thurincin H 99.19% gene cluster, complete sequence Bacillus cereus Q1 plasmid 100.00 98.35% CP000228.1 pBc239, complete sequence22 Bacillus cereus strain AR156 100.00 CP015592.1 plasmid pAR460, complete 95.93% sequence 23 Bacillus tropicus strain AOA- 100.00 95.12% CP049019.1 CPS1 chromosome 24 Bacillus anthracis strain 100.00 CP026608.1 HDZK-BYSB7 chromosome, 95.12% complete genome 25 Bacillus anthracis strain 100.00 CP031643.1 MCCC 1A01412 chromosome, 95.12% complete genome 26 Bacillus thuringiensis strain 100.00 CP01 plete genome 95. 3000.1 XL6, com 12% 27Bacillus sp. SD-4 chromosome100.0095.12%CP083987.128 Bacillus sp. CRB-7 100.00 CP083749.1 chromosome, complete 95.12% genome 29 Bacillus cereus strain BC07 100.00 CP072769.1 chromosome, complete 95.12% genome

[0090] Table C: Selected BLASTn results for Peptide CXC-1 gene sequence 3 Gene 3 Sequence [SEQ ID NO: 10]: atggaaacaccagtagtacaaccaagggactggacttgttggagttgtttagtatgtgcagcatgttctgtggaattattaaatt tagttactgcggcaacaggggctagtactgcaagctaa S. Query Percent # Description coverage Identity Accession (%) (%) # 1 Bacillus thuringiensis strain 100.00 HER1410 plasmid pLUSID1, 100.00 CP050184.1 complete sequence 2 Bacillus thuringiensis strain HM- 100.00 311 plasmid p1, complete 100.00 CP040783.1 sequence 3 Bacillus thuringiensis strain 100.00 QZL38 plasmid p.2, complete 100.00 CP032609.1 sequence 4 Bacillus cereus strain HBL-AI 100.00 plasmid unnamed1, complete 100.00 CP023246.1 sequence 5 Bacillus cereus C1L plasmid 100.00 pC1L1, complete sequence 100.00 CP022446.1Bacillus thuringiensis strain Bt185 100.00 plasmid pBT1850294, complete 100.00 CP014284.1 sequence Bacillus thuringiensis strain YC-10 100.00 plasmid pYC1, complete 100.00 CP011350.1 sequence Bacillus thuringiensis serovar 100.00 kurstaki strain HD 1 plasmid 100.00 CP009998.1 unnamed1, complete sequence Bacillus thuringiensis serovar 100.00 galleriae strain HD-29 plasmid 100.00 CP010090.1 pBMB426, complete sequence Bacillus thuringiensis serovar 100.00 kurstaki str. YBT-1520 plasmid 100.00 CP007616.1 pBMB400, complete sequence Bacillus thuringiensis serovar 100.00 kurstaki str. HD-1 plasmid 100.00 CP004877.1 pBMB431, complete sequence Bacillus thuringiensis serovar 100.00 kurstaki str. YBT-1520 plasmid 100.00 CP004860.1 pBMB422, complete sequence Bacillus thuringiensis strain ABTS- 100.00 1857 plasmid pCH_181-a, 100.00 CP083157.1 complete sequence Bacillus thuringiensis strain GC-91 100.00 plasmid pCH_186-a, complete 100.00 CP083145.1 sequence Bacillus thuringiensis strain SA11 100.00 plasmid pCH_164-a, complete 100.00 CP083117.1 sequence Bacillus thuringiensis strain ABTS- 100.00 351 plasmid pCH_183-a, 100.00 CP083102.1 complete sequence Bacillus thuringiensis strain B401 100.00 plasmid pP05_2-a, complete 100.00 CP083086.1 sequence Bacillus thuringiensis strain Bt- 100.00 GS57 plasmid unnamed2, 100.00 CP043233.1 complete sequence Bacillus thuringiensis strain SF361 100.00 bacteriocin, thurincin H gene 100.00 FJ977580.1 cluster, complete sequence Bacillus thuringiensis thuricin17 100.00 (tucA1), thuricin17 (tucA2), and 100.00 FJ159242.1 thuricin17 (tucA3) genes, complete cds21 Bacillus cereus Q1 plasmid 100.00 pBc239, complete sequence 99.19 CP000228.1

[0091] Table D: Selected BLASTp results for Peptide CXC-1 gene sequence 2 Search 1 - Signal and mature peptide sequence ETPVVQPRDWTCWSCLVCAACSVELLNLVTAATGASTAS [SEQ ID NO: 11] Description Query Percent coverage Identity Hypothetical protein BCQ_P1102 (Bacillus cereus 100 % 100 % Q1) Hypothetical protein bthur0007_54070 (Bacillus 100 % 100 % thuringiensis serovar monterrey BGSC4AJ1) Hypothetical protein C621_0220570 (Bacillus 89 % 100 % thuringiensis serovar azawai str. Leapi01)

[0092] Table E: Selected BLASTp results for Peptide CXC-1 gene sequence 2 Search 2 - Mature peptide sequence only DWTCWSCLVCAACSVELLNLVTAATGASTAS [SEQ ID NO: 12] Description Query Percent coverage Identity Hypothetical protein BCQ_P1102 (Bacillus cereus 100 % 100 % Q1) Hypothetical protein bthur0007_54070 (Bacillus 100 % 100 % thuringiensis serovar monterrey BGSC4AJ1) Hypothetical protein C621_0220570 (Bacillus 87 % 100 % thuringiensis serovar azawai str. Leapi01)

[0093] 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 to be within the scope of this invention, and covered by the claims appended hereto. The invention is further illustrated by the following examples, which should not be construed as further or specifically limiting.EXAMPLES

[0094] Example 1: Production and purification of Peptide CXC-1 from bacterial culture of Bacillus thuringiensis strain CXC-1

[0095] Production of Peptide CXC-1

[0096] Bacillus thuringiensis strain CXC-1 was cultured in King’s B liquid medium at 28^ 20C on an orbital shaker for at least 72 hours, rotating at 150 rev min-1. The composition of this medium was as follows: protein peptone #3 – 20 g; K2HPO4– 1.5 g; MgSO4– 0.75 g; glycerol – 15 mL; distilled water – 1000 mL. The entire culture was extracted by adding 40% n-butanol. The butanol-water mixture was shaken for 30 min and kept overnight at room temperature. The separated butanol phase was collected and evaporated at 450C using the rotary evaporator. The dried extract was resuspended in 20% acetonitrile and used for the purification of Peptide CXC-1.

[0097] Purification of Peptide CXC-1

[0098] Butanol-soluble compounds in 20% acetonitrile were loaded on PrepSep C18™ cartridge (Fisher Scientific, cat#11-131-12) and fractionated using 35 % (acetonitrile:water, v / v), 43% and 100% acetonitrile. These fractions were collected. Aliquots of 20 µL were used for the HPLC analyses to quantify Peptide CXC-1 in fractions.

[0099] Peptide CXC-1 was not detected in a fraction of 35% acetonitrile but was abundant in the fraction of 43% acetonitrile. Small quantities of Peptide CXC-1 was present in 100% acetonitrile fraction. [000100] Multiple repetitions (10 times) of the procedure of purification of Peptide CXC- 1 showed that only 1.0 ± 0.4% and 0.3 ± 0.2% of total loaded bacteriocin were eluted with 35% and 100% acetonitrile, respectively. The maximum of 98.7 ± 0.3% was detected in a fraction of 43% acetonitrile.[000101] Example 2: Characterization of Peptide CXC-1 [000102] Tandem mass spectrometry (MS / MS) analysis revealed presence of two major peaks and confirmed that the peptide was reasonably pure (e.g., about >95% purity) even though other proteins and peptides were also present in the bacterial extract including, but not limited to, ESAT-6-like protein (Accession: WP_000918601), Heavy metal-binding domain-containing protein (Accession: WP_000637511), Non-hemolytic enterotoxin NHE subunit A (Accession: WP_000751828.1), Non-hemolytic enterotoxin NHE subunit B (Accession: WP_000162964.1), WXG100 family type VII secretion target (Accession: WP_002025379.1) and Alanine dehydrogenase (Accession: WP_001219742.1), as identified through shotgun proteomics analyses. [000103] Identification of B and Y fragment ions was obtained through CID MS / MS for Peptide CXC-1. Table F displays assignment of fragment ions that match the MS / MS spectra ((CID (+3), CID (+2) and HCD (+3) spectra (superimposed) with the matching B and Y fragments ions)). Table F: List of internal fragments (IF) Internal fragment (IF) Observed m / z Calculated m / z Error (ppm) CAACSVELL 890.4111 890.4111 0.02 CAACSVEL 777.3255 777.3270 -1.96 CAACSVE 664.2442 664.2430 1.88 [000104] Particularly the MS / MS confirmed the peptide amino acid sequence DWTCWSCLVCAACSVELLNLVTAATGASTAS (SEQ ID NO: 1). The MS / MS sequencing provided direct evidence of most of the amino acids for Peptide CXC-1’s sequence DWTCWSCLVCAACSVELLNLVTAATGASTAS for the parent ion mass peak of 3137.3603 Daltons (C134H212N34O45S4) (direct evidence in bold; remaining residues confirmed by inference through bioinformatics analyses). [000105] The NMR analysis suggests that the peptide is a sactipeptide comprising four (4) sulfur to alpha carbon thioether linkages, The linkages are as follows: ^ Linkage 1: Cys4 - Ser28; ^ Linkage 2: Cys7 - Thr25;^ Linkage 3: Cys10 - Thr22; and ^ Linkage 4: Cys13 - Asn19 [000106] Based on all that information, it was possible to prepared 3D models the structure of Peptide CXC-1, including its four Cys-to-alpha carbon bonds / linkage, as depicted in Figure 2 and Figure 3. [000107] HPLC quantification for β-exotoxin production under standard growth conditions of CXC-1 revealed that this strain did not produce detectable amounts of β- exotoxin (data not shown). These preliminary results suggest that CXC-1 produces lower amount and / or lower concentration of β-exotoxin compared to BtNEB17, and thus that bacterial strain CXC-1 is different and distinguishable from BtNEB17. [000108] Example 3: Treatment of tomatoes with a combination of Peptide CXC-1 and copper hydroxide [000109] Methodology: Peptide CXC-1 may be evaluated in field trials alongside a commercial copper hydroxide crop protection product as follow. In such trial, 2255 variety tomatoes are manually transplanted into 6' wide rows spaced 19" apart. Peptide CXC-1 is applied as a foliar spray application alone or prior application of copper each week for 10 weeks. Copper is also applied alone and an untreated check is included for reference. Plots are replicated four times in a randomized block design. [000110] Results: In embodiments, the use of Peptide CXC-1 provides the following benefits, compared to a treatment where tomatoes are treated with only copper: a. Peptide CXC-1 combined with copper increases total harvested tomato count by at least 10%, or by at least 20%, or by at least 30%; b. Peptide CXC-1 combined with copper increases marketable tomato weight harvested by at least 2.5%, fy at least 5%, or by at least 10%; c. Peptide CXC-1 combined with copper increases estimated marketable yield (lbs / acre) by at least 2.5%, by at least 5%, or by at least 10%; d. Peptide CXC-1 combined with copper provides t gross income return upon harvest by at least 2.5%, or by at least 5%, or by at least 10% higher than control;e. Peptide CXC-1 combined with copper increases weight of tomatoes harvested (e.g. by at least 2.5%, or by at least 5%, or by at least 10%); and f. Peptide CXC-1 combined with copper yields provides at least 5%, at least 10%, or at least 20%, or at least 25% higher number of extra-large tomatoes harvested. [000111] Conclusion: Peptide CXC-1 is expected to increase tomato weight, marketable yield and gross harvest income in tomatoes when combined with copper fungicide crop protection products which contain copper hydroxide. Example 4: Corn field treatment with Peptide CXC-1 [000112] Methodology: Peptide CXC-1 may be tested on corn crops. For more certainty, the trial may be held at two locations and can feature two planting dates (normal planting date and a late planting date). [000113] Results: Peptide CXC-1 is expected to increase Bushels / Acre, for instance an average yield increase of at least 1%, or at least 2.5% or at least 5%. [000114] Conclusion: Such study should demonstrate that Peptide CXC-1 is efficacious in increasing corn yields. Example 5: Soybean field treatment with Peptide CXC-1 [000115] Methodology: Peptide CXC-1 may be tested on soybean crops. For more certainty, the trial may be held at two locations and may feature two planting dates (normal planting date and a late planting date). [000116] Results: Peptide CXC-1 is expected to provide an average increase of more than 1.5 Bushel / acre (e.g. at least 0.5 Bushel / acre, or at least at least Bushel / acre), and at least 1%, or at least 2%, or at least 3% average yield increase. [000117] Conclusion: Such study should demonstrate that Peptide CXC-1 is efficacious in increasing soybean yields.Example 6: Rice field treatment with Peptide CXC-1 [000118] Methodology: Peptide CXC-1 may be tested on rice crops The trial may be held at two locations and feature two planting dates (normal planting date and a late planting date). [000119] Results: Peptide CXC-1 is expected to provide an average increase of at least at least 0.5 hundredweight (CWT), or at least at least 1 hundredweight (CWT), or at lease 2 hundredweight (CWT), and at least 0.5%, or at least 1%, or at least 2% average yield increase. [000120] Conclusion: Such study should demonstrate that Peptide CXC-1 is efficacious in increasing rice yields. Example 7: Mitigation of plant stress by Peptide CXC-1Methodology: Peptide CXC-1 may be tested to measure its efficacy in plant stress mitigation when applied as a seed treatment on corn or soybean alongside commercial herbicides. For instance, Peptide CXC-1 may be applied alongside CruiserMaxx Vibrance™ as well as Acceleron™ Seed Applied Solutions. [000122] Results: Peptide CXC-1 is expected to provide an increase of at least 0.5 bushels / acre, or at least 1 bushels / acre in soybean, and at least 0.5 bushels / acre, or at least 1 bushels / acre, or at least 2 bushels / acre in corn, as compared to respective control applications without Peptide CXC-1. [000123] Conclusion: Such study should demonstrate that Peptide CXC-1 mitigates stress when applied as a seed treatment to both corn and soybean crops. Such study should also show that Peptide CXC-1 effectively returns yield that would otherwise be lost be herbicidal stress. Example 8: Mitigation of plant stress by Peptide CXC-1Methodology: Peptide CXC-1 may be tested as a growth-promoting inoculant for soybeans. Such study may be conducted across different locations (e.g., in Brazil) toobtain distinct tropical climates and soil types, wherein Peptide CXC-1 is applied via seed treatment. Evaluations may include germination, Emergency Speed Index (ESI), crop stand, plant vigor, plant height, and masses of aerial parts and roots, both fresh and dry. The experimental design may be a randomized block with four treatments and six replications, with plots measuring at least 54 m². [000125] Results: Peptide CXC-1 is expected to provide significantly increased various growth and yield parameters compared to the control For instance, Peptide CXC-1 may increase seed emergence by at least 1%, 2.5%, 5%, 10% 15% or more over control. Relative to the control, Peptide CXC-1 should increase plant vigour (e.g. by at least 10%, at least 20%, at least 30%, at least 40%, at least 40%, or more), increase in plant height (e.g. by at least 1%, at least 2%, at least 3%, or more), increase in crop stand (e.g., by at least 1%, at least 2%, at least 3%, or more), and increase in dry weight (e.g. by at least 10%, at least 20%, at least 30%, at least 40%, at least 40%, or more). [000126] Conclusion: Such study should demonstrate that Peptide CXC-1 provides faster seed emergence, greater plant vigour, height, crop stand and harvested dry weight. Example 9: Treatment of cannabis with Peptide CXC-1 [000127] Methodology: Peptide CXC-1 may be tested to assess the impact of different treatments on the growth of the "Mandarin cookies" marijuana variety. Cannabis plants may be given different foliar treatments and different soil treatments. The plants are consistently watered every two days and they receive fertilization as required. Peptide CXC-1 was further be administered as either a soil or foliar inoculum, for instance at a dose of 5 ml per plant. Such experiment may have a duration of four weeks and be carried out inside a growth chamber, maintaining conditions at 24 / 26°C with an 18- hour light and 6-hour dark cycle. The soil substrate chosen for such study may be Canna coco, which is a standard within the Canadian cannabis industry. [000128] Results: Such study should demonstrate that foliar spray application of Peptide CXC-1 provides at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25% increase of fresh plant weight over the control; at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, increase ofdry weight over control; and at least 2.5%, or at least 5%, at or at least 10%, of plant height increase over control. [000129] Conclusion: Such study should demonstrate that Peptide CXC-1 is effective in cannabis plants, increasing fresh plant weight, dry weight and increased plant height. Example 10: Effect of Peptide CXC-1 on Wheat Growth and Yield [000130] Methodology: A replicated field trial was conducted during the ^2024–25 season to evaluate the effect of Peptide CXC-1 on wheat as a foliar spray. Treatments included: T1: untreated control; and T2: 150L / acre (spray volume) of an aqueous solution comprising Peptide CXC-1 at a concentration of about 1x10-11M. Assessments were made 10^days after spray (DAS) for leaf green-colour rating, plant height and crop vigour, and at harvest for 50% heading days, effective tillers per metre row, plant height, shoot and root biomass, plant dry weight, filled grains per spike, 1,000-grain weight, grain shining, and grain yield (quintals per acre). [000131] ^Results: Compared to the untreated control, Peptide CXC-1 produced the following improvements: -^Leaf green colour rating at 10^days increased by 15.0% -^Plant height rating at 10^days increased by 46.9% -^Crop vigour rating at 10^days increased by 41.2% -^50% heading was delayed by 1.15% -^Effective tillers per metre row increased by 10.6% -^Final plant height increased by 2.0% -^Shoot biomass per plant increased by 9.4% -^Root biomass per plant increased by 17.9% -^Dry weight per plant increased by 13.4% -^Filled grains per spike increased by 6.1% -^1,000-grain weight increased by 5.4% -^Grain shining rating increased by 35.5% -^Grain yield increased by 8.9%[000132] Conclusion: Application of Peptide CXC-1 significantly enhanced early vigour, biomass accumulation, grain-quality attributes and overall yield in wheat, demonstrating its potential as an effective biostimulant in wheat production. * * * [000133] 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. [000134] The singular forms “a”, “an” and “the” include corresponding plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide" includes one or more of such peptides 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. [000135] 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. [000136] 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 besuggested to persons skilled in the art and are to be included within the present invention and scope of the appended claims.

Claims

CLAIMS:

1. A purified polypeptide selected from the group consisting of: (a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; (b) a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; (c) a polypeptide which is a fragment of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, the fragment comprising at least 25, 26, 27, 27, 28, or 29 consecutive amino acids of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; wherein said purified peptide is a sactipeptide comprising one, two, three or four Cys-to-alpha carbon bond(s); wherein said purified polypeptide has plant biostimulant activity.

2. The purified polypeptide of claim 1, wherein said peptide is encoded by a nucleotide sequence comprising SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or by a nucleotide sequence comprising SEQ ID NO:

4.

3. The purified polypeptide of claim 1 or 2, wherein said Cys-to-alpha carbon bond(s) consists of a Sulfur to Alpha-Carbon Thioether Linkage.

4. The purified polypeptide of any one of claims 1 to 3, wherein said peptide is characterized by a LC / MS mass of 3137.3603 Daltons (C134H212N34O45S4).

5. The purified polypeptide of any one of claims 1 to 4, wherein said sactipeptide comprises Formula 1:wherein the lines represent Cys-to-alpha carbon bonds.

6. The purified polypeptide of any one of claims 1 to 5, said peptide comprises a 3D structure as depicted in Figure 2 and / or Figure 3.

7. The purified polypeptide of any one of claims 1 to 6, wherein the plant biostimulant activity is at least one of promoting plant growth, increasing production of plant stress- response proteins, and inducing expression of plant defense-related enzymes.

8. The purified polypeptide of any one of claims 1 to 7, wherein said polypeptide possesses one or more of the following properties: ^ increasing plant growth by at least 2%, 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25% or more; ^ stimulating the plant root system 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 antibiosis and biocontrol of plant pathogens ^ stimulating production of antibiotics; ^ enhancing early seedling growth of soybean and corn by at least 5%, 10%, 15%, 20%, 25% or more; ^ increasing nodule number by at least 5%, 10%, 15%, 20%, 25% or more; ^ increasing plant dry weight increased (by at least 5%, 10%, 15%, 20%, 25% or more; ^ increasing root irrigation nodule number by at least 5%, 10%, 15%, 20%, 25% or more; ^ increasing concentration of root irrigation in shoot tissue by at least 5%, 10%, 15%, 20%, 25% or more; ^ increasing leaf photosynthetic rates when applied on leaves by at least 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25% or more; ^ increasing photosynthetic rate by at least 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25% or more;^ increasing crop yields by at least 2%, 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25% or more; ^ protecting crops from abiotic stress; ^ improving plant resistance to disease(s); ^ reducing the need for synthetic fertilizers and pesticides yields by at least 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25% or more; ^ act as a signaling compound; ^ increasing farm revenues by at least 3%, 4%, 5%, 6%, 7%, 10%, 15%, 20%, 25% or more; ^ improving global food security; ^ cost efficient and economical to apply.

9. The purified polypeptide of any one of claims 1 to 8, wherein said polypeptide is produced by Bacillus thuringiensis strain deposited at the International Depositary Authority of Canada (IDAC) on July 3rd, 2024 under Accession No.030724-01.

10. A purified polypeptide, wherein said purified peptide consists of a sactipeptide comprising one, two, three or four Cys-to-alpha carbon bond(s), wherein said peptide is encoded by a nucleotide sequence comprising SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or by a nucleotide sequence comprising SEQ ID NO:4, and wherein said purified polypeptide has plant biostimulant activity.

11. The purified polypeptide of any one of claims 1 to 10, wherein said purified peptide is different from polypeptide “Thuricin 17” produced by Bacillus thurigiensis NEB17 and is different from Thuricin H produced by Bacillus thurigiensis SF361.

12. A sactipeptide comprising the amino acid sequence of SEQ ID NO: 1, wherein said sactipeptide comprises one, two, three or four Cys-to-alpha carbon bond(s).

13. The sactipeptide of claim 12, wherein said Cys-to-alpha carbon bond(s) consists of a Sulfur to Alpha-Carbon Thioether Linkage.

14. The sactipeptide of claim 12 or 13, wherein said sactipeptide is characterized by a LC / MS mass of 3137.3603 Daltons (C134H212N34O45S4).

15. The sactipeptide of any one of claims 12 to 14, wherein said sactipeptide comprises Formula 1:wherein the lines represent Cys-to-alpha carbon bonds.

16. An isolated sactipeptide represented by Formula 1:Formula 1 wherein the lines represent Sulfur to Alpha-Carbon Thioether Linkage; and wherein said sactipeptide comprises a LC / MS mass of 3137.36 Daltons (C134H212N34O45S4).

17. The sactipeptide of claim 16, wherein said sactipeptide has plant biostimulant activity.

18. The sactipeptide of claim 16 or 17, wherein said sactipeptide is produced by Bacillus thuringiensis strain deposited at the International Depositary Authority of Canada (IDAC) on July 3rd, 2024 under Accession No.030724-01.

19. The sactipeptide of any one of claims 12 to 18, wherein said sactipeptide is different from polypeptide Thuricin 17 produced by Bacillus thurigiensis NEB17 and is different from Thuricin H produced by Bacillus thurigiensis SF361.

20. A bacterial extract comprising a plurality of polypeptides as defined in any one of claims 1 to 11 and / or comprising a plurality of sactipeptides as defined in any one of claims 12 to 19.

21. A composition for plant biostimulation comprising a polypeptide as defined in any one of claims 1 to 11 and / or comprising a sactipeptide as defined in any one of claims 12 to 19.

22. A method for plant biostimulation, comprising applying to a plant the polypeptide as defined in any one of claims 1 to 11 and / or the sactipeptide as defined in any one of claims 12 to 19.

23. The method of claim 22, wherein said plant is selected from the group consisting of soybean, peanut, pea, bean, alfalfa clover, lupine, sainfoin, trefoil, tomato, corn; peach tree, apple tree, plum tree, pear tree, mango tree, spruce, pine, fir, maple, oak, poplar, grain cereals, rice, turf grass, cannabis, fruits and canola.

24. The method of claim 22 or 23, wherein application of said purified polypeptide provides at least one of the following benefits to the plant: ^ an increase of nodule number; ^ an increase of plant dry weight or yield; ^ an increase of root irrigation nodule number ^ an increase of root irrigation concentration; ^ an increase of leaf photosynthetic rate; ^ an increase of leaf greenness; ^ an increase of photosynthetic rate; ^ an increase of plant growth; ^ an increase of production of plant stress-response proteins, ^ an increase of seed germination; ^ an increase of expression of plant defense-related enzymes; ^ an increase of nitrogen fixation; and ^ an increase of leaf area.

25. The method of any one of claims 22 to 24, wherein said polypeptide is applied on plant leaves, stems, roots, seeds or combinations thereof.

26. The method of any one of claims 22 to 24, wherein said polypeptide is applied before, during and / or after planting.

27. The method of any one of claims 22 to 24, wherein said polypeptide is applied in the growing environment of the plant.

28. The method of any one of claims 22 to 24, wherein said polypeptide is applied as a free-flowing powder or in a liquid form.

29. The method of any one of claims 22 to 28, wherein said polypeptide is applied at a concentration of about 10-9M, about 10-10 M, or about 10-11M.

30. The method of any one of claims 22 to 29, wherein said polypeptide is applied in combination with at least one of an organic fertilizer, a chemical fertilizer, flavonoids, rhizobacteria, and Plant Growth Promoting Rhizobacteria (PGPR).

31. An isolated polynucleotide encoding the polypeptide as defined in any one of claims 1 to 11 and / or the sactipeptide as defined in any one of claims 12 to 19, or the complement thereto.

32. The isolated polynucleotide as defined in claim 31, said polynucleotide comprising the sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:

7.

33. An isolated polynucleotide that hybridizes under stringent conditions to the polynucleotide of claim 31 or 32.

34. A fragment of the isolated polynucleotide as defined in any one of claims 31 to 33, said fragment having a length of at least 10 nucleotides.

35. A vector comprising the polynucleotide according to any one of claims 31 to 33, or comprising the fragment of claim 34.

36. A host cell genetically modified to comprise the polynucleotide according to any one of claims 31 to 33, to comprise the fragment of claim 34, and / or to comprise the vector of claim 35.

37. An isolated bacterium consisting of a Bacillus thuringiensis strain deposited at the International Depositary Authority of Canada (IDAC) on July 3rd, 2024 under Accession No.030724-01.

38. The isolated bacterium of claim 37, comprising the polynucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:

7.

39. The isolated bacterium of claim 37 or 38, encoding the polypeptide as defined in any one of claims 1 to 11 and / or encoding the sactipeptide as defined in any one of claims 12 to 16.

40. The isolated bacterium of any one of claims 37 to 39, wherein said bacterium is different and distinguishable from Bacillus thurigiensis BtNEB17 and different and distinguishable from Bacillus thurigiensis SF361.

41. The isolated bacterium of any one of claims 37 to 40, wherein said isolated bacterium produces a lower amount and / or a lower concentration of β-exotoxin(s) compared to bacterium BtNEB17.

42. A purified polypeptide selected from the group consisting of: (a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; (b) a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity over the entire length of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; (c) a polypeptide which is a fragment of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, the fragment comprising at least 25, 26, 27, 27, 28, or 29 consecutive amino acids of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; wherein said purified peptide is produced by any one of the bacterial strains listed in Tables A, B, and C.

43. The purified polypeptide of claim 42, wherein said bacterial strain is a Bacillus thuringiensis strain, a Bacillus cereus strain, a Bacillus tropicus strain, a Bacillus anthracis strain, or a Bacillus sp strain.

44. The purified polypeptide of claim 42 or 43, wherein said polypeptide is selected from the group consisting of the peptides and proteins identified in Table D and Table E.

45. The purified polypeptide of any one of claims 42 to 44, wherein said purified polypeptide comprises a plant biostimulant activity substantially similar to that of Peptide CXC-1.

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