Biostimulatory and biodefensive peptides and their agricultural uses

Abstract

The present invention relates to novel isolated peptides with plant biostimulatory and biodefense activities, and compositions containing the peptides. Preferably, the peptides of the present invention are derived from Solanum lycopersicon (tomato) plants and are produced by recombinant or synthetic methods. The present invention also relates to the use of the peptides and / or compositions for enhancing plant tolerance to biotic and / or abiotic stresses.

Description

[Technical Field]

[0001] The present invention relates to the field of agriculture, more particularly to agricultural methods aimed at promoting plant growth and improving productivity. In particular, the present invention relates to novel peptides having biostimulant and bioprotective activity against biotic stress in plants. [Background technology]

[0002] In agriculture, promoting plant growth, health, and productivity is essential. Over the years, agricultural methods have developed various approaches to combat damage to plants and crops caused by biotic stresses, i.e., attacks by microorganisms such as viruses, bacteria, and fungi, and microorganisms such as nematodes, insects, and mites, as well as abiotic stresses such as water shortages, high temperatures, extreme cold, and high salinity.

[0003] Traditionally, biotic and abiotic stresses in plants have been controlled by the use of fertilizers and pesticides, the introduction of physical soil amendments, etc. However, the use of pesticides can have serious long-term environmental impacts, such as excessive use, persistence in the environment, and the residue of harmful substances in edible parts of plants, causing significant damage to human health. In addition, crop protection agents can lose their effectiveness over time due to the emergence of resistant strains in pathogen populations.

[0004] To limit the use of risky strategies, many efforts have been made to develop and implement ecologically sensitive and sustainable approaches in an attempt to improve crop productivity by promoting plant growth and stress tolerance.

[0005] U.S. Patent Nos. US 5,378,819, US 5,883,076 and US 6,022,739 describe the isolation of the plant peptide hormone systemin and its involvement in the activation of defense genes in Solanum lycopersicon (tomato) plants in response to damage caused by chewing pests or mechanical wounding.

[0006] Systemin is an 18-amino acid peptide hormone located at the end of the carboxy-terminal region of a 200-amino acid precursor called prosystemin (ProSys). Upon plant wounding, the precursor undergoes proteolysis, likely via phytaspase, an aspartate-specific protease of the subtilase family, to release systemin. This peptide is released into the apoplast and activates defense signals through interaction with the membrane receptor SYR1 (Narvaez-Vasquez and Orozco-Cardenas, (2008) “Systemins and AtPeps: Defense-related peptide signals”; In Induced plant resistance to herbivory (pp. 313-328). Springer, Dordrecht; Wang L. et al., (2018) “The systemin receptor SYR1 enhances resistance of tomato against herbivorous insects”, Nature Plants, 4(3), 152-156).

[0007] Under physiological conditions, the prosystemin gene is expressed at femtomolar levels in plant leaves, petals, and stems, but not in roots (Pearce G. et al., (1991) "A polypeptide from tomato leaves induces wound-inducible proteinase inhibitor protein", Science, 253(5022), 895-897; Narvaez-Vasquez, J., and Ryan, CA, (2004) "The cellular localization of prosystemin: a functional role for phloem parenchyma in systemic wound signaling", Planta, 218(3), 360-369). Conversely, wounds caused by mechanical damage or attack by chewing pests increase prosystemin gene expression.

[0008] The role of prosystemin / systemin in tomato plant defense mechanisms has been widely recognized through studies of transgenic plants in which the gene encoding prosystemin was overexpressed or silenced. In particular, overexpression of prosystemin has been shown to increase the synthesis of protease inhibitor proteins in the insect gut, significantly reducing digestive capacity and subsequent nutrient absorption (McGurl B. et al., (1994) “Overexpression of the ProSystemin gene in transgenic tomato plants generates a systemic signal that constitutively induces proteinase inhibitor synthesis”, Proceedings of the National Academy of Sciences, 91(21), 9799-9802). In contrast, underexpression of the prosystemin gene almost completely suppresses the production of protease inhibitors after wounding, making the plants more susceptible to tobacco hornworm (Manduca sexta) larvae (Orozco-Cardenas et al., (1993) "Expression of an antisense prosystemin gene in tomato plants reduces resistance toward Manduca sexta larvae", Proceedings of the National Academy of Sciences, 90(17), 8273-8276).

[0009] Recent studies have confirmed that plants that constitutively express the prosystemin gene can protect themselves from numerous biotic stresses by activating a wide range of defense signals (Coppola M. et al., (2015) “Prosystemin overexpression in tomato enhances resistance to different biotic stresses by activating genes of multiple signaling pathways”, Plant molecular biology reporter, 33(5), 1270-1285).In particular, these plants can emit a mixture of volatile compounds that attract predators and parasitoids of herbivorous insects, thereby improving plant defense indirectly (Corrado G. et al., (2007), “Systemin regulates both systemic and volatile signaling in tomato plants”, Journal of chemical ecology, 33(4), 669-681), and are resistant to attack by necrotrophic fungi and aphids (El Oirdi et al., (2011) “Botrytis cinerea manipulates the antagonistic effects between immune pathways to promote disease development in tomato”, Plant Cell 23, 2405-2421), and are more resistant to viral infections (Bubici G. et al., (2017) “Prosystemin overexpression induces transcriptional modifications of defense-related and receptor-like kinase genes and reduces the susceptibility to Cucumber mosaic virus and its satellite RNAs in transgenic tomato plants”, PloSone, 12(2), e0171902) and at the same time, tolerate salt stress conditions (Orsini F. et al., (2010) “Systemin-dependent salinity tolerance in tomato: evidence of specific convergence of abiotic and biotic stress responses”, Physiologia plantarum, 138(1), 10-21).

[0010] The systemin-depleted portion of the prosystemin precursor protein also functions to induce defense gene activation. Corrado G. et al. (2016) “The expression of the tomato prosystemin in tobacco induces alterations irrespective of its functional domain,” Plant Cell, Tissue and Organ Culture (PCTOC), 125(3), 509-519, conducted a study on tobacco plants containing a structurally distinct functional ortholog instead of the prosystemin gene. Transforming these plants with a systemin-depleted prosystemin coding sequence resulted in the synthesis of the missing precursor protein, activating a series of defense-related genes and increasing resistance to Botrytis cinerea.

[0011] Although research into the effects of endogenous expression / overexpression of prosystemin proteins is promising, agronomic approaches based on plant genetic engineering have undoubted limitations, such as the need for complex, laborious, and expensive technical procedures, which consequently limit their scope of application, and which are accompanied by challenging legal and regulatory issues.

[0012] Therefore, there is a need to provide a method that effectively maintains and improves the condition and health of plants and enhances crop quality, which is easy to implement and has a low environmental impact.

[0013] This need and others have been met by the present invention, which provides an isolated peptide as set forth in claim 1 of the accompanying claims, a biostimulatory and bioprotective composition as set forth in claim 8, and a method for increasing tolerance to biotic and / or abiotic stress in plants as set forth in claim 13.

[0014] The accompanying independent and dependent claims form an integral part of this description. Summary of the Invention

[0015] As will be explained in more detail in the experimental section below, the inventors have surprisingly produced peptides which, when isolated from the prosystemin polypeptide and then exogenously administered to plants, for example by foliar spraying or irrigation, advantageously confer growth biostimulatory activity on the plant without direct biocidal action against pathogens, and at the same time induce defense mechanisms against biotic and abiotic stresses.

[0016] Without wishing to be bound by theory, the inventors believe that the resistance induced in plants, particularly tomato plants, against the action of harmful insects and fungi after treatment with the peptides of the invention is mediated by the activation of genes involved in the control of the plant's endogenous defense mechanisms (as shown in Figure 9).

[0017] The results shown in Figures 17 to 20 also surprisingly show that the peptides according to the invention are able to induce important signals for tolerance to exogenous stresses such as high salinity in treated plants.

[0018] Accordingly, one object of the present invention is an isolated peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 23-26 and a fragment of SEQ ID NO: 2 having a length of at least 8 amino acids, and having biostimulatory and bioprotective activity against biotic and abiotic stresses in plants, the peptide optionally having a histidine tail linked to the amino or carboxy terminus.

[0019] Research by the present inventors has revealed that the peptides of the present invention (hereinafter referred to as PS1-70 (SEQ ID NO: 1) and PS1-120 (SEQ ID NO: 2)) are contained in the amino (N-) terminal region of the prosystemin polypeptide, more specifically in the portion of the precursor that does not contain the hormone systemin.

[0020] In further studies carried out using a bioinformatics approach based on the presence of repeated amino acid motifs, the inventors also identified peptides consisting of the amino acid sequences DDAQEKPKVEHEEG (SEQ ID NO: 23), DKETPSQDI (SEQ ID NO: 24), DDAQEKLKVEYEEEEYEKEKIVEKETPSQDI (SEQ ID NO: 25) and DDAQEKPKVEHEEGDDKETPSQDI (SEQ ID NO: 26).

[0021] As described in Example 2, the present inventors found it particularly difficult to identify the target peptide of the present invention, requiring complex investigations due to its unusual electrophoretic migration profile. Subsequent analysis of the amino acid sequence of the peptide revealed that the sequence contains many amino acid residues known to promote structural disorder.

[0022] The term "fragment" as used herein with respect to an amino acid sequence means a contiguous sequence of amino acid residues that represents a portion of a longer amino acid sequence.

[0023] According to one embodiment, the isolated peptide consists of a fragment of the amino acid sequence of SEQ ID NO:2 that is at least 9 amino acids in length, at least 10 amino acids in length, at least 15 amino acids in length, at least 20 amino acids in length, at least 25 amino acids in length, at least 30 amino acids in length, at least 35 amino acids in length, at least 40 amino acids in length, at least 45 amino acids in length, at least 50 amino acids in length, at least 55 amino acids in length, at least 60 amino acids in length, at least 65 amino acids in length, at least 70 amino acids in length, at least 75 amino acids in length, at least 80 amino acids in length, at least 85 amino acids in length, at least 90 amino acids in length, at least 95 amino acids in length, at least 100 amino acids in length, at least 110 amino acids in length, or at least 120 amino acids in length.

[0024] In a preferred embodiment, the isolated peptide is 9 amino acids in length, 10 amino acids in length, 11 amino acids in length, 12 amino acids in length, 13 amino acids in length, 14 amino acids in length, 15 amino acids in length, 16 amino acids in length, 17 amino acids in length, 18 amino acids in length, 19 amino acids in length, 20 amino acids in length, 21 amino acids in length, 22 amino acids in length, 23 amino acids in length, 24 amino acids in length, 25 amino acids in length, 26 amino acids in length, 27 amino acids in length, 28 amino acids in length, 29 amino acids in length, 30 amino acids in length, 31 amino acids in length, 32 amino acids in length, 33 amino acids in length, 34 amino acids in length, 35 amino acids, 36 amino acids, 37 amino acids, 38 amino acids, 39 amino acids, 40 amino acids, 41 amino acids, 42 amino acids, 43 amino acids, 44 amino acids, 45 amino acids, 46 amino acids, 47 amino acids, 48 ​​amino acids, 49 amino acids, 50 amino acids, 51 amino acids, 52 amino acids, 53 amino acids, 54 amino acids, 55 amino acids, 56 amino acids, 57 amino acids, 58 amino acids, 59 amino acids, 60 amino acids, 61 amino acids, 62 amino acids, 63 amino acids, 64 amino acids, 6 5 amino acids, 66 amino acids, 67 amino acids, 68 amino acids, 69 amino acids, 70 amino acids, 71 amino acids, 72 amino acids, 73 amino acids, 74 amino acids, 75 amino acids, 76 amino acids, 77 amino acids, 78 amino acids, 79 amino acids, 80 amino acids, 81 amino acids, 82 amino acids, 83 amino acids, 84 amino acids, 85 amino acids, 86 amino acids, 87 amino acids, 88 amino acids, 89 amino acids, 90 amino acids, 91 amino acids, 92 amino acids, 93 amino acids, 94 amino acids, 9 The fragments may be 5, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 amino acids in length.

[0025] In particular, the following specific amino acid sequences are preferred: EKETPSQDI (SEQ ID NO: 3), EKETISQYI (SEQ ID NO: 4), DDMQEEPKVKLHHEKG (SEQ ID NO: 5), DDTQEIPKMEHEEG (SEQ ID NO: 6), DDAQEKLKVEYEEE (SEQ ID NO: 7), DDMQEEPKVKLHHEKGGDEKEKIIEKETPSQDI (SEQ ID NO: 8) and DDTQEIPKMEHEEGGYVKEKIVEKETISQYI (SEQ ID NO: 9).

[0026] In one embodiment, the isolated peptide subject of the present invention may optionally be linked to a histidine tail at the amino terminus (N-terminus) or carboxy terminus (C-terminus).

[0027] In the present invention, the term "conjugated" refers to the presence of a covalent bond, whether preceded by a linker or not, between the N-terminal amino acid of a peptide of the invention and the C-terminal amino acid of a histidine tail (as illustrated in Figures 2B and 3B), or vice versa, between the C-terminal amino acid of a peptide of the invention and the N-terminal amino acid of a histidine tail.

[0028] As is known in the art, histidine tail attachment is routinely used in protein chemistry to simplify protein purification procedures on matrices containing transition metal ions, and the use of anti-histidine tail antibodies has also become a useful tool in localization and immunoprecipitation studies.

[0029] Methods for producing peptides linked to histidine tails are known and described in the state of the art, for example by expressing recombinant protein products.

[0030] According to another aspect, the isolated peptide subject of the present invention comprises an acetylated amino-terminus (N-terminus) and / or an amidated carboxy-terminus (C-terminus). As widely described in the art, such modifications advantageously make it possible to increase the stability of the peptide and its resistance to enzymatic degradation by aminopeptidases, exopeptidases and synthetases.

[0031] A further object of the present invention is an isolated nucleic acid sequence encoding an isolated peptide as defined above.

[0032] Preferably, the isolated nucleic acid sequence comprises or consists of a nucleotide sequence selected from the nucleotide sequences of SEQ ID NO:10 and SEQ ID NO:11.

[0033] Also included within the scope of the present invention are expression vectors comprising the nucleic acid sequences defined above, and optionally further comprising a promoter sequence and a polyadenylation signal sequence, as well as host cells comprising said expression vectors.

[0034] Recombinant expression vectors used for producing peptides or proteins are known and described in the art, and their selection and use are within the skill of those skilled in the art. Such vectors can be prokaryotic or eukaryotic. Non-limiting examples include the PET series (Novagen) such as pET15 or pET30, and the pGEX series (GE Healthcare) prokaryotic vectors.

[0035] Examples of eukaryotic vectors include the pPIC series of vectors used in Pichia pastoris yeast cells.

[0036] Preferably, the cell system used to express the expression vector of the present invention is selected from prokaryotic systems, such as E. coli cells.

[0037] Alternatively, the expression cell line may be a eukaryotic system, for example yeast cells such as Saccharomyces cerevisiae and Pichia pastoris.

[0038] A further object of the present invention is a method for producing the peptides of the present invention, by which transformed host cells are cultured under suitable conditions for a time sufficient for the expression of the peptides of the present invention. Generally, suitable culture conditions and times will vary depending on the cell line used, and may include, for example, the composition of the culture medium, pH, relative humidity, O 2 and CO The selection of the most appropriate culture conditions and times for use in the methods of the present invention is within the knowledge and skill of one of ordinary skill in the art.

[0039] In a preferred embodiment, the method according to the invention further comprises the step of recovering the produced peptide from the cell culture, which can be carried out using protein purification methods that are part of the prior art, for example by one or more chromatographic steps, such as affinity chromatography or size exclusion chromatography or ion exchange chromatography, or by ultrafiltration, dialysis and / or lyophilization.

[0040] Suitable alternative methods for producing the peptides according to the invention include, for example, chemical synthesis procedures or techniques for proteolytic cleavage of precursor proteins, for example by using specific proteases or chemical materials, etc. The selection of the most appropriate method to use within the scope of the present invention to produce the peptides is well within the skill of the person skilled in the art.

[0041] Due to the above-mentioned advantageous characteristics, the peptides of the invention are particularly suitable for use in agricultural applications aimed at improving plant growth and crop yields while allowing correct management of the soil and the environment. In particular, the action exerted by the peptides according to the invention does not have a direct biocidal effect and therefore advantageously does not cause harmful consequences for populations of useful pollinating insects.

[0042] The use of small molecules such as peptides represents a further advantage of the present invention, as they are suitable for designing and / or modifying to maintain or enhance specific activities, and their small size, unlike long proteins such as full-length prosystemin, allows for significant reductions in synthesis and purification costs.

[0043] Therefore, one object of the present invention is to provide a biostimulatory and bioprotective composition against biotic and abiotic stresses in plants, comprising at least one peptide as defined above, or any combination thereof, and at least one adjuvant, stabilizer and / or preservative.

[0044] Preferably, at least one adjuvant, stabilizer and / or preservative in the composition is one conventionally used in agricultural technology, e.g., to improve uniformity of distribution on plants or seeds and / or to avoid excessive foaming.

[0045] Non-limiting examples of adjuvants suitable for use in the compositions of the present invention include moisturizers, humectants and anti-foaming agents.

[0046] Exemplary antifoaming agents include mixtures of siloxanes, sorbitol and silicones.

[0047] Exemplary moisturizers or humectants include surfactant compounds such as sodium lauryl sulfate and betaine, mixtures of terpenes and alcohols, and the like.

[0048] Examples of stabilizers in the composition of the present invention include pH adjusters including citric acid, acetic acid, and sodium hydroxide.

[0049] Examples of preservatives suitable for use in the biostimulatory and bioprotective compositions of the present invention include dehydroacetic acid, benzoic acid, ethylhexylglycerin, and phenoxyethanol.

[0050] The selection of at least one adjuvant, stabilizer and / or preservative suitable for use in the compositions of the present invention is within the skill of one in the art.

[0051] In one embodiment, the composition of the invention comprises at least a peptide having the amino acid sequence of SEQ ID NO:1 and a peptide having the amino acid sequence of SEQ ID NO:2.

[0052] In another embodiment, a composition of the invention comprises a peptide having the amino acid sequence of SEQ ID NO:3 in combination with peptides having the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:8.

[0053] In yet another embodiment, the composition of the present invention comprises the following combination of peptides: SEQ ID NO: 1 The amino acid sequence of A peptide having ; a peptide having the amino acid sequence of SEQ ID NO: 2; a peptide having the amino acid sequence of SEQ ID NO: 3; a peptide having the amino acid sequence of SEQ ID NO: 4; a peptide having the amino acid sequence of SEQ ID NO: 5; a peptide having the amino acid sequence of SEQ ID NO: 6; a peptide having the amino acid sequence of SEQ ID NO: 7; A peptide having the amino acid sequence of SEQ ID NO: 8; and A peptide having the amino acid sequence of SEQ ID NO:9.

[0054] In a further embodiment, the composition of the invention comprises the following peptide combinations: a peptide having the amino acid sequence of SEQ ID NO: 3; a peptide having the amino acid sequence of SEQ ID NO: 4; a peptide having the amino acid sequence of SEQ ID NO: 5; a peptide having the amino acid sequence of SEQ ID NO: 6; a peptide having the amino acid sequence of SEQ ID NO: 7; A peptide having the amino acid sequence of SEQ ID NO: 8; and A peptide having the amino acid sequence of SEQ ID NO:9.

[0055] In yet another embodiment, the composition of the present invention comprises the following combination of peptides: a peptide having the amino acid sequence of SEQ ID NO: 1; a peptide having the amino acid sequence of SEQ ID NO: 2; a peptide having the amino acid sequence of SEQ ID NO: 3; a peptide having the amino acid sequence of SEQ ID NO: 4; a peptide having the amino acid sequence of SEQ ID NO: 5; a peptide having the amino acid sequence of SEQ ID NO: 6; a peptide having the amino acid sequence of SEQ ID NO: 7; a peptide having the amino acid sequence of SEQ ID NO: 8; a peptide having the amino acid sequence of SEQ ID NO: 9; a peptide having the amino acid sequence of SEQ ID NO: 23; a peptide having the amino acid sequence of SEQ ID NO: 24; A peptide having the amino acid sequence of SEQ ID NO: 25; and A peptide having the amino acid sequence of SEQ ID NO:26.

[0056] According to the official definition of the European Biostimulants Industry Council (EBIC), a “biostimulant” is a substance and / or microorganism that, when applied to plants or their rhizosphere, functions to stimulate natural processes and improve / promote nutrient uptake, nutrient efficiency, crop quality, and resistance to biotic stress.

[0057] Within the scope of the present invention, the term "bioprotector" means a substance and / or a microorganism which, after administration to a plant or to the rhizosphere, induces the activation of the plant's natural defenses against biotic and abiotic stresses.

[0058] As used herein, the term "plant" refers to a living multicellular plant organism.

[0059] Preferably, the plant belongs to a family selected from the group consisting of Solanaceae, e.g., Solanum lycopersicon and Solanum melongena, Vitaceae, e.g., Vitis vinifera, Rosaceae, Apple, e.g., Malus domestica, Olive, e.g., Olea europaea, and combinations thereof.

[0060] With respect to biological stresses, non-limiting examples include herbivorous insects, plant pathogenic fungi, plant pathogenic bacteria, and viruses.

[0061] In the present specification, the herbivorous insect is preferably selected from the group consisting of Lepidoptera, for example, Spodoptera littoralis and Tuta absoluta, Aphids, for example, Macrosiphum euphorbiae, Homoptera, for example, Bemisia tabaci and Trialeurodes vaporariorum, and combinations thereof.

[0062] The plant pathogenic fungus is preferably selected from the group consisting of Botrytis cinerea, Alternaria alternata, Alternaria solani, and combinations thereof.

[0063] The plant pathogenic bacterium is preferably the bacterium Pseudomonas syringae.

[0064] The virus is preferably selected from tomato spotted wilt virus and cucumber mosaic virus.

[0065] Biotic stresses include, but are not limited to, low temperatures causing freezing, high temperatures, drought, high light intensity, low light intensity, excess salt, excess water, and combinations thereof.

[0066] Preferably, the compositions of the present invention also include a buffer. Among the buffers suitable for use in the biostimulating and bioprotective compositions of the present invention, phosphate buffers, more preferably phosphate buffered saline, are particularly preferred. However, it will be understood that other buffers may be used in the present invention, and their selection is within the skill of one of ordinary skill in the art.

[0067] Preferably, at least one peptide is present in the biostimulatory and bioprotective compositions of the present invention in a concentration range of 0.02 picomolar (pM) to 100 pM, more preferably 0.02 pM to 0.08 pM, or 0.085 pM to 0.1 pM, or 0.095 to 0.25 pM, or 1 pM to 100 pM.

[0068] According to a preferred embodiment of the present invention, the biostimulating and bioprotective composition also includes a microorganism selected from the group consisting of mycorrhizal fungi, saprophytic fungi, plant growth-promoting bacteria, spores of Bacillus thuringiensis, and any combination thereof.

[0069] As is known in the art, mycorrhizal fungi in the soil form symbiotic relationships with the roots of many crop plants, providing mutual benefits to the organisms involved: specifically, mycorrhizal fungi can metabolize mineral elements present in the soil even if they are fixed in the soil's absorbent capacity, and plants can provide the symbiotic fungi with sugars produced by photosynthesis.

[0070] Within the scope of the present invention, it is preferred that the mycorrhizal fungus is selected from the group consisting of Gigaspora fasciculatus, Glomus constrictum, Glomus tortuosum, Glomus geosporum, Gigaspora margarita, Acaulospora scrobicurata and any combination thereof.

[0071] Within the scope of the present invention, preferably the saprophytic fungus belongs to the genus Trichoderma.

[0072] Saprophytic fungi are known for their beneficial activity in decomposing plant and animal remains in the soil.

[0073] According to the present invention, a biostimulating and bioprotective composition comprising a combination of at least one peptide as defined above and a saprophytic fungus belonging to the genus Trichoderma is highly preferred since the combination has a significant synergistic effect against plant pathogenic fungi, as shown in Figure 15.

[0074] Within the scope of the present invention, the plant growth-promoting bacteria are preferably selected from Burkholderia cepacia and Pseudomonas fluorescens.

[0075] Bacillus thuringiensis is a spore-forming fungus found naturally in soil and is known to produce, under adverse conditions, spores and parasites (commonly called crystals) containing insecticidal endotoxins, which are released from the parasite after ingestion by susceptible insects and cause lysis of the intestinal epithelial cells, resulting in paralysis and death of the insects.

[0076] More preferably, the spores in the composition according to the invention are derived from the bacterium Bacillus thuringiensis Aizawa strain.

[0077] According to the present invention, the biostimulating and bioprotective composition may be in the form of a lyophilizate. In this embodiment, the composition of the present invention is stable at room temperature for at least 3 months.

[0078] In another embodiment, the compositions of the present invention may be in the form of an aqueous liquid composition or phosphate buffered saline, in which case the compositions of the present invention can be used as is or diluted before use.

[0079] Also within the scope of the present invention is a method for increasing the tolerance of plants to biotic and / or abiotic stresses, comprising applying to the plant, plant part, plant propagation material, and / or plant growing locus a biostimulatory and bioprotective composition as defined above.

[0080] According to the methods of the present invention, the biostimulatory and bioprotective compositions can be applied to a variety of plants in various forms or parts of the plant, such as, for example, leaves, stems, branches, stalks, bark, flowers, flower buds, fruits, roots, seeds, bulbs, tubers and / or shoots.

[0081] As used herein, the term "propagation material" refers to any plant material from which a plant or plant part may be derived, including, but not limited to, seeds, seedlings, cuttings, scions, rootstocks, excisions, bulbs, tubers, and combinations thereof.

[0082] Additionally or alternatively, the compositions of the present invention can be applied to the plant growing locus.

[0083] In one embodiment, the plant is grown in soil and application of the composition of the present invention can be, for example, over the entire growing surface, in and / or around one or more furrows, in the seed hole, in the area under the stem or trunk, and / or in the area between the roots.

[0084] In another embodiment, the plants are grown in soil or soil-free. Hydroponic techniques refer, by way of non-limiting example, to soil-free or soil-free cultivation methods in which the soil is replaced with an inert substrate, such as expanded clay, coconut fiber, rock wool, or zeolite, and the plants are grown in water and, for example, Mg(NO 3 ) 2 -6H 2 O, Ca(NO 3 ) 2 -4H 2 O, KNO 3 、K 2 SO 4 , K.H. 2 PO The plants absorb nutrients from a solution containing inorganic elements such as 4. A major advantage of hydroponic cultivation is that it does not use pesticides, herbicides, or plant protection agents, making it possible to produce crops with consistent quality and hygiene throughout the year.

[0085] The biostimulatory and bioprotective compositions of the present invention can be applied to plants, plant parts, plant propagation material and / or plant growing loci by conventional methods, such as spraying, atomizing, dusting, scattering or irrigating (by hand, tractor, airplane, etc.).

[0086] According to a preferred embodiment, the compositions of the invention are applied to the plants or plant parts, preferably the leaves, by spraying or misting.

[0087] According to another preferred embodiment, the compositions of the invention are applied by irrigation, ie directly to the soil, for example in the form of an irrigation solution or by injection into the soil.

[0088] In the case of hydroponics, the method of the present invention provides that the biostimulatory and bioprotective compositions are administered to the plants in a nutrient solution.

[0089] According to one aspect of the invention, the method comprises applying the composition at least two times, preferably four times, more preferably five times.

[0090] In this embodiment, the time interval between a single application, e.g., a first application, a second application, a third application, a fourth application, or a fifth application to a plant and a subsequent application can range from about 3 weeks to about 4 weeks.

[0091] A further object of the present invention is the use of an isolated peptide as defined above, or a biostimulatory and bioprotective composition as defined above, for increasing the tolerance of plants to biotic and / or abiotic stresses.

[0092] The following experimental section is provided for illustrative purposes only and is not intended to limit the scope of the invention as defined in the appended claims. In the experimental section, reference is made to the accompanying drawings. [Brief explanation of the drawings]

[0093] [Figure 1] FIG. 1 shows a schematic representation of the cloning vector pETM11 that we used for the recombinant production of the peptides of the present invention. [Figure 2]Figure 2 is a schematic diagram of the insert containing the nucleotide sequence encoding peptide PS1-70 after cloning into the vector pETM11. (A) The nucleotide sequence of the recombinant insert (SEQ ID NO: 12) obtained by Sanger sequencing is shown. The nucleotide sequence encoding peptide PS1-70 correctly inserted into the cloning vector (SEQ ID NO: 10) is underlined in black; the sequence encoding the histidine tail (His-tag) (SEQ ID NO: 14) is highlighted in bold capital letters; the tobacco etch virus (TEV) protease recognition site (SEQ ID NO: 16), located downstream of the histidine tail and enabling its removal, is highlighted in italicized capital letters underlined in gray. The sequences of the forward primer P11F1 (SEQ ID NO: 17) and reverse primer P11R1 (SEQ ID NO: 18) used for amplification and cloning of the nucleotide sequence encoding peptide PS1-70 are highlighted in light gray and dark gray, respectively. (B) Amino acid sequence of peptide PS1-70 (bold, SEQ ID NO: 1) linked at the N-terminal position to the C-terminus of the histidine tail (underlined sequence, SEQ ID NO: 15). [Figure 3]Figure 3 is a schematic diagram of the insert containing the nucleotide sequence encoding peptide PS1-120 after cloning into the vector pETM11. (A) The nucleotide sequence of the recombinant insert (SEQ ID NO: 13) obtained by Sanger sequencing. The nucleotide sequence encoding peptide PS1-120 correctly inserted into the cloning vector (SEQ ID NO: 11) is underlined in black; the sequence encoding the histidine tail (His-tag) (SEQ ID NO: 14) is highlighted in bold; the tobacco etch virus (TEV) protease recognition site (SEQ ID NO: 16), placed downstream of the histidine tail to enable its removal, is highlighted in gray, italicized capital letters. The sequences of the forward primer P11F1 (SEQ ID NO: 17) and reverse primer P11R3 (SEQ ID NO: 19) used for amplification and cloning of the nucleotide sequence encoding peptide PS1-120 are highlighted in light gray and dark gray, respectively. (B) Amino acid sequence of peptide PS1-120 (bold, SEQ ID NO: 2) linked at the N-terminal position to the C-terminus of a histidine tail (underlined sequence, SEQ ID NO: 15). [Figure 4] FIG. 4 is a table showing the nucleotide sequences and characteristics of the primers used for amplifying and cloning the nucleotide sequences encoding peptides PS1-70 and PS1-120, respectively. [Figure 5] Figure 5 shows the results of affinity chromatography (IMAC), electrophoretic analysis of elution fractions on 15% SDS-PAGE, and Western blot analysis of purified PS1-70 and PS1-120 peptides. (A1, B1) Chromatographic profiles of the first stage of purification of peptides PS1-70 and PS1-120. Elution was performed with 150 mM and 50 mM imidazole, respectively. (A2 and B2) Polyacrylamide gel analysis of elution fractions; M: molecular weight marker; closed squares: elution fractions containing peptides PS1-70 and PS1-120. (A3 and B3) Identification of peptides PS1-70 and PS1-120 by Western blot analysis; M: molecular weight marker; closed squares: PS1-70 and PS1-120 peptides. [Figure 6]Figure 6 shows the results of size-exclusion chromatography (SEC) and electrophoresis of the eluted fractions on 15% SDS-PAGE. (A1, B1) Chromatographic profiles of the second stage of purification of peptides PS1-70 and PS1-120, with peaks at 12.16 ml and 10.92 ml, respectively. (A2 and B2) Polyacrylamide gel analysis of the eluted fractions; M: molecular weight marker; black squares: eluted fractions containing peptides PS1-70 and PS1-120. (A3, B3) Deconvoluted masses of peptides PS1-70 and PS1-120.

[0094] [Figure 7] Figure 7 shows the amino acid composition profiles of peptides PS1-70 (A) and PS1-120 (B). The graphs in the figure show that the amino acid sequences of both peptides contain significantly more structurally irregular amino acids (dark gray) than amino acids with regular secondary structures (light gray). [Figure 8] In Figure 8, graphs A and B show the results of light scattering experiments performed by SEC-MALS-QELS at pH 8 for peptides PS1-70 (A) and PS1-120 (B), as described in Examples 1 and 2. The peaks in the curves represent the monomeric proteins in solution. Figure 8 (C, D) shows dichroic spectra recorded at 20°C using purified peptides PS1-70 (C) and PS1-120 (D) at concentrations of 4.4 μM and 3.5 μM, respectively, in 10 mM phosphate buffer. The horizontal axis represents wavelength (nm), and the vertical axis represents the mean residual molar ellipticity value. [Figure 9] Figure 9 shows the relative quantification of induced gene expression in tomato plants 6 hours (A, C) and 24 hours (B, D) after foliar application of peptides PS1-70 and PS1-120 at 100 pM and 100 fM concentrations. Analysis was performed for the Lox C, AOS, Pin I, and Pin II genes. Letters a, b, and c indicate the statistical significance of the data (ANOVA), and each letter represents a statistical group. [Figure 10]Figure 10 shows the effect of treatment of tomato plant leaves with peptides PS1-70 and PS1-120 on S. littoralis lepidopteran larvae. Histograms (A, C) show the mean weight change in grams measured over the following days for larvae fed on leaves treated with 100 pM and 100 fM of peptides PS1-70 and PS1-120, respectively, and the relevant controls. The letters a and b indicate the statistical significance of the data (ANOVA), and each letter represents a statistical group. Graphs (B, D) show the daily mortality recorded for larvae fed on tomato plant leaves treated with the peptides of the invention and the relevant controls (Log-Rank test; ***p<0.0001). [Figure 11] Figure 11 shows the effect of treatment of tomato plant leaves with peptides PS1-70 and SEQ ID NOs: 3, 5, and 8 on S. littoralis lepidopteran larvae. Histogram (A) shows the change in mean weight in grams of larvae fed leaves treated with the peptides and the relevant controls, measured on days 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19. The letters a and b indicate the statistical significance of the data (ANOVA), with each letter representing a statistical group. Graph (B) shows the daily mortality recorded for larvae fed tomato plant leaves treated with the peptides of the invention and the relevant controls (log-rank test; ***p<0.0001). [Figure 12] Figure 12 shows that the necrotic area caused by B. cinerea was reduced in tomato leaves (A, B), eggplant leaves (C), and vine leaves (D) after treatment with PS1-70 and PS1-120 peptides compared to untreated controls. The average necrotic area was measured 1, 3, 5, and 8 days after inoculation with the pathogen. The letters a, b, c, and d indicate the statistical significance of the data (ANOVA), and each letter represents a statistical group.

[0095] [Figure 13]Figure 13 shows the reduction in necrotic areas caused by necrotic B. cinerea fungi on tomato plant leaves after treatment with PS1-70, SEQ ID NOs: 3, 5, and 8 compared to untreated controls. The average necrotic area was measured 1, 3, 5, and 8 days after inoculation with the pathogen. The letters a, b, c, and d indicate the statistical significance of the data (ANOVA), with each letter representing a statistical group. [Figure 14] Figure 14 shows the reduction of necrotic areas caused by the scurvy fungus A. alternata on tomato plant leaves after treatment with peptides PS1-70 and PS1-120 (A), and PS1-70, SEQ ID NOs: 3, 5, and 8 (B), compared to untreated controls. The average necrotic area was measured 1, 3, 5, and 8 days after inoculation with the pathogen. The letters a and b indicate the statistical significance of the data (ANOVA), and each letter represents a statistical group. [Figure 15] Figure 15 shows the effects of combined treatment of 4-week-old plants developed from seeds co-cultured with Trichoderma hartianum T22 spores with peptides PS1-70, PS1-120, and systemin (Sys) on the survival of S. littoralis lepidopteran larvae (A1, A2, and A3) and on the colonization of leaves by necrotrophic B. cinerea (B1) and A. alternata (B2) fungi. Larval survival was measured daily (Log-Rank test; ***p<0.0001). Each letter represents a statistical group. The mean necrotic area was measured 1, 3, 5, and 8 days after inoculation. The letters a, b, c, d, e, and f indicate the statistical significance of the data (ANOVA). Each letter represents a statistical group. [Figure 16] Figure 16 shows the reduction in necrotic area caused by necrotic B. cinerea fungi on leaves of 4-week-old tomato plants developed from seeds treated with a 100 fM suspension of peptides PS1-70 and PS1-120, as well as the respective controls. The average necrotic area was measured 1, 3, and 5 days after inoculation with the pathogen. The letters a and b indicate the statistical significance of the data (ANOVA), and each letter represents a statistical group. [Figure 17]Figure 17 shows relative quantification of induced gene expression in tomato plants irrigated with 100 pM PS1-70 peptide in the absence (A) of salt (0 mM NaCl) and the presence (B) of salt (80 mM NaCl). Analysis was performed for the cat1, tft1, Sam, HSFA2, HSP70, HSP90, MPK1, and WRKY40 genes. Asterisks indicate statistical significance of data by Student's t-test (*p<0.05; **p<0.01; ***p<0.001). [Figure 18] Figure 18 shows relative quantification of induced gene expression in tomato plants irrigated with peptides PS1-70, PS1-120, and SEQ ID NO:5 (100 fM) in the absence (0 mM NaCl), presence (150 mM NaCl), and relevant controls. Analysis was performed on the CAT2 (A), SAM (B), and APX2 (C) genes. Asterisks indicate statistical significance of data by Student's t-test (*p<0.05; **p<0.01; ***p<0.001). [Figure 19] Figure 19 shows the average proline content in plant leaves irrigated with peptides PS1-70, PS1-120, and SEQ ID NO: 5 (100 fM) in the absence of salt (0 mM NaCl), the presence of salt (150 mM NaCl), and relevant controls. The letters a, b, and c indicate the statistical significance of the data (ANOVA), and each letter represents a statistical group. [Figure 20] Figure 20 shows the effect of drench treatment with peptides PS1-70, PS1-120, and SEQ ID NO: 5 at a concentration of 100 fM on biological parameters of tomato plants. Histogram (A) shows the root area, expressed in square centimeters, of plants treated with the peptide control of the present invention and the relevant control in the absence (0 mM NaCl) and presence (150 mM NaCl) of salt. Histogram (B) shows the change in fresh weight of the aboveground part (expressed in grams) of plants treated with the peptide control of the present invention and the relevant control in the absence (0 mM NaCl) of salt. Asterisks indicate statistical significance by Student's t-test (*p<0.05). [Figure 21]21 is a table showing the results of evaluating the direct toxic effects of increasing concentrations of the peptides of the present invention assayed against S. littoralis larvae, as shown in Example 4. The survival rates to the pupal stage were recorded for larvae that had been injected or painted epidermally with PS1-70 and PS1-120 peptides. [Figure 22] Figure 22 shows the evaluation of the direct toxic effect of peptides of the invention, assayed at increasing concentrations when added to the growth media of two different fungi: B. cinerea, (A) PS1-70 and PS1-120, and (B) PS1-70, SEQ ID NOS: 3, 5, and 8, and Trichoderma T22, (C) PS1-70 and PS1-120. Fungal growth 24 hours after addition of the peptides of the invention was measured as medium turbidity (absorbance at 600 nm). The letters a and b indicate the statistical significance of the data (ANOVA), and each letter represents a statistical group. DETAILED DESCRIPTION OF THE INVENTION

[0096] Example 1. Preparation of peptides according to the present invention Cloning, expression and purification To isolate the nucleotide sequence encoding the peptide of the present invention, a PCR reaction was set up using the amplification primer pair having the sequences shown in the table of Figure 4 and the cDNA encoding full-length prosystemin as a template.

[0097] The amplicon was digested with NcoI and XhoI restriction enzymes and then cloned into the pETM11 vector, which had been previously digested with the same enzymes. pETM11 (kindly provided by EMBL, Heidelberg) is a prokaryotic expression vector that allows the addition of a six-histidine tail (His-tag) to the amino (N-) terminal portion of cloned proteins and contains a TEV (Tobacco Etch Virus) protease recognition site downstream of the His-tag sequence for removal of the latter (Figure 1).

[0098] The integrity of the cloned fragments and the absence of mutations that may have occurred during the amplification reaction were confirmed by sequencing the resulting constructs (Figures 2A and 3A). After initial screening, large-scale expression of the inventive peptides PS1-70 and PS1-120 was carried out in Escherichia coli BL21(DE3) strain in LB and 2-YT medium at 22°C for 16 hours in the presence of 2 mM IPTG. Figures 2B and 3B show the amino acid sequences of the resulting peptides PS1-70 (SEQ ID NO: 1) and PS1-120 (SEQ ID NO: 2), respectively, highlighted in bold and indicating the underlined histidine tails attached to the N- and C-termini, respectively.

[0099] Post-expression peptide purification was performed by affinity chromatography (IMAC) (Figure 5) and size exclusion chromatography (SEC) (Figure 6) at room temperature using an FPLC-AKTA (GE Healthcare), resulting in a cell culture yield of 2 mg / L.

[0100] Peptide synthesis The peptides of the present invention having the sequences of SEQ ID NOs: 3, 5, and 8 were prepared by solid-phase chemical synthesis using standard protocols (Chandrudu S. et al., "Chemical methods for peptide and protein production"; Molecules. 2013 Apr 12;18(4):4373-88). This procedure used a resin to obtain peptides with amidated carboxy termini. At the end of the synthesis, the amino termini of the peptides were also modified by acetylation. Purification was performed by reversed-phase HPLC.

[0101] Example 2: Structural analysis of the peptide of the present invention During the process of identifying the peptides of the present invention, we encountered considerable difficulties due to the unique properties of the PS1-70 and PS1-120 peptides, primarily the significant presence of amino acid residues in their primary sequences that promote structural perturbation (Fig. 7). Both peptides, PS1-70 and PS1-120, exhibited unusual migration patterns when subjected to SDS-PAGE electrophoresis, migrating with apparent molecular weights of 20-25 kDa relative to their actual weights (MW PS1-70 = 11 kDa; MW PS1-120 = 17 kDa). Mass spectrometry again accurately confirmed the molecular weights of the two recombinant peptides (Fig. 6, A3, B3).

[0102] Furthermore, size-exclusion chromatography (SEC) showed retention volumes of 12.16 ml for peptide PS1-70 and 10.92 ml for PS1-120 (Figure 6), indicating that they are oligomeric or poorly folded proteins. Light scattering experiments performed by SEC-MALS-QELS revealed that, regardless of retention volume, the peptides of the present invention existed in solution as monodisperse monomeric proteins with molecular weights of 9.36 ± 0.6 kDa for PS1-70 and 19.98 ± 1.5 kDa for PS1-120, consistent with theoretical values ​​(Figures 8A and 8B).

[0103] The secondary structure of peptides PS1-70 and PS1-120 was then analyzed by circular dichroism (CD). The Far-UV CD spectra obtained for both test peptides showed negative molar ellipticity values ​​at 198 and 190 nm. However, the ellipticity values ​​observed at 200 and 222 nm indicate some secondary structure (Figures 8C and 8D). These features are typical of disordered proteins with large unstructured regions. This is likely due to both a high abundance of acidic residues (negatively charged at physiological pH) that cause essential repulsion and a paucity of hydrophobic residues that generally aid in the correct folding of proteins.

[0104] Example 3: Induction of plant defense gene expression by the peptide of the present invention To test the biological activity of the peptides of the present invention, we applied peptides PS1-70 and PS1-120 to tomato plants (Solanum lycopersicon) and measured the expression of defense genes in these plants. The peptides were assayed at picomolar (pM) and femtomolar (fM) concentrations in 1X PBS buffer (0.14 M NaCl, 0.0027 M KCl, 0.01 M phosphate buffer, pH 7.4) by applying 2 μl of an aqueous composition containing the peptide to several sites on the upper surface of expanded leaves of 4-week-old tomato plants.

[0105] Leaf samples were collected 6 and 24 hours after application of the peptides of the present invention, and RNA was extracted and gene expression analysis was performed. In particular, we selected two early-expressing genes, lipoxygenase C (Lox C) and allene oxide synthase (AOS), which are active in the octadecanoid biosynthetic pathway leading to the production of jasmonic acid (JA), and two late-expressing genes, proteinase I inhibitor (Pin I) and proteinase II inhibitor (Pin II), known to be plant defense-related genes.

[0106] All assayed genes were significantly overexpressed after exogenous application of both peptides of the invention at both concentrations tested (FIG. 9).

[0107] Example 4. Peptides of the present invention promote tolerance to biotic stress in plants To demonstrate that the peptides of the present invention can promote plant resistance to pathogens, the present inventors conducted a test to evaluate the effects on herbivorous insects or plant pathogenic fungi resulting from treating plants with peptides having the amino acid sequences of SEQ ID NO: 1 (PS1-70), SEQ ID NO: 2 (PS1-120), SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 8. Specifically, changes in the weight gain and survival rate of larvae of Spodoptera littoralis, a lepidopteran pest that causes severe damage to tomatoes, and plant colonization by the necrotrophic plant pathogen Botrytis cinerea and the parasitic fungus Alternaria alternata, which cause gray mold disease in tomato, were monitored.

[0108] Experiments with Spodoptera littoralis larvae Briefly, S. littoralis larvae were reared on artificial diet at 25°C under a 16-hour light / 8-hour dark cycle at 70% relative humidity until they completed their first molt. For the bioassay, 150 larvae from each experiment were reared on tomato leaves for the entire second instar period and acclimated to the different diets. Third-instar larvae were then fed plants containing peptides PS1-70 and PS1-120 at concentrations of 100 pM and 100 fM in 1X PBS buffer. Larvae fed on plants treated with 1X PBS buffer alone served as controls. Bioassays were performed under the same environmental conditions in 32-well plastic trays containing 1.5% (w / v) agar and 0.005% (w / v) methyl parahydroxybenzoate, which helps create a humid environment to maintain intracellular pressure in tomato leaves. Each experimental group consisted of 32 larvae. Larval survival was monitored daily, and body weight was monitored every other day. Larvae fed on leaves treated with the peptides of the present invention exhibited a weight loss throughout the bioassay period compared with larvae fed on control leaves, with significant differences observed as early as day 3 for both concentrations. Notably, at day 15 of the bioassay, at a 100 pM concentration, the average weight of larvae fed on control leaves was 38 mg, while the average weights of larvae fed on leaves treated with peptides PS1-70 and PS1-120 were 15 mg and 20 mg, respectively (Figure 10A). In a bioassay performed using a 100 fM concentration of the peptides of the present invention, the average weight of larvae fed on control leaves was 44 mg, while the average weights of larvae fed on leaves treated with peptides PS1-70 and PS1-120 were 11 mg and 12 mg, respectively (Figure 10C). Furthermore, the survival rate of larvae fed on leaves treated with the peptides of the present invention was significantly reduced in both assays compared with larvae fed on control leaves (Figures 10B and 10D). Indeed, on day 15 of the bioassay, a survival rate of 96.87% was observed in larvae fed with control leaves, while survival rates of 34.37% and 31.25%, respectively, were observed in larvae fed with leaves treated with the peptide of the present invention at a concentration of 100 pM (Figure 10B).In a bioassay performed at a concentration of 100 fM, on day 13, the survival rate of larvae fed with control leaves was 100%, while the survival rates of larvae fed with leaves treated with the peptides of the present invention were 0% and 21.87%, respectively (Figure 10D).

[0109] We performed the same assay by feeding S. littoralis larvae leaves treated with peptide PS1-70 and peptides having the sequences of SEQ ID NOS: 3, 5, and 8 at femtomolar concentrations. Larvae fed treated leaves showed significantly reduced body weights from day 3 compared with control leaves (FIG. 11). Notably, on day 13 of the bioassay, the average body weight of larvae fed control leaves was 59 mg, whereas those treated with peptide PS1-70 and peptides having the sequences of SEQ ID NOS: 8, 3, and 5 had average body weights of 22 mg, 13 mg, 24 mg, and 21 mg, respectively (FIG. 11A). Furthermore, the survival rate of larvae fed peptide-treated leaves was significantly reduced compared with larvae fed control leaves (FIG. 11B). Indeed, on day 13 of the bioassay, 100% survival rate was observed in larvae fed with control leaves, while survival rates of 31.25%, 15.62%, 43.75% and 15.62% were observed in those fed with leaves treated with peptides PS1-70, SEQ ID NO:8, SEQ ID NO:3 and SEQ ID NO:5, respectively (Figure 11B).

[0110] Experiments with Botrytis cinerea and Alternaria alternata To perform assays for the plant pathogenic Botrytis cinerea and Alternaria alternata, we used spores of these organisms obtained from culture on a solid PDA (potato dextrose agar) sporulation substrate. 10 6 20 μl of the conidial suspension was inoculated at a concentration of spores / ml and incubated at 25°C under diffused light for 15 days to ensure complete sporulation. The spores were then collected in 5 ml of sterile water, filtered through glass wool to remove mycelia, washed with sterile distilled water, and then centrifuged at room temperature to recover the spores. The spore concentration suitable for inoculation ( 10 5 ~ 10 7spores / ml) was determined by serial dilution using a Burker cell counting chamber for spore counting.

[0111] Assays were performed on detached compound leaves from each treated and control plant, each of which was marked with three markers to guide subsequent spore dispersal and to detect pathogens causing necrotic lesions.

[0112] 2 μl of a composition containing the peptides PS1-70 and PS1-120 of the present invention at a concentration of 100 pM or 100 fM was applied, or 2 μl of 1× PBS was applied to control leaves. After 6 hours, which is the time required for peptide recognition, the leaves were detached, and 10 μl of spore suspension was inoculated near and between the previously marked areas. Monitoring was performed by measuring the necrotic area ( mm 2 The necrotic areas recorded in control leaves were much larger than those detected in plants treated with the peptides of the present invention. This difference increased as a function of time after inoculation. The resistance-inducing effect of picomolar treatment was observed at 18 pmol for both peptides. mm 2 Unlike the treated leaves, where values ​​not exceeding 33 were recorded, the control leaves showed a high value of 33 from the first day to the 8th day after inoculation. mm 2 The values ​​reached 0.01 and were already statistically significant (Figure 12A).

[0113] Similar results were observed after application of the peptide target of the present invention at femtomolar concentrations. In particular, it was observed that from day 1 of inoculation with the plant pathogen, the occurrence of necrotic lesions in treated leaves was strongly suppressed compared to control leaves. This reduction was maintained until day 8, and in control leaves, it was suppressed by 20%. mm 2 and 6.69 in leaves treated with PS1-70 and PS1-120, respectively. mm 2 and 5.19 mm 2 reached a value of 0.05 (Figure 12B).

[0114] We also conducted studies aimed at determining the effect of exogenously applied picomolar and femtomolar concentrations of peptides of the present invention on the development of necrotic bacteria in Solanum melongena (eggplant) plants (Figure 12C) and Vitis vinifera (grape) plants (Figure 12D).

[0115] As shown in Figure 12C, the experiments performed showed a significant reduction in fungal colonization of treated plants compared to control plants, with the most pronounced effect observed after treatment with the lowest concentration of the peptide of the present invention. The positive effect of the treatment was statistically significant from day 3 to day 8 after inoculation of the pathogen, with a 11% reduction in fungal colonization in control leaves. mm 2 The value reached 8.4 in leaves treated with peptides PS1-70 and PS1-120 at 100 pM. mm 2 and 6.0 mm 2 The value of 6.2 was recorded in the leaves treated with the peptide at a concentration of 100 fM. mm 2 and 4.10 mm 2 The positive effect of the treatment was already statistically significant from day 1 to day 8 after inoculation, while the control leaves showed a 43 mm 2 and 16.7 in leaves treated with peptides PS1-70 and PS1-120 at 100 pM, respectively. mm 2 and 13.6 mm 2 In leaves treated with the peptide at a concentration of 100 fM, the value reached 12 mm 2 and 11.8 mm 2 (Figure 12D). Similar results were obtained in olive trees.

[0116] Furthermore, the effect of applying femtomolar concentrations of peptides having the sequences of SEQ ID NOs: 3, 5 and 8 to tomato leaves was evaluated on the development of B. cinerea disease.

[0117] As shown in Figure 13, the necrotic areas recorded in the control leaves were much larger than those detected in plants treated with the peptide of the invention. The positive effect of the treatment was statistically significant already on day 1 of pathogen inoculation and continued until day 8, with a necrotic area of ​​11.72 in the control leaves. mm 2 and 7.69 in leaves treated with peptides PS1-70, SEQ ID NO:8, SEQ ID NO:3 and SEQ ID NO:5 at a concentration of 100 fM, respectively. mm 2 , 7.67 mm 2 , 8.89 mm 2 and 6.81 mm 2 reached a value of (Figure 13).

[0118] The present inventors also performed an assay aimed at measuring the effect of the peptides of the present invention on the development of necrotrophic Alternaria alternata fungi when applied exogenously to tomato plants (Figure 14).

[0119] As shown in Figures 14A and 14B, the assays performed showed a significant reduction in fungal colonization of treated plants compared to control plants. The positive effect of the treatment was statistically significant from day 1 to day 8 of pathogen inoculation, where the fungal colonization was significantly reduced in control leaves. mm 2 and 8.4 recorded on leaves treated with peptides PS1-70 and PS1-120 at a concentration of 100 fM. mm 2 and 8.0 mm 2 The values ​​of 17 were different from those of the treated leaves (Fig. 14A). mm 2 A value of 8.3 was recorded on the control leaves. mm 2 , 7.9 mm 2 , 8.9 mm 2 and 7.8 mm 2were recorded on leaves treated with peptides PS1-70, SEQ ID NO:8, SEQ ID NO:3 and SEQ ID NO:5 at a concentration of 100 fM each (FIG. 14B).

[0120] Activity of the peptides of the present invention in combination with Trichoderma T22 spores Furthermore, the present inventors conducted studies to determine the effect of a combination of the peptides of the present invention and Trichoderma T22 spores at femtomolar concentrations on tomato plants in reducing the survival rate of S. littoralis larvae (A1, A2, A3) and the occurrence of necrotrophic B. cinerea (B1) and A. alternata (B2) fungi (Figure 15).

[0121] Tomato seeds were soaked in a spore suspension of Trichoderma harzianum T22 strain (1X 10 7 The leaves were treated with either 100 fM of the peptides systemin (Sys), PS1-70, or PS1-120 (spores / ml) or water, dried, and then germinated in the dark on sterile absorbent paper at 24°C. Leaves from 4-week-old plants were treated with 2 μl of a composition containing the peptides systemin (Sys), PS1-70, or PS1-120, or 1X PBS (control leaves). After 6 h, the time required for peptide recognition, the leaves were detached from the plants and subjected to both assays with S. littoralis larvae and two necrotrophic fungi, B. cinerea and A. alternata.

[0122] In the assay using S. littoralis larvae, larval survival was monitored daily. To observe the development of the two plant pathogenic fungi, spore suspension (1X spore suspension) was applied to pre-treated tomato leaves. 10 6 The necrotic area was measured 1, 3, 5, and 8 days after inoculation of 10 μl of spores / ml. mm 2 (shown in ).

[0123] Figure 15 shows that treatment with the tested peptides in combination with Trichoderma T22 resulted in a surprising synergistic effect in reducing larval development and survival, and provided significantly greater protection against colonization by the two plant pathogenic fungi, compared to treatment with Trichoderma T22 alone or the peptides alone. Evidence of these effects increased over time. Furthermore, the efficacy of the peptides of the present invention, alone or in combination with Trichoderma T22 spores, was shown to be superior to that of the systemin peptide.

[0124] Seed protection effect of the peptide of the present invention The present inventors also evaluated the protective effect of direct seed treatment with the peptides of the present invention. Tomato seeds were treated with a composition containing the peptide at a concentration of 100 fM or with 1X PBS as a control, dried, and then germinated in the dark at 24°C on sterile absorbent paper. Leaves from 4-week-old plants were detached, and 10 μl of spore suspension was inoculated between the leaves and near the previously marked spots. As shown in Figure 16, the assay showed that fungal colonization in leaves of plants developed from seeds treated with PS1-70 and PS1-120 was significantly reduced compared to control plants.

[0125] The positive effect of the treatment was statistically significant from day 1 to day 8 after inoculation, and was 11% for control leaves. mm 2 The values ​​reached 7.1 in leaves of plants produced from seeds treated with peptides PS1-70 and PS1-120 at 100 fM, respectively. mm 2 and 7.4 mm 2 The values ​​reached (Figure 16). The present inventors performed further tests to examine whether peptides PS1-70 and PS1-120 have a direct toxic effect on the pathogenic organisms tested. As can be seen from the data reported in the table of Figure 21, oral or injection administration of the peptides of the present invention into the cuticle of S. littoralis larvae did not affect their survival or development.

[0126] Furthermore, as shown in Figures 22A and 22B, the addition of peptides PS1-70 and PS1-120 of the present invention to the growth medium did not inhibit the growth of B. cinerea. Furthermore, when Trichoderma harzianum T22 strain was used, the addition of peptides PS1-70 and PS1-120 at concentrations of 100 pM and 100 fM did not affect the growth of the fungus, as shown in Figure 22C. Instead, the addition of peptide PS1-70 to the growth medium increased the growth of T22 fungus, suggesting that the fungus may utilize this protein as an amino acid source.

[0127] The above experimental results demonstrate that the peptide targets of the present invention have biological activity and can promote plant resistance to harmful insects and fungi when applied exogenously.

[0128] Example 5. Peptides of the present invention promote plant tolerance to abiotic stress The present inventors have also conducted experiments aimed at verifying the effect of the peptides of the present invention in promoting plant tolerance to various abiotic stresses.

[0129] Through this experiment, the inventors first discovered that applying the peptides of the present invention to tomato plants increases their biomass and favors the production of larger fruits with more seeds. Subsequent experiments, as shown in Figure 17, confirmed that application of peptide PS1-70 protects tomato plants from salt stress. Briefly, 48 hours after irrigation with 100 pM of PS1-70 peptide, the treated plants, unlike control plants, were able to activate a series of genes and transcription factors that respond to salt stress, thereby demonstrating that the peptides of the present invention can stimulate a state of alertness in plants known as priming. Furthermore, under moderate salt stress (80 mM NaCl), tomato plants treated with the peptides of the present invention were shown to be more tolerant to salt than untreated control plants, particularly with greater induction of genes that respond to this stress (Figure 17B). Experiments were also conducted to verify the effect of peptides PS1-70, PS1-120, and SEQ ID NO: 5 on promoting tolerance to high-salt stress conditions when administered at femtomolar concentrations. As shown in Figure 18, 8 days after irrigation with peptides PS1-70, PS1-120, and SEQ ID NO:5, treated plants, unlike control plants, were able to activate a series of salt stress-responsive genes, thus confirming that the peptides of the present invention can stimulate a primed defense state even at femtomolar concentrations (see Figures 18A1, 18B1, and 18C1). Furthermore, tomato plants treated with the above peptides and irrigated 24 hours later with high salt stress levels (150 mM NaCl) for 7 days were more salt tolerant than untreated control plants, and in particular showed a significant induction of the same genes that respond to this stress (Figures 18A2, 18B2, and 18C2).

[0130] The average proline content in these plants was also assessed. Proline is an osmoprotectant that is produced in free form within plant cells in response to salt stress and water deficit. In plants exposed to salt stress, this amino acid has been shown to be involved in osmotic regulation, membrane protection from free radicals, cytoplasmic pH regulation, and enzyme protection from denaturation.

[0131] As shown in Figure 19, plants treated with the peptides of the present invention in the presence of high salt stress (150 mM NaCl) showed a lower average proline content than saline-treated control plants, thus indicating a lower perception of salt-induced osmotic stress.

[0132] Several biological parameters measured after 14 days of continuous salt stress (150 mM) and 15 days after irrigation with 100 fM concentrations of PS1-70, PS1-120, and peptides of SEQ ID NO: 5, and their respective controls, are shown in Figure 20. Figure 20A shows that peptide-treated plants were more tolerant to stress than untreated control plants, particularly in terms of increased root surface area (Figure 20A). Furthermore, in the absence of salt, plants treated with the peptide target of the present invention were able to promote aboveground growth of plant parts, unlike control plants, confirming that the peptide of the present invention has biostimulatory effects even at femtomolar concentrations (Figure 20B). Further aspects of the present invention are described below: [Section 1] An isolated peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 23-26 and a fragment of SEQ ID NO: 2 having a length of at least 8 amino acids, and having biostimulatory and biodefensive activity against abiotic and biotic stresses in plants, wherein the peptide may have a histidine tail linked to the amino or carboxy terminus. [Section 2] Item 2. The isolated peptide according to Item 1, wherein the fragment is selected from the group consisting of the amino acid sequences of SEQ ID NOs: 3 to 9. [Section 3] 3. The isolated peptide of paragraph 1 or 2, wherein the amino terminus is modified by acetylation and / or the carboxy terminus is modified by amidation. [Section 4] Item 4. An isolated nucleic acid sequence encoding the peptide of any one of items 1 to 3. [Section 5] Item 5. An expression vector comprising the nucleic acid sequence according to Item 4. [Section 6] A host cell comprising the expression vector according to item 5. [Section 7] A method for producing the peptide according to Item 1 or 2, comprising culturing the host cell according to Item 6 under appropriate conditions for a period of time sufficient for expression of the peptide, and optionally comprising recovering the peptide from the culture. [Section 8] A composition for biostimulation and bioprotection of plants against abiotic and biotic stresses, comprising at least one peptide according to any one of items 1 to 3, or a combination thereof, and at least one adjuvant, stabilizer and / or preservative. [Section 9] 9. The biostimulating and bioprotective composition of paragraph 8, wherein the at least one peptide is present at a concentration ranging from 0.01 picomolar (pM) to 100 pM. [Section 10] Item 10. The biostimulating and bioprotective composition of item 8 or 9, further comprising a microorganism selected from the group consisting of mycorrhizal fungi, saprophytic fungi, plant growth-promoting fungi, Bacillus thuringiensis spores, and any combination thereof. [Section 11] Item 11. The biostimulating and bioprotective composition according to any one of Items 8 to 10, which is an aqueous liquid composition. [Section 12] Item 11. The biostimulating and bioprotective composition of any one of items 8 to 10, in a lyophilized form. [Section 13] 13. A method for increasing resistance to biotic and / or abiotic stress in a plant, comprising applying the biostimulatory and bioprotective composition of any one of paragraphs 8 to 12 to the plant, plant part, plant propagation material and / or plant growing site. [Section 14] 14. The method of claim 13, wherein the biostimulatory and bioprotective composition is applied by spraying, by irrigation, or in a hydroponic solution. [Section 15] Item 15. The method according to Item 13 or 14, wherein the plant is a plant belonging to the Solanaceae, Vitaceae, Rosaceae, or Apocynaceae family. [Section 16] Use of the isolated peptide of any one of paragraphs 1 to 3 and / or the biostimulatory and bioprotective composition of any one of paragraphs 8 to 12 to increase tolerance to abiotic and / or biotic stress in plants.

Claims

1. An isolated peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 5 and 8, and having biostimulatory and bioprotective activity against abiotic and biotic stresses in plants, wherein the peptide may have a histidine tail linked to the amino or carboxy terminus.

2. 2. The isolated peptide of claim 1, wherein the amino terminus is modified by acetylation and / or the carboxy terminus is modified by amidation.

3. 3. An isolated nucleic acid comprising a nucleic acid sequence encoding the peptide of claim 1 or 2.

4. An expression vector comprising the nucleic acid of claim 3.

5. A host cell comprising the expression vector of claim 4.

6. A method for producing the peptide according to claim 1, comprising the step of culturing the host cell according to claim 5 under appropriate conditions for a time sufficient for expression of the peptide, and optionally comprising the step of recovering the peptide from the culture.

7. A composition for biostimulation and bioprotection of plants against abiotic and biotic stresses, comprising at least one peptide according to claim 1 or 2, or a combination thereof, and at least one adjuvant, stabilizer and / or preservative.

8. 8. The biostimulatory and bioprotective composition of claim 7, wherein the at least one peptide is present at a concentration ranging from 0.01 picomolar (pM) to 100 pM.

9. 9. The biostimulatory and bioprotective composition of claim 7 or 8, further comprising a microorganism selected from the group consisting of mycorrhizal fungi, saprophytic fungi, plant growth-promoting fungi, Bacillus thuringiensis spores, and any combination thereof.

10. The biostimulating and bioprotective composition according to any one of claims 7 to 9, which is an aqueous liquid composition.

11. 10. The biostimulating and bioprotective composition of any one of claims 7 to 9, in lyophilized form.

12. 12. A method for increasing resistance to biotic and / or abiotic stress in a plant, comprising applying a biostimulatory and bioprotective composition according to any one of claims 7 to 11 to the plant, plant part, plant propagation material and / or plant growing site.

13. 13. The method of claim 12, wherein the biostimulatory and bioprotective composition is applied by spraying, by irrigation, or in a hydroponic solution.

14. The method according to claim 12 or 13, wherein the plant is a plant belonging to the Solanaceae, Vitaceae, Rosaceae or Apocynaceae family.

15. Use of an isolated peptide according to claim 1 or 2 and / or a biostimulatory and bioprotective composition according to any one of claims 7 to 11 for increasing tolerance to abiotic and / or biotic stress in plants.

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