Biostimulants and bioprotective peptides and their use in agriculture

Abstract

The present invention relates to novel isolated peptides having biostimulating and bioprotective activity against abiotic and biotic stresses in plants, and to compositions comprising said peptides. Preferably, the peptides according to the invention are derived from tomato (Solanum lycopersicum) plants and are produced recombinantly or synthetically. Also described is the use of said peptides and / or said compositions in increasing resistance to biotic and / or abiotic stresses in plants.

Description

Technical Field

[0001] The present invention belongs to the field of agriculture, and more specifically, to agricultural processes for promoting the growth and productivity of plants. Specifically, the present invention relates to novel peptides having biostimulant and bioprotective activities conferring resistance to abiotic and biotic stresses in plants. Background Art

[0002] In the field of agriculture, it is necessary to promote the growth, health, and productivity of plants. Over the years, different methods have been developed in agricultural technology to combat and contain the damage caused to plants and crops by biotic stresses (i.e., attacks by microorganisms such as viruses, bacteria, fungi, and large organisms including nematodes, insects, and mites) and abiotic stresses (such as water shortage, overheating, extreme cold, or high salinity).

[0003] Traditionally, biotic and abiotic stresses in plants have been controlled by using fertilizers and pesticides and by introducing physical soil amendments. However, the use of agrochemicals can have serious long-term environmental consequences if these products are overused or remain in the environment, and can cause significant damage to human health due to toxic residues in the edible parts. In addition, crop protection products may lose their effectiveness over time due to the emergence of strains resistant to their action in pathogen populations.

[0004] To limit the use of hazardous strategies and to try to increase the productivity of crops by promoting plant growth and stress resistance, many efforts have been made to develop and implement ecologically prudent and sustainable methods.

[0005] U.S. Patents US5,378,819, US5,883,076, and US6,022,739 describe the isolation of the plant peptide hormone systemin in response to damage caused by chewing insects or mechanical damage, and the involvement of said peptide in the activation of defense genes in tomato (Solanum lycopersicum) plants.

[0006] Systemin is an 18 - amino - acid peptide hormone located at the C - terminal end of the carboxyl - terminal region of a 200 - amino - acid precursor called prosystemin (ProSys). After plant wounding, the prosystemin precursor undergoes proteolysis, which may be mediated by phytaspase (an aspartic - specific protease of the subtilisin family), and this phytaspase can release systemin. This peptide is released into the apoplast and activates defense signaling through interaction with the membrane receptor SYR1 (Narvàez - Vàsquez and Orozco - Càrdenas, (2008) “Systemins and AtPeps: Defense - related peptide signals”; In Induced plant resistance to herbivory (pp.313 - 328). Springer, Dordrecht; Wang 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 the leaves, petals, and stems of plants, but not in the roots (Pearce G. et al., (1991) “A polypeptide from tomato leaves induces wound - inducible proteinase inhibitor protein”, Science, 253(5022), 895 - 897; Narváez - Vásquez, J., and Ryan, C.A., (2004) “The cellular localization of prosystemin: a functional role for phloem parenchyma in systemic wound signaling”, Planta, 218(3), 360 - 369). In contrast, in the case of wounding caused by mechanical damage or attack by chewing insects, the prosystemin gene increases its expression.

[0008] The role of prosystemin / systemin in the defense mechanism of tomato plants has been widely demonstrated by studying the overexpression or silencing of the systemin-encoding gene in transgenic plants. Specifically, after overexpression of prosystemin, an increase in the synthesis of proteinase inhibitor was detected in the gut of insects, thereby greatly reducing their digestive ability 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). Conversely, insufficient expression of the prosystemin gene results in almost complete inhibition of proteinase inhibitor production after wounding, leading to greater susceptibility of the plant to larvae of the tobacco hornworm (Manduca sexta) (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 constitutively expressing the prosystemin precursor gene can defend against multiple biotic stresses by activating a wide range of defense signals (Coppola M et al., (2015) “Prosystemin overexpression into tomato enhances resistance to different biotic stresses by activating genes of multiple signaling pathways”, Plant molecular biology reporter, 33(5), 1270 - 1285).Specifically, these plants are able to release a mixture of volatile compounds that attract predators and parasitoids of herbivorous insects, thus enhancing the plant's indirect defense (Corrado G. et al., (2007), “Systemin regulates both systemic and volatile signaling in tomato plants”, Journal of chemical ecology, 33(4), 669-681), resist necrotrophic fungi (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 aphid attacks, better tolerate certain 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”, PloS one, 12(2), e0171902), and simultaneously 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] It is also known that portions of the precursor protein of prosystemin lacking the systemin protein are also capable of inducing the activation of defense genes. The study described in Corrado G et al. (2016) "The expression of the tomato prosystemin intobacco induces alterations irrespective of its functional domain", Plant Cell, Tissue and Organ Culture (PCTOC), 125(3), 509-519 was conducted in tobacco plants that contained structurally distinct, functional orthologs rather than the prosystemin precursor gene. The study showed that by transforming these plants with a sequence encoding a prosystemin lacking systemin, the missing precursor protein was synthesized and led to the activation of a series of defense-related genes and a higher tolerance to the fungal pathogen Botrytis cinerea.

[0011] Although the studies on the effects of endogenous expression / overexpression of the prosystemin precursor protein have yielded promising results, agricultural methods based on plant genetic engineering techniques undoubtedly have limitations that require complex, laborious, and expensive technical processes, thus restricting their scope of application. These limitations are also accompanied by difficult legislative and regulatory issues.

[0012] Therefore, there is a need to provide methods that are aimed at effectively maintaining and improving plant well-being and health and enhancing crop quality while being easy to implement and having a minimal impact on the environment. Summary of the Invention

[0013] The present invention now meets this need and other needs by providing an isolated peptide as described below, a biostimulant and bioprotective composition as described below, and a method for enhancing resistance to biotic and / or abiotic stress in plants as described below.

[0014] The appended independent and dependent claims form an integral part of this specification.

[0015] As will be described in more detail in the experimental section below, the inventors have unexpectedly isolated and subsequently generated a peptide from the prosystemin precursor polypeptide that, when exogenously applied to plants (e.g., by spraying on leaves or irrigation), is capable of advantageously performing growth biostimulant activity on the plants and simultaneously triggering defense mechanisms against biotic and abiotic stress without any direct biocidal effect on the pathogens.

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

[0017] In Figures 17 - 20 The results presented also show that the peptides according to the invention are also surprisingly able to trigger key signals of resistance to abiotic stress (e.g., high salinity) in the treated plants.

[0018] Accordingly, an 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, SEQ ID NO:2, SEQ ID NO:23 - 26, and fragments of SEQ ID NO:2 of at least 8 amino acid length, and having biostimulating and bioprotective activities against abiotic and biotic stresses in plants, said peptide optionally conjugated with a histidine tail at the amino or carboxyl terminus.

[0019] Studies conducted by the inventors have 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, and more specifically, are contained in the precursor portion without the hormone systemin.

[0020] In the context of further studies using bioinformatics methods based on the presence of repetitive amino acid motifs, the inventors have also identified peptides consisting of the following 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 Experimental Example 2, due to the abnormal electrophoretic migration curve, the inventors had particular difficulty in identifying the peptide objects of the present invention and required complex investigations. Subsequent analysis of the amino acid sequences of the peptides actually revealed the presence of an important component of amino acid residues known to promote structural disorder in the sequences.

[0022] As used herein with reference to amino acid sequences, the term "fragment" refers to a continuous sequence of amino acid residues representing 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 SEQ ID NO.2 that is at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, or at least 120 amino acids in length.

[0024] In a preferred embodiment, the isolated peptide consists of a fragment of the amino acid sequence SEQ ID NO.2 that is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 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] The following specific amino acid sequences are particularly 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] According to one embodiment, the isolated peptide of the invention is optionally conjugated to a histidine tail at the amino terminus (N-terminus) or carboxyl terminus (C-terminus).

[0027] In the context of the present invention, the term "conjugated" refers to the presence of a covalent bond between the amino acid at the N-terminus of the peptide of the present invention and the amino acid at the C-terminus of the histidine tail (as Figure 2 illustrated in Figure 3 B), and vice versa, the presence of a covalent bond between the amino acid at the C-terminus of the peptide of the present invention and the amino acid at the N-terminus of the histidine tail, whether or not there is a linker in front.

[0028] As is known in the art, conjugation with a histidine tail is commonly used in protein science because it simplifies the protein purification process on matrices containing transition metal ions, and the use of anti-histidine tail antibodies is also a useful tool in localization and immunoprecipitation studies.

[0029] Methods for making peptides conjugated with a histidine tail are known in the prior art and have been described, for example, by expressing recombinant protein products.

[0030] According to another embodiment, the isolated peptide object of the present invention comprises an acetylated modified amino-terminal (N-terminal) end and / or an amidated modified carboxyl-terminal (C-terminal) end. As widely described in the art, the above modifications advantageously allow for the stability of the peptide and an increase in its resistance to enzymatic degradation by aminopeptidases, exopeptidases, and synthetases.

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

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

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

[0034] Recombinant expression vectors for making peptides or proteins are known in the prior art and have been described, and thus their selection and use are within the skills of those of ordinary skill in the art. Such vectors can be prokaryotic or eukaryotic. Non-limiting examples of prokaryotic vectors include the PET series (Novagen) (e.g., pET15 or pET30) and the pGEX series (GE Healthcare).

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

[0036] Preferably, the cell system of the expression vector for expressing the present invention is selected from prokaryotic systems, such as Escherichia coli (E. coli) bacterial cells.

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

[0038] Another object of the present invention is a method for preparing the peptide of the present invention. According to the method, the transformed host cells are cultured under suitable conditions for a sufficient time to express the peptide of the present invention. Typically, the suitable culture conditions and time depend on the cell system used and can be related to, for example, the composition of the culture medium, pH, relative humidity, gas components of O2 and CO2, and temperature. The selection of the most suitable culture conditions and time for the method of the present invention is entirely within the knowledge and skills of a person of ordinary skill in the art.

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

[0040] Suitable alternative methods for manufacturing the peptide according to the present invention include, for example, chemical synthesis processes or techniques for protease cleavage of precursor proteins, for example, by using specific proteases or chemical reagents. The selection of the most suitable method for producing the peptide within the scope of the present invention falls within the technical scope of a person of ordinary skill in the art.

[0041] Due to the above advantageous features, the peptide according to the present invention is particularly suitable for agricultural practices aimed at improving plant growth and crop yields, while also allowing proper management of the soil and the environment. In particular, since the peptide according to the present invention does not have any direct killing effect, the action performed by the peptide advantageously does not have harmful consequences for useful pollinator populations.

[0042] The use of small molecules such as peptides represents another advantage of the present invention because they are better suited for design and / or modification in order to preserve or amplify specific activity. In addition, unlike longer proteins (e.g., full-length systemin precursor), the small size of peptides significantly reduces the synthesis and purification costs.

[0043] Accordingly, an object of the present invention is a biostimulant and bioprotective composition against abiotic and biotic stresses in plants, the biostimulant and bioprotective composition comprising at least one peptide as defined above or any combination thereof, and at least one adjuvant, stabilizer and / or preservative. At least one adjuvant, stabilizer and / or preservative in the composition is preferably conventionally used in agrotechnology to allow, for example, better uniform distribution on plants or seeds and / or to avoid excessive foaming.

[0044] Among the adjuvants suitable for use in the compositions according to the invention, mention is made, by way of non-limiting examples, of humectants, wetting agents and defoamers.

[0045] Exemplary defoamers include mixtures of siloxanes, sorbitol and silicon.

[0046] Exemplary humectants or wetting agents include surfactant compounds such as sodium dodecyl sulfate and betaine, mixtures of terpenes and alcohols.

[0047] Stabilizers in the compositions of the present invention include, for example, pH regulators, which include citric acid, acetic acid, sodium hydroxide.

[0048] Examples of preservatives suitable for use in the biostimulant and bioprotective compositions of the present invention include dehydroacetic acid, benzoic acid, ethylhexylglycerin and phenoxyethanol.

[0049] The selection of at least one adjuvant, stabilizer and / or preservative suitable for use in the compositions according to the invention falls within the skills of a person of ordinary skill in the art.

[0050] In one embodiment, the composition of the present 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.

[0051] In another embodiment, the composition of the present invention comprises a combination of a peptide having the amino acid sequence SEQ ID NO.3 and peptides having the amino acid sequences SEQ ID NO.5 and SEQ ID NO.8.

[0052] In yet another embodiment, the composition of the present invention comprises a combination of the following peptides:

[0053] A peptide having the amino acid sequence of SEQ ID NO.1;

[0054] A peptide having the amino acid sequence of SEQ ID NO.2;

[0055] A peptide having the amino acid sequence of SEQ ID NO.3;

[0056] A peptide having the amino acid sequence of SEQ ID NO.4;

[0057] A peptide having the amino acid sequence of SEQ ID NO.5;

[0058] A peptide having the amino acid sequence of SEQ ID NO.6;

[0059] A peptide having the amino acid sequence of SEQ ID NO.7;

[0060] A peptide having the amino acid sequence of SEQ ID No.8; and

[0061] A peptide having the amino acid sequence of SEQ ID NO.9.

[0062] In another embodiment, the composition of the present invention comprises a combination of the following peptides:

[0063] A peptide having the amino acid sequence of SEQ ID NO.3;

[0064] A peptide having the amino acid sequence of SEQ ID NO.4;

[0065] A peptide having the amino acid sequence of SEQ ID NO.5;

[0066] A peptide having the amino acid sequence of SEQ ID NO.6;

[0067] A peptide having the amino acid sequence of SEQ ID NO.7;

[0068] A peptide having the amino acid sequence of SEQ ID NO.8;

[0069] A peptide having the amino acid sequence of SEQ ID NO.9;

[0070] In another embodiment, the composition of the present invention comprises the following peptide combinations:

[0071] A peptide having the amino acid sequence of SEQ ID NO.1;

[0072] A peptide having the amino acid sequence of SEQ ID NO.2;

[0073] A peptide having the amino acid sequence of SEQ ID NO.3;

[0074] A peptide having the amino acid sequence of SEQ ID NO.4;

[0075] A peptide having the amino acid sequence of SEQ ID NO.5;

[0076] A peptide having the amino acid sequence of SEQ ID NO.6;

[0077] A peptide having the amino acid sequence of SEQ ID NO.7;

[0078] A peptide having the amino acid sequence of SEQ ID NO.8;

[0079] A peptide having the amino acid sequence of SEQ ID NO.9;

[0080] A peptide having the amino acid sequence of SEQ ID NO.23;

[0081] A peptide having the amino acid sequence of SEQ ID NO.24;

[0082] A peptide having the amino acid sequence of SEQ ID NO.25; and

[0083] A peptide having the amino acid sequence of SEQ ID NO.26.

[0084] According to the official definition established by the European Biostimulant Industry Council (EBIC), the term "biostimulant" refers to substances and / or microorganisms that, when applied to plants or the rhizosphere, act to stimulate natural processes to improve / promote nutrient uptake, nutrient use efficiency, crop quality, and tolerance to abiotic stress.

[0085] Within the scope of the present invention, the term "bioprotector" refers to substances and / or microorganisms that, after being applied to plants or the rhizosphere, induce the activation of the natural defenses of plants against biotic and abiotic stress.

[0086] As used herein, the term "plant" refers to a living multicellular plant organism.

[0087] Preferably, the plant belongs to a family selected from the group consisting of: Solanaceae, such as tomatoes and eggplants (Solanum melongena); Vitaceae, such as grapes (Vitis vinifera); Rosaceae, such as apples (Malus domestica); Oleaceae, such as olives; and combinations thereof.

[0088] With reference to biotic stress, herbivorous insects, phytopathogenic fungi, phytopathogenic bacteria, and viruses are mentioned by way of non-limiting examples.

[0089] In this case, the herbivorous insects are preferably selected from the group consisting of: Lepidoptera insects, such as Spodoptera littoralis and Tuta absoluta; plant mites, such as aphids, for example Macrosiphum euphorbiae; Homoptera insects, such as Bemisia tabaci and Trialeurodes vaporariorum; and combinations thereof.

[0090] The phytopathogenic fungi are preferably selected from the group consisting of Botrytis cinerea, Alternaria alternata, Alternaria solani, and combinations thereof.

[0091] The phytopathogenic bacteria are preferably Pseudomonas syringae bacteria.

[0092] The viruses are preferably selected from Tomato spotted wilt virus and Cucumber Mosaic Virus.

[0093] Among the abiotic stresses, although not exclusively, mention is made, for example, of low temperatures that cause, for example, freezing, high temperatures, drought, high light intensity, low light intensity, excessive salinity, excessive moisture, and combinations thereof.

[0094] Preferably, the composition according to the invention further comprises a buffer. Among the buffers suitable for the biostimulant and bioprotective compositions of the invention, phosphate buffers are particularly preferred, and phosphate buffered saline is even more preferred. However, it should be understood that other buffers can be used in the present invention, and the selection of other buffers falls within the skills of a person of ordinary skill in the art.

[0095] Preferably, the at least one peptide is present in the biostimulant and bioprotective composition according to the invention at a concentration of from 0.02 picomoles (pM) to 100 pM, more preferably from 0.02 pM to 0.08 pM, or from 0.085 pM to 0.1 pM, or from 0.095 pM to 0.25 pM, or from 1 pM to 100 pM.

[0096] According to a preferred embodiment of the present invention, the biostimulant and bioprotective composition further comprises microorganisms selected from the group consisting of: mycorrhizal fungi, saprophytic fungi, plant growth promoting bacteria, Bacillus thuringiensis spores, and any combination thereof.

[0097] As is known in the art, subterranean mycorrhizal fungi establish symbionts with the roots of many agricultural crops, which is mutually beneficial to the organisms involved. More specifically, mycorrhizal fungi are able to metabolize mineral elements present in the soil even if they are fixed to the soil's absorption capacity, while the plant provides the symbiotic fungi with sugars produced through photosynthesis.

[0098] Within the scope of the present invention, the mycorrhizal fungi are preferably selected from the group consisting of: Gigaspora fasciculatus, Glomus constrictum, Glomus tortuosum, Glomus geosporum, Gigaspora margarita, Acaulospora scrobicurata, and any combination thereof.

[0099] Within the scope of the present invention, the saprophytic fungi preferably belong to the genus Trichoderma.

[0100] It is known that the beneficial activity of saprophytic fungi in degrading dead plants and dead animals in the soil is known.

[0101] According to the present invention, it is highly preferred that the biostimulant and bioprotective composition comprising at least one peptide as defined above be combined with saprophytic fungi belonging to the genus Trichoderma, because the combination (as Figure 15 shown) has a significant synergistic effect on plant pathogens.

[0102] Within the scope of the present invention, the bacteria promoting plant growth are preferably selected from Burkholderia cepacia and Pseudomonas fluorescens.

[0103] Bacillus thuringiensis is a spore-forming bacterium that naturally occurs in the soil and is known to produce spores and parasporal bodies (commonly referred to as crystals) containing endotoxins with insecticidal effects under adverse conditions. After being ingested by sensitive insects, these endotoxins are released from the parasporal bodies and cause lysis of the intestinal epithelial cells, resulting in insect paralysis and death.

[0104] More preferably, the spores in the composition according to the invention are from the bacterium Bacillus thuringiensis subspecies aizawai.

[0105] According to the invention, the biostimulant and bioprotective composition can be in the form of a lyophilisate. In such an embodiment, the composition according to the invention is stable at room temperature for at least 3 months.

[0106] In another embodiment, the composition of the invention can be in the form of a water-based liquid composition or phosphate-buffered saline. In this form, the composition according to the invention can be used as such or diluted before use.

[0107] A method for increasing the resistance to biotic and / or abiotic stress in plants, which method comprises the step of applying the biostimulant and bioprotective composition as defined above to plants, parts of plants, plant propagation materials and / or plant growth sites, is also within the scope of the invention.

[0108] According to the method of the invention, the biostimulant and bioprotective composition can be applied in various forms to various plants or parts of plants, such as leaves, leaf buds, branches, stems, bark, flowers, flower buds, fruits, roots, seeds, bulbs, tubers and / or buds.

[0109] As used herein, the term "propagation material" means any plant material from which a plant or part of a plant can be derived. By way of non-limiting example, seeds, seedlings, cuttings, scions, rhizomes, explants, bulbs, tubers, and combinations thereof are mentioned.

[0110] Additionally or alternatively, the composition according to the invention can be applied to plant growth sites.

[0111] In one embodiment, the plants are grown in soil and the application of the composition according to the invention can be carried out, for example, over the entire planting surface, in and / or around one or more furrows, in sowing holes, in the area under the stem or trunk, and / or in the area between the roots.

[0112] In another embodiment, the plants grow from the soil or are grown hydroponically. Among the methods outside the soil or hydroponically, the hydroponic cultivation technique is mentioned as a non-limiting example, in which the soil is replaced by an inert substrate, such as expanded clay, coconut fiber, rock wool or zeolite, and the plants absorb nutrients through a solution composed of water and inorganic elements (for example, Mg(NO3)2·6H2O, Ca(NO3)2·4H2O, KNO3, K2SO4, KH2PO4), and the purpose of these inorganic elements is to provide all substances required for the normal mineral nutrition of the plant organism. A great advantage of hydroponic cultivation practice is that, due to the non-use of pesticides, herbicides and plant protection products, this cultivation technique allows for a constant and controlled yield throughout the year, both from the quality perspective and from the hygiene and cleanliness perspective.

[0113] The biostimulant and bioprotective composition of the present invention can be applied to plants, parts of plants, plant propagation materials and / or plant growth sites by conventional methods, such as by spraying, atomizing, misting, spreading or irrigation (by hand, using a tractor, by plane, etc.).

[0114] According to a preferred embodiment, the composition of the present invention is applied by spraying or atomizing on the plant or part of the plant (preferably on the leaves).

[0115] According to another preferred embodiment, the composition of the present invention is applied by irrigation (i.e., directly into the soil), for example in the form of an irrigation liquid or by injection into the soil.

[0116] In the case of hydroponic cultivation, the method according to the present invention provides for applying the biostimulant and bioprotective composition to the plants in the nutrient solution.

[0117] According to an embodiment of the present invention, the method comprises applying the composition at least twice, preferably 4 times, more preferably 5 times.

[0118] In such an embodiment, the time interval between one application on the plant (for example, the first, second, third, fourth or fifth application) and a subsequent application can be in the range of about 3 weeks to about 4 weeks.

[0119] Another object of the present invention is to use the previously defined isolated peptide or the previously defined biostimulant and bioprotective composition to enhance the resistance to biotic and / or abiotic stresses in plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] The following experimental section is for illustrative purposes only and does not limit the scope of the present invention defined in the appended claims. In the experimental section, reference is made to the accompanying drawings, in which:

[0121] Figure 1Shows a schematic diagram of the cloning vector pETM11 used by the present inventors for the recombinant production of the peptides of the present invention.

[0122] Figure 2 Shows a schematic diagram of an insert containing the nucleotide sequence encoding peptide PS1-70 after cloning into the vector pETM11. (A) Sequence of the recombinant insert (SEQ ID NO.12) obtained by Sanger sequencing. The nucleotide sequence encoding peptide PS1-70 (SEQ ID NO.10) correctly inserted into the cloning vector is underlined in black; the sequence encoding the histidine tail (His-tag) (SEQ ID NO.14) is highlighted in bold uppercase letters; the Tobacco Etch Virus (TEV) protease recognition site (SEQ ID NO.16) located downstream of the histidine tail to allow removal of the latter is highlighted in gray underlined uppercase italic. The sequences of the forward primer P11F1 (SEQ ID NO.17) and reverse primer P11R1 (SEQ ID NO.18) used for amplifying and cloning the nucleotide sequence encoding peptide PS1-70 are highlighted in light gray and dark gray respectively. B) The amino acid sequence of peptide PS1-70 (bold, SEQ ID NO.1) is conjugated to the C-terminus of the histidine tail (underlined sequence, SEQ ID NO.15) at the N-terminal position.

[0123] Figure 3 Shows a schematic diagram of an insert containing the nucleotide sequence encoding peptide PS1-120 after cloning into the vector pETM11. (A) Sequence of the recombinant insert (SEQ ID NO.13) obtained by Sanger sequencing. The nucleotide sequence encoding peptide PS1-120 (SEQ ID NO.11) correctly inserted into the cloning vector 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) located downstream of the histidine tail to allow removal of the latter is highlighted in gray underlined uppercase italic. The sequences of the forward primer P11F1 (SEQ ID NO.17) and reverse primer P11R3 (SEQ ID NO.19) used for amplifying and cloning the nucleotide sequence encoding peptide PS1-120 are highlighted in light gray and dark gray respectively. (B) The amino acid sequence of peptide PS1-120 (bold, SEQ ID NO.2) is conjugated to the C-terminus of the histidine tail (underlined sequence, SEQ ID NO.15) at the N-terminal position.

[0124] Figure 4Table showing the nucleotide sequences and characteristics of primers for amplifying and cloning nucleotide sequences encoding peptides PS1-70 and PS1-120, respectively.

[0125] Figure 5 Results of affinity chromatography (IMAC), results of analyzing elution fractions by 15% SDS-PAGE electrophoresis, and results of Western blot analysis performed on purified PSA-70 and PS1-120 peptides are shown. (A1 and B1) Chromatograms of the first step of purification of peptides PS1-70 and PS1-120 eluted with 150 mM and 50 mM imidazole, respectively. (A2 and B2) Polyacrylamide gel analysis of elution fractions; M: molecular weight marker; black rectangles: 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; black rectangles: PS1-70 peptide and PS1-120 peptide.

[0126] Figure 6 Results of size exclusion chromatography (SEC) and results of analyzing elution fractions by 15% SDS-PAGE electrophoresis are shown. (A1 and B1) Chromatograms of the second step of purification of peptides PS1-70 and PS1-120, where peptides PS1-70 and PS1-120 have elution peaks at elution volumes of 12.16 ml and 10.92 ml, respectively. (A2 and B2) Polyacrylamide gel analysis of elution fractions; M: molecular weight marker; black rectangles: elution fractions containing peptides PS1-70 and PS1-120. (A3 and B3) Deconvoluted masses of peptides PS1-70 and PS1-120.

[0127] Figure 7 Distribution of the amino acid composition of peptides PS1-70 (A) and PS1-120 (B) is shown. The curves in the figure indicate that the amino acid sequences of the peptides both contain a significant representation of amino acids that promote structural disorder (dark gray) compared to amino acids that promote an ordered secondary structure (light gray).

[0128] In Figure 8 curves A and B show the results of light scattering experiments by SEC-MALS-QELS on peptides PS1-70 (A) and PS1-120 (B) at pH 8.0 as described in Examples 1 and 2. The peaks of the curves represent monomeric proteins in solution. Figure 8(C, D) show the dichroic spectra of purified PS1-70 peptide (C) and PS1-120 peptide (D) recorded at 20 °C in 10 mM phosphate buffer using PS1-70 and PS1-120 peptides at concentrations of 4.4 μM and 3.5 μM, respectively. The horizontal axis shows the wavelength (nm), and the vertical axis shows the mean residue molar ellipticity value.

[0129] Figure 9 Relative quantification of induced gene expression in tomato plants at 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 is shown. Analysis was performed on the Lox C, AOS, Pin I, and Pin II genes. Letters a, b, c indicate the statistical significance of the data (ANOVA), and each letter represents a statistical group.

[0130] Figure 10 The effects of treating tomato plant leaves with peptides PS1-70 and PS1-120 on the larvae of the Lepidoptera insect Spodoptera littoralis are shown. The bar graphs (A, C) show the changes in the average weight (expressed in grams) of larvae fed with leaves treated with peptides PS1-70 and PS1-120 at 100 pM and 100 fM, respectively, and the relevant controls, measured over subsequent days. Letters a, b indicate the statistical significance of the data (ANOVA), and each letter represents a statistical group. The line graphs (B, D) show the mortality rates of larvae fed with tomato plant leaves treated with the peptides of the present invention and the relevant controls, recorded daily (log-rank test; ***p < 0.0001).

[0131] Figure 11 The effects of treating tomato plant leaves with peptide PS1-70 and SEQ ID NO.3, SEQ ID NO.5, and SEQ ID NO.8 on the larvae of the Lepidoptera insect Spodoptera littoralis are shown. The bar graph (A) shows the changes in the average weight (expressed in grams) of larvae fed with leaves treated with the above-mentioned peptides and the relevant controls, measured at 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 days. Letters a, b indicate the statistical significance of the data (ANOVA), and each letter represents a statistical group. The graph (B) shows the mortality rates of larvae fed with tomato plant leaves treated with the peptides of the present invention and the relevant controls, recorded daily (log-rank test; ***p < 0.0001).

[0132] Figure 12Shows the reduction in the necrotic area produced by the necrotrophic Botrytis cinerea fungus on tomato plant leaves (A, B), eggplant leaves (C), and grape plant leaves (D) after treatment with PS1-70 and PS1-120 peptides compared to untreated controls. The mean necrotic area was measured at 1, 3, 5, and 8 days after inoculation with the pathogen. Letters a, b, c, d indicate the statistical significance of the data (ANOVA), and each letter represents a statistical group.

[0133] Figure 13 Shows the reduction in the necrotic area produced by the necrotrophic Botrytis cinerea fungus on tomato plant leaves after treatment with PS1-70, SEQ ID NO.3, SEQ ID NO.5, and SEQ ID NO.8 compared to untreated controls. The mean necrotic area was measured at 1, 3, 5, and 8 days after inoculation with the pathogen. Letters a, b, c, d indicate the statistical significance of the data (ANOVA), and each letter represents a statistical group.

[0134] Figure 14 Shows the reduction in the necrotic area produced by the necrotrophic Alternaria alternata fungus on tomato plant leaves after treatment with the peptides PS1-70 and PS1-120 (A) and with PS1-70, SEQ ID NO.3, SEQ ID NO.5, and SEQ ID NO.8 (B) compared to untreated controls. The mean necrotic area was measured at 1, 3, 5, and 8 days after inoculation with the pathogen. Letters a, b indicate the statistical significance of the data (ANOVA), and each letter represents a statistical group.

[0135] Figure 15 Shows the effects of combined treatment with the peptides PS1-70, PS1-120, and systemin (Sys) and spores of the Trichoderma harzianum T22 strain on 4-week-old plants (grown from seeds co-infected with T. harzianum spores), on the survival of Spodoptera littoralis Lepidoptera insect larvae (A1, A2, and A3), and on the colonization of the necrotrophic Botrytis cinerea (B1) and Alternaria alternata (B2) fungi in the leaves. Larval survival was measured daily (log-rank test; ***p < 0.0001), and each letter represents a statistical group. The mean necrotic area was measured at 1, 3, 5, and 8 days after inoculation with the pathogen. Letters a, b, c, d, e, f indicate the statistical significance of the data (ANOVA), and each letter represents a statistical group.

[0136] Figure 16Shows the reduction in the necrotic area produced by the necrotrophic Botrytis cinerea fungus on the leaves of 4-week-old tomato plants in seeds treated with suspensions of peptides PS1-70 and PS1-120 at 100 fM and the corresponding controls. The average necrotic area was measured 1, 3, and 5 days after inoculation with the pathogen. Letters a, b indicate the statistical significance of the data (ANOVA), and each letter represents a statistical group.

[0137] Figure 17 Shows the relative quantification of gene expression induced in tomato plants irrigated with peptide PS1-70 at a concentration of 100 pM under conditions of absence of salt (A) (0 mM NaCl) and presence of salt (B) (80 mM NaCl). Analyses were performed on the cat1, tft1, Sam, HSFA2, HSP70, HSP90, MPK1, and WRKY40 genes. Asterisks indicate the statistical significance of the data by Student's t-test (*p < 0.05; **p < 0.01; ***p < 0.001).

[0138] Figure 18 Shows the relative quantification of gene expression induced in tomato plants irrigated with peptides PS1-70, PS1-120, and SEQ ID NO.5 (100 fM) under conditions of absence of salt (0 mM NaCl), presence of salt (B) (150 mM NaCl), and the relevant controls. Analyses were performed on the CAT2 (A), SAM (B), and APX2 (C) genes. Asterisks indicate the statistical significance of the data by Student's t-test (*p < 0.05; **p < 0.01; ***p < 0.001).

[0139] Figure 19 Shows the average proline content in the leaves of plants irrigated with peptides PS1-70, PS1-120, and SEQ ID NO.5 (100 fM) under conditions of absence of salt (0 mM NaCl), presence of salt (150 mM NaCl), and the relevant controls. Letters a, b, c indicate the statistical significance of the data (ANOVA), and each letter represents a statistical group.

[0140] Figure 20Shows the effect of irrigation treatment with peptides PS1-70, PS1-120, and SEQ ID NO.5 at a concentration of 100 fM on the biometric parameters of tomato plants. The bar graph (A) shows the root area (expressed in square centimeters) of plants treated with the peptides of the present invention and the relevant controls in the absence of salt (0 mM NaCl) and in the presence of salt (150 mM NaCl), as well as the relevant controls. The bar graph (B) shows the change in the fresh weight (expressed in grams) of the above-ground parts in plants treated with the peptides of the present invention and the relevant controls in the absence of salt (0 mM NaCl). Asterisks indicate the statistical significance of the data by Student's t-test (*p < 0.05).

[0141] Figure 21 Shows a table of the evaluation results of the direct toxic effect of the peptides of the present invention measured on Spodoptera littoralis larvae at increasing concentrations (as shown in Example 4). Peptides PS1-70 and PS1-120 were injected or applied to the epidermis, and the survival rate of the larvae at the chrysalis stage was recorded.

[0142] Figure 22 Shows the evaluation of the direct toxic effect of the peptides of the present invention measured at increasing concentrations when added to the growth media of the following two different fungi: Botrytis cinerea (A) PS1-70 and PS1-120, (B) PS1-80, SEQ ID NO.3, SEQ ID NO.5, and SEQ ID NO.8, and Trichoderma T22, (C) PS1-90 and PS1-10. The growth of the fungi was measured as the turbidity level (absorbance at 600 nm) of the media 24 hours after adding the peptides of the present invention. Letters a, b indicate the statistical significance of the data (ANOVA), and each letter represents a statistical group. Detailed Description

[0143] Example 1: Production of the Peptide According to the Invention

[0144] Cloning, Expression, and Purification

[0145] To isolate the nucleotide sequence encoding the peptide of the present invention, a PCR reaction was established using amplification primer pairs and using the cDNA encoding the full-length systemin precursor as a template. The sequences of the amplification primer pairs are shown in the table in Figure 4 shown.

[0146] The amplicons were digested with the NcoI and XhoI restriction enzymes and subsequently cloned into the pETM11 vector, which had been previously digested with the same enzymes. pETM11 (provided by EMBL, Heidelberg) is a prokaryotic expression vector that is capable of adding a tail of six histidines (His-tag) at the amino (N-) terminal part of the cloned protein and has a TEV (Tobacco Etch Virus) protease recognition site downstream of the His-tag sequence to allow removal of the latter ( Figure 1 ).

[0147] The integrity of the cloned fragment and the absence of possible mutations that occurred during the amplification reaction were confirmed by the construct obtained by sequencing ( Figure 2 A and 3A). After initial screening, the peptides PS1-70 and PS1-120 of the present invention were expressed at a large scale for 16 h in Escherichia coli BL21(DE3) strain in LB and 2-YT media at 22 °C with 2 mM IPTG. Figure 2 B and Figure 3 B respectively show the amino acid sequences of the obtained peptides PS1-70 (SEQ ID NO.1) and PS1-120 (SEQ ID NO.2) (highlighted in bold), with each peptide conjugated to the C-terminal of the histidine tail at the N-terminus (highlighted in underline).

[0148] After expression, purification of the peptides was performed at room temperature on Figure 5 by affinity chromatography (IMAC) ( Figure 6 ) and size exclusion chromatography (SEC) ( ) (GE Healthcare), and the yield obtained was 2 mg / L cell culture.

[0149] Peptide Synthesis

[0150] The peptides of the present invention having the sequences SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:8 were produced 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 process involves the use of a resin, which allows for obtaining peptides modified by amidation at the carboxyl terminus. At the end of the synthesis, the amino terminus of the peptide was also modified by acetylation. Purification was performed by reverse-phase HPLC.

[0151] Example 2: Analysis of the Peptide Structure According to the Invention

[0152] During the process of identifying the peptides of the present invention, the inventors encountered considerable difficulties due to the special characteristics of the PS1-70 and PS1-120 peptides, mainly the significant presence of amino acid residues that promote structural disorder in their primary sequences ( Figure 7 ). When subjected to SDS-PAGE electrophoresis, both peptides PS1-70 and PS1-120 showed abnormal migration, migrating with an apparent molecular weight of 20 - 25 kDa relative to their actual weights (MW PS1-70 = 11 kDa; MW PS1-120 = 17 kDa), and mass spectrometry confirmed again the exact molecular weights of the two recombinant peptides ( Figure 6 , A3 and B3).

[0153] In addition, during size exclusion chromatography (SEC), peptides PS1-70 and PS1-120 showed retention volumes of 12.16 ml and 10.92 ml, respectively ( Figure 6 ), indicating oligomers or low-density proteins. Light scattering experiments performed by SEC-MALS-QELS showed that regardless of the retention volume, the peptides of the present invention exist in solution as monodisperse monomeric proteins, with the molecular weights of the peptides being 9.36 ± 0.6 kDa for PS1-70 and 19.98 ± 1.5 kDa for PS1-120, consistent with the theoretical results ( Figure 8 A, Figure 8 B).

[0154] Then, the secondary structures of peptides PS1-70 and PS1-120 were analyzed by circular dichroism (CD). For both tested peptides, the obtained far-UV CD spectra showed negative molar ellipticity values at 198 nm and 190 nm. However, the ellipticity values observed at 200 nm and 222 nm indicated the presence of some secondary structures ( Figure 8 C, Figure 8 D). The above characteristics are typical of disordered proteins with a large unstructured portion. This may be related both to the large number of acidic residues (negatively charged at physiological pH) that cause intramolecular repulsion and to the low content of hydrophobic residues that usually contribute to the correct folding of proteins.

[0155] Example 3: Inducing Defense Gene Expression in Plants by the Peptide According to the Invention

[0156] To test the biological activity of the peptides of the present invention, the inventors conducted studies to measure the expression of defense genes in tomato plants after application of peptides PS1-70 and PS1-120. 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 the aqueous composition containing the peptide to several points on the upper side of the expanded leaves of 4-week-old tomato plants.

[0157] Leaf samples were taken 6 hours and 24 hours after application of the peptides of the present invention for RNA extraction and subsequent gene expression analysis. In particular, four genes known to be associated with plant defense were selected and tested: two early-expressed genes active in the octadecanoid biosynthetic pathway leading to the formation of jasmonic acid (JA), such as lipoxygenase C gene (Lox C) and allene oxide synthase gene (AOS); and two late-expressed genes, such as protease I inhibitor (Pin I) and protease II inhibitor (Pin II) genes.

[0158] After exogenous application of the two peptides of the present invention at the two test concentrations, all the genes detected were significantly overexpressed ( Figure 9 ).

[0159] Example 4: The Peptide According to the Invention Promotes Resistance to Biotic Stress in Plants

[0160] To demonstrate that the peptides of the present invention are capable of promoting plant resistance to pathogenic organisms, the inventors conducted studies to evaluate the effects on herbivorous insects or plant pathogenic fungi of treating plants with peptides having amino acid sequences 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. More specifically, the inventors monitored two different parameters, namely the change in body weight gain and survival rate of the larvae of Spodoptera littoralis (a lepidopteran insect that causes considerable damage to tomato plants), and the colonization of the plant by the plant pathogenic necrotrophic fungus Botrytis cinerea and the parasitic fungus Alternaria alternata, the Botrytis cinerea fungus being the agent causing tomato gray mold.

[0161] Experiment on Spodoptera littoralis Larvae

[0162] In short, Spodoptera litura larvae were fed with artificial diet until the first molt was completed in a climate chamber at 25 °C and 70% relative humidity (RH), with a photoperiod of 16 h light and 8 h dark. For bioassays, 150 larvae of each treatment were reared on tomato leaves throughout the second instar to acclimatize to the different diet. Newly molted third instar larvae were fed with plants until the molting period, and on the plants, a composition containing PS1-70 and PS1-120 peptides was applied at concentrations of 100 pM and 100 fM in 1X PBS buffer. Larvae fed with plants treated only with 1X PBS buffer were used as controls. Bioassays were conducted in 32-well plastic trays containing 1.5% (w / v) agar and 0.005% (w / v) methylparaben under the same environmental conditions, which helped create a humid environment for maintaining cell turgor of tomato leaves. Each experimental group consisted of 32 larvae. Larval survival was monitored daily and body weight was monitored every other day. Compared with the larvae fed with control leaves, the larvae fed with leaves treated with the peptides of the present invention showed a decrease in body weight throughout the bioassay period, and significant differences were observed as early as day 3 for both concentrations. In particular, on day 15 of the bioassay, for the 100 pM concentration, the larvae fed with control leaves had an average weight of 38 mg, while the larvae fed with leaves treated with peptides PS1-70 and PS1-120 had average weights of 15 mg and 20 mg, respectively ( Figure 10 A). In the bioassay conducted with the peptides of the present invention at a concentration of 100 fM, on day 13, the larvae fed with control leaves had an average weight of 44 mg, while the larvae fed with leaves treated with peptides PS1-70 and PS1-120 had average weights of 11 mg and 12 mg, respectively ( Figure 10 C). In both experiments, it was also observed that the survival rate of the larvae fed with plant leaves treated with the peptides of the present invention decreased significantly compared with that of the larvae fed with control plant leaves ( Figure 10 B and 10D). In fact, on day 15 of the bioassay, a survival rate of 96.87% was observed for the larvae fed with control leaves, and survival rates of 34.37% and 31.25% were observed for the larvae fed with leaves treated with the peptides of the present invention at a concentration of 100 pM, respectively ( Figure 10 B). In the bioassay conducted at a concentration of 100 fM, on day 13, a survival rate of 100% was observed for the larvae fed with control leaves, while survival rates of 0% and 21.87% were observed for the larvae fed with leaves treated with the peptides of the present invention, respectively ( Figure 10 D).

[0163] The present inventors performed the same assay protocol by feeding Spodoptera littoralis larvae with leaves treated with peptide PS1-70 and with leaves treated with peptides having femtomolar concentrations of sequences SEQ ID No.3, SEQ ID No.5 and SEQ ID No.8. Larvae fed with the treated leaves showed a significant decrease in weight starting from the 3rd day compared to the control leaves( Figure 11 ). In particular, on the 13th day of the bioassay, the average weight of the larvae fed with the control leaves was 59 mg, while the average weights of the larvae fed with leaves treated with peptide PS1-70, SEQ IDNO.8, SEQ ID No.3 and SEQ ID No.5 were 22 mg, 13 mg, 24 mg and 21 mg, respectively( Figure 11 A). A significant decrease in the survival rate of the larvae fed with the peptide-treated leaves was also observed compared to the larvae fed with the control leaves (Supp Figure 11 B). In fact, on the 13th day of the bioassay, a 100% survival rate was observed for the larvae fed with the control leaves, while survival rates of 31.25%, 15.62%, 43.75% and 15.62% were observed for the larvae fed with leaves treated with PS1-70, SEQ ID NO.8, SEQ ID No.3 and SEQ ID No.5, respectively( Figure 11 B).

[0164] Experiments on Botrytis cinerea and Alternaria alternata

[0165] To perform the assays on the phytopathogenic Botrytis cinerea and Alternaria fungi of plants, the present inventors used the spores of this microorganism obtained from cultures on solid PDA (Potato Dextrose Agar) sporulation substrates. Plates were inoculated with 20 μl of conidial suspension at a concentration of 1×10 6 spores / ml and incubated at 25 °C for 15 days in the presence of diffused light to obtain complete sporulation. The spores were then collected in 5 ml of sterile water and, to remove the mycelium, filtered through glass wool, washed with sterile distilled water and collected by centrifugation at room temperature. The appropriate inoculation spore concentration (10 5 -10 7 spores / ml) was determined by serial dilution using a Bürker chamber for spore counting.

[0166] Assays were performed on the compound leaves taken from each treated tomato plant and each control plant by making marks on each primary leaf of the compound leaf to guide subsequent spore application and detection of necrotic areas formed by the pathogen.

[0167] Leaves were treated by applying 2 μl of a composition containing the peptides PS1-70 and PS1-120 of the present invention at concentrations of 100 pM and 100 fM, or by applying 1X PBS (for control leaves). Six hours later (i.e., the time required to sense the peptide), the leaves were detached from the plants and inoculated with 10 μl of a spore solution near the nerve gap and the previously marked points. Monitoring was carried out by measuring the necrotic area (expressed in mm 2 at 1, 3, 5, and 8 days after inoculation with the pathogen. The necrotic areas recorded on the control leaves were much larger than those detected on the plants treated with the peptides of the present invention. This difference increased over time after inoculation. From the first day to the eighth day after inoculation with the pathogen, the resistance induction effect of the picomolar treatment was already statistically significant, reaching a value of 33 mm 2 on the control leaves, and the recorded values for both polypeptides did not exceed 18 mm 2 ( Figure 12 A).

[0168] Similar results were observed after applying the femtomolar concentration of the peptides of the present invention to the objects. In particular, as early as the first day after inoculating the plant pathogen, a significant reduction in the development of the necrotic area on the treated leaves was observed compared to the necrotic area on the control leaves. This reduction continued until the eighth day, with the control leaves reaching a value of 20 mm 2 , and the leaves treated with PS1-70 and PS1-120 reaching values of 6.69 mm 2 and 5.19 mm 2 respectively ( Figure 12 B).

[0169] The inventors also carried out tests aimed at determining the effect of exogenous application of the peptides of the present invention at picomolar and femtomolar concentrations on the development of necrotic fungi on plants of the Solanaceae family (Solanum melongena, eggplant) Figure 12 C) and on plants of the Vitaceae family (grapevine) Figure 12 D).

[0170] As Figure 12 shown in 2 C, the tests carried out showed that the fungal colonization of the treated plants was significantly reduced compared to the control plants, and the most significant effect was observed after treatment with the lowest concentration of the peptides of the present invention. From the third day to the eighth day after inoculation with the pathogen, the positive effect of the treatment was statistically significant, reaching a value of 11 mm 2 on the control leaves, and the values recorded on the leaves treated with the peptides PS1-70 and PS1-120 at a concentration of 100 pM were 8.4 mm 2 and 6.0 mm2 and 4.10 mm 2 ( Figure 12 C). Similar results were also obtained on grape plants ( Figure 12 D). From day 1 to day 8 after inoculation with the pathogen, the positive effect of the treatment was statistically significant, reaching 43 mm on the control leaves, and 16.7 mm and 13.6 mm 2 on the leaves treated with 100 pM concentrations of PS1-70 and PS1-120 peptides, respectively, and 12 mm and 11.8 mm 2 on the leaves treated with 100 fM concentrations of said peptides, respectively ( 2 ( 2 D). Similar results were also obtained on olive trees. 2 ) Figure 12 The effect of applying peptides having SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.8 at femtomolar concentrations on the growth of Botrytis cinerea fungi on tomato leaves was also evaluated.

[0171] As

[0172] shown, the necrotic areas recorded on the control leaves were much larger than those detected on the plants treated with the peptides of the present invention. From day 1 after inoculation with the pathogen, the positive effect of the treatment was statistically significant and continued until day 8, reaching 11.72 mm Figure 13 on the control leaves, and 7.69 mm, 7.67 mm, 8.89 mm and 6.81 mm 2 on the leaves treated with 100 fM concentrations of PS1-70, SEQ ID NO.8, SEQ ID NO.3 and SEQ ID NO.5, respectively ( 2 ( 2 ) 2 ). 2 ) Figure 13 )

[0173] The inventors also conducted tests aimed at determining the effect of exogenous application of the peptides of the present invention on the growth of Alternaria alternata fungi resistant to tomato plants ( Figure 14 )

[0174] As Figure 14 shown in FIGS. 14A and 14B, the tests conducted showed that the fungal colonization of the treated plants was significantly reduced compared to the control plants. From day 1 to day 8 after inoculation with the pathogen, the positive effect of the treatment was statistically significant, reaching 24 mm 2 on the control leaves, and the values recorded on the leaves treated with 100 fM concentrations of PS1-70 and PS1-120 peptides were 8.4 mm, different from the treated leaves2 and 8.0 mm 2 ( Figure 14 A); the value recorded on the control leaf was 17 mm 2 , and the values recorded on the leaves treated with PS1-70, SEQ ID NO.8, SEQ ID NO.3, and SEQ ID NO.5 peptides were 8.3 mm 2 , 7.9 mm 2 , 8.9 mm 2 and 7.8 mm 2 .

[0175] Activity of the Peptide According to the Invention in Combination with Trichoderma T22 Spores

[0176] The present inventors conducted further studies in order to determine the effect of treating tomato plants with a combination of the peptides of the present invention at femtomolar concentrations and Trichoderma harzianum T22 spores on reducing the survival rate of Spodoptera littoralis larvae (A1, A2, A3) and the growth of necrotrophic fungi (B1) and Alternaria alternata fungi (B2) ( Figure 15 ).

[0177] Tomato seeds were treated with a suspension of spores of Trichoderma harzianum T22 strain (1X10 7 spores / ml) or with water as a control, dried in the dark on sterile filter paper at 24 °C and germinated. Four-week-old plant leaves were treated by applying 2 μl of a composition containing systemin (Sys), PS1-70 or PS1-120 peptides at a concentration of 100 fM or 1X PBS (for control leaves). Six hours later (i.e., the time required to perceive the polypeptide), the leaves were separated from the plants for assays with Spodoptera littoralis larvae and two necrotrophic fungi Botrytis cinerea and Alternaria alternata.

[0178] For the assay of Spodoptera littoralis larvae, the survival of the larvae was monitored daily. On the other hand, in order to monitor the growth of two phytopathogenic fungi, after inoculating 10 μl of a spore solution (1X10 6 spores / ml) on the previously treated tomato leaves, the necrotic areas (expressed in mm 2 ) were measured on days 1, 3, 5, and 8.

[0179] Figure 15 It is shown that the combination treatment of the tested peptides with Trichoderma harzianum T22 produces a surprising synergistic effect in counteracting the growth and survival of the larvae and has significantly higher protection against the colonization of two phytopathogenic fungi compared to the use of Trichoderma harzianum T22 alone or the peptides alone. The evidence of these effects increases over time. In addition, the effects produced by the peptides of the present invention (used alone or in combination with Trichoderma harzianum T22 spores) have been shown to be superior to the peptide systemin.

[0180] Seed Protection Effect of the Peptide According to the Invention

[0181] The inventors also evaluated the protective effect conferred by directly treating seeds with the peptides of the present invention. Tomato seeds were treated with a peptide-containing composition at a concentration of 100 fM or with 1X PBS (as a control), dried in the dark on sterile absorbent paper at 24 °C and germinated. Leaves were isolated from 4-week-old plants and 10 μl of a spore solution was inoculated near the nerve gap and the previously marked points. As Figure 16 shown, this test demonstrated that fungal colonization on the leaves of plants grown from seeds treated with PS1-70 and PS1-120 was significantly reduced compared to the control plants.

[0182] From day 1 to day 8 after inoculation with the pathogen, the positive effect of the treatment was statistically significant, reaching 11 mm on the control leaves 2 and 7.1 mm 2 and 7.4 mm 2 on the leaves of plants grown from seeds treated with the peptides PS1-70 and PS1-120 at a concentration of 100 fM ([[]] Figure 16 ). The inventors conducted further studies to investigate whether the peptides PS1-70 and PS1-120 have a direct toxic effect on the tested pathogenic organisms. From Figure 21 the data reported in the table, it can be seen that the peptides of the present invention have no effect on the survival and development of Spodoptera littoralis larvae when administered orally or by injection into the cuticle.

[0183] In addition, as Figure 22 A and Figure 22 B show, when the peptides PS1-70 and PS1-120 of the present invention were added to the growth medium, the development of Botrytis cinerea fungi was not disturbed. Further studies were conducted on the Trichoderma harzianum T22 strain, and as Figure 22 C shows, the addition of the peptides PS1-70 and PS1-120 at concentrations of 100 pM and 100 fM had no effect on the development of the fungi. On the contrary, when the peptide PS1-70 was added to the growth medium, an increase in the growth of T22 fungi was observed, indicating that the fungi may use this protein as a source of amino acids.

[0184] In summary, the above experimental results indicate that the peptide objects of the present invention are biologically active and their exogenous application promotes resistance to harmful insects and fungi in plants.

[0185] Example 5: The Peptide According to the Invention Promotes Resistance to Abiotic Stress in Plants

[0186] During their research, the inventors also established experiments aimed at verifying the efficacy of the peptides of the present invention in promoting the resistance of plants to various abiotic stresses.

[0187] Through the experiments conducted, the inventors first found that applying the peptides of the present invention to tomato plants leads to an increase in their biomass and favors the production of larger berries with a greater number of seeds. As shown in Figure 17 , the results of subsequent experiments showed that the application of the peptide PS1-70 protected tomato plants from the effects of salt stress. Briefly, after irrigating with the PS1-70 peptide at a concentration of 100 pM for 48 hours, unlike the control plants, the treated plants were able to activate a series of genes and transcription factors in response to salt stress, thus demonstrating that the peptides of the present invention are capable of stimulating the alarm state (termed priming) of the plants. In addition, under conditions of moderate salt stress (80 mM of NaCl), the tomato plants treated with the peptides of the present invention were more salt-tolerant than the untreated control plants, particularly showing a stronger induction of genes in response to this stress ( Figure 17 B). Experiments were also established to verify the efficiency of the peptides PS1-70, PS1-120, and SEQ ID NO.5 in promoting tolerance to high salt stress conditions when applied at femtomolar concentrations. As shown in Figure 18 , after irrigating with the peptides PS1-70, PS1-120, and SEQ ID NO.5 for 8 days, unlike the control plants, the treated plants were able to activate a series of genes in response to salt stress, thus confirming that the peptides of the present invention are capable of stimulating the primed defense state even at femtomolar concentrations ( Figure 18 A1, 18B1, and 18C1). In addition, compared to the untreated control plants, tomato plants treated with the above polypeptides and irrigated for 7 days after 24 hours under high salt stress levels (150 mM of NaCl) were more salt-tolerant, particularly showing a stronger induction of the same genes in response to this stress ( Figure 18 A2, Figure 18 B2, and Figure 18 C2).

[0188] The average proline content of these plants was also evaluated. Proline is an osmoprotectant that is produced in free form in plant cells in response to salt stress and water deficiency. In fact, in salt-stressed plants, this amino acid is involved in the regulation of the osmotic potential, protects cell membranes from free radicals, regulates cytoplasmic pH, and prevents enzyme denaturation.

[0189] As shown in Figure 19 , under high salt stress (150 mM of NaCl), compared to the salinized control plants, the plants treated with the peptides of the present invention showed a lower average proline content, thus indicating a lower perception of salt-induced osmotic stress by them.

[0190] In Figure 20Several biometric parameters are shown, measured after 14 days of continuous salt stress (150 mM) and 15 days of irrigation with peptides PS1-70, PS1-120, and SEQ ID NO.5 at a concentration of 100 fM and the corresponding controls. Figure 20 A shows that peptide-treated plants exhibit greater tolerance to stress compared to untreated control plants, particularly having a larger root surface ( Figure 20 A). Additionally, under salt-free conditions, unlike control plants, plants treated with the peptides of the present invention are able to promote the growth of the aerial parts of the plant, thus confirming that the peptides of the present invention have biostimulant effects even at femtomolar concentrations ( Figure 20 B). Sequence Listing <110> Materias s.r.l. <120> Biostimulant and Bioprotective Peptides and Their Use in Agriculture <130> E0135467 <160> 26 <170> BiSSAP 1.3.6 <210> 1 <211> 70 <212> PRT <213> Artificial Sequence <220> <223> Peptide PS1-70 <400> 1 Met Gly Thr Pro Ser Tyr Asp Ile Lys Asn Lys Gly Asp Asp Met Gln 1 5 10 15 Glu Glu Pro Lys Val Lys Leu His His Glu Lys Gly Gly Asp Glu Lys 20 25 30 Glu Lys Ile Ile Glu Lys Glu Thr Pro Ser Gln Asp Ile Asn Asn Lys 35 40 45 Asp Thr Ile Ser Ser Tyr Val Leu Arg Asp Asp Thr Gln Glu Ile Pro 50 55 60 Lys Met Glu His Glu Glu 65 70 <210> 2 <211> 120 <212> PRT <213> 人工序列 <220> <223> 肽PS1-120 <400> 2 Met Gly Thr Pro Ser Tyr Asp Ile Lys Asn Lys Gly Asp Asp Met Gln 1 5 10 15 Glu Glu Pro Lys Val Lys Leu His His Glu Lys Gly Gly Asp Glu Lys 20 25 30 Glu Lys Ile Ile Glu Lys Glu Thr Pro Ser Gln Asp Ile Asn Asn Lys 35 40 45 Asp Thr Ile Ser Ser Tyr Val Leu Arg Asp Asp Thr Gln Glu Ile Pro 50 55 60 Lys Met Glu His Glu Glu Gly Gly Tyr Val Lys Glu Lys Ile Val Glu 65 70 75 80 Lys Glu Thr Ile Ser Gln Tyr Ile Ile Lys Ile Glu Gly Asp Asp Asp 85 90 95 Ala Gln Glu Lys Leu Lys Val Glu Tyr Glu Glu Glu Glu Tyr Glu Lys 100 105 110 Glu Lys Ile Val Glu Lys Glu Thr 115 120 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 3 Glu Lys Glu Thr Pro Ser Gln Asp Ile 1 5 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 4 Glu Lys Glu Thr Ile Ser Gln Tyr Ile 1 5 <210> 5 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 5 Asp Asp Met Gln Glu Glu Pro Lys Val Lys Leu His His Glu Lys Gly 1 5 10 15 <210> 6 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 6 Asp Asp Thr Gln Glu Ile Pro Lys Met Glu His Glu Glu Gly 1 5 10 <210> 7 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 7 Asp Asp Ala Gln Glu Lys Leu Lys Val Glu Tyr Glu Glu Glu 1 5 10 <210> 8 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 8 Asp Asp Met Gln Glu Glu Pro Lys Val Lys Leu His His Glu Lys Gly 1 5 10 15 Gly Asp Glu Lys Glu Lys Ile Ile Glu Lys Glu Thr Pro Ser Gln Asp 20 25 30 Ile <210> 9 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 9 Asp Asp Thr Gln Glu Ile Pro Lys Met Glu His Glu Glu Gly Gly Tyr 1 5 10 15 Val Lys Glu Lys Ile Val Glu Lys Glu Thr Ile Ser Gln Tyr Ile 20 25 30 <210> 10 <211> 210 <212> DNA <213> Artificial Sequence <220> <223> Sequence encoding PS1-70 <400> 10 atgggaactc cttcatatga tatcaaaaac aaaggagatg acatgcaaga agaaccaaag 60 gtgaaacttc accatgagaa gggaggagat gaaaaggaaa aaataattga aaaagagact 120 ccatcccaag atatcaacaa caaagatacc atctcttcat atgttttaag agatgataca 180 caagaaatac caaagatgga acatgaggag 210 <210> 11 <211> 360 <212> DNA <213> Artificial sequence <220> <223> Sequence encoding PS1-120 <400> 11 atgggaactc cttcatatga tatcaaaaac aaaggagatg acatgcaaga agaaccaaag 60 gtgaaacttc accatgagaa gggaggagat gaaaaggaaa aaataattga aaaagagact 120 ccatcccaag atatcaacaa caaagatacc atctcttcat atgttttaag agatgataca 180 caagaaatac caaagatgga acatgaggag ggaggatatg taaaggagaa aattgttgaa 240 aaggagacta tatcccaata tatcatcaag attgaaggag atgatgatgc acaagaaaaa 300 ctaaaggttg agtatgagga ggaagaatat gaaaaagaga aaatagttga aaaagagact 360 <210> 12 <211> 436 <212> DNA <213> Artificial sequence <220> <223> PS1-70 recombinant insert <400> 12 gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca 60 attcccctct agaaataatt ttgtttaact ttaagaagga gatataccat gaaacatcac 120 catcaccatc accccatgag cgattacgac atccccacta ctgagaatct ttattttcag 180 ggcgccatgg gaactccttc atatgatatc aaaaacaaag gagatgacat gcaagaagaa 240 ccaaaggtga aacttcacca tgagaaggga ggagatgaaa aggaaaaaat aattgaaaaa 300 gagactccat cccaagatat caacaacaaa gataccatct cttcatatgt tttaagagat 360 gatacacaag aaataccaaa gatggaacat gaggagtagt aactcgagca ccaccaccac 420 caccactgag atccgg 436 <210> 13 <211> 513 <212> DNA <213> Artificial sequence <220> <223> PS1-120 recombinant insert <400> 13 ataattttgt ttaactttaa gaaggagata taccatgaaa catcaccatc accatcaccc 60 catgagcgat tacgacatcc ccactactga gaatctttat tttcagggcg ccatgggaac 120 tccttcatat gatatcaaaa acaaaggaga tgacatgcaa gaagaaccaa aggtgaaact 180 tcaccatgag aagggaggag atgaaaagga aaaaataatt gaaaaagaga ctccatccca 240 agatatcaac aacaaagata ccatctcttc atatgtttta agagatgata cacaagaaat 300 accaaagatg gaacatgagg agggaggata tgtaaaggag aaaattgttg aaaaggagac 360 tatatcccaa tatatcatca agattgaagg agatgatgat gcacaagaaa aactaaaggt 420 tgagtatgag gaggaagaat atgaaaaaga gaaaatagtt gaaaaagaga cttagtaact 480 cgagcaccac caccaccacc actgagatcc ggc 513 <210> 14 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Histidine tag for coding sequence <400> 14 catcaccatc accatcac 18 <210> 15 <211> 26 <212> PRT <213> Artificial sequence <220> <223> Histidine tag <400> 15 Met Lys His His His His His His Pro Met Ser Asp Tyr Asp Ile Pro 1 5 10 15 Thr Thr Glu Asn Leu Tyr Phe Gln Gly Ala 20 25 <210> 16 <211> 21 <212> DNA <213> Artificial sequence <220> <223> TEV site <400> 16 gagaatcttt attttcaggg c 21 <210> 17 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Forward primer P11F1 <400> 17 cgcgcgccat gggaactcct tcatatgata tca 33 <210> 18 <211> 41 <212> DNA <213> Artificial sequence <220> <223> Reverse primer P11R1 <400> 18 cgcgcgctcg agttactact cctcatgttc catctttggt a 41 <210> 19 <211> 48 <212> DNA <213> Artificial sequence <220> <223> Reverse primer P11R3 <400> 19 cgcgcgctcg agttactaag tctctttttc aactattttc tctttttc 48 <210> 20 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Sequence between the histidine tag and the TEV site <400> 20 cccatgagcg attacgacat ccccactact 30 <210> 21 <211> 96 <212> PRT <213> Artificial sequence <220> <223> Peptide PS1-70 with a histidine tag <400> 21 Met Lys His His His His His His Pro Met Ser Asp Tyr Asp Ile Pro 1 5 10 15 Thr Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Gly Thr Pro Ser Tyr 20 25 30 Asp Ile Lys Asn Lys Gly Asp Asp Met Gln Glu Glu Pro Lys Val Lys 35 40 45 Leu His His Glu Lys Gly Gly Asp Glu Lys Glu Lys Ile Ile Glu Lys 50 55 60 Glu Thr Pro Ser Gln Asp Ile Asn Asn Lys Asp Thr Ile Ser Ser Tyr 65 70 75 80 Val Leu Arg Asp Asp Thr Gln Glu Ile Pro Lys Met Glu His Glu Glu 85 90 95 <210> 22 <211> 146 <212> PRT <213> Artificial Sequence <220> <223> Peptide PS1-120 with Histidine Tag <400> 22 Met Lys His His His His His His Pro Met Ser Asp Tyr Asp Ile Pro 1 5 10 15 Thr Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met Gly Thr Pro Ser Tyr 20 25 30 Asp Ile Lys Asn Lys Gly Asp Asp Met Gln Glu Glu Pro Lys Val Lys 35 40 45 Leu His His Glu Lys Gly Gly Asp Glu Lys Glu Lys Ile Ile Glu Lys 50 55 60 Glu Thr Pro Ser Gln Asp Ile Asn Asn Lys Asp Thr Ile Ser Ser Tyr 65 70 75 80 Val Leu Arg Asp Asp Thr Gln Glu Ile Pro Lys Met Glu His Glu Glu 85 90 95 Gly Gly Tyr Val Lys Glu Lys Ile Val Glu Lys Glu Thr Ile Ser Gln 100 105 110 Tyr Ile Ile Lys Ile Glu Gly Asp Asp Asp Ala Gln Glu Lys Leu Lys 115 120 125 Val Glu Tyr Glu Glu Glu Glu Tyr Glu Lys Glu Lys Ile Val Glu Lys 130 135 140 Glu Thr 145 <210> 23 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Peptide <400> 23 Asp Asp Ala Gln Glu Lys Pro Lys Val Glu His Glu Glu Gly 1 5 10 <210> 24 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptide <400> 24 Asp Lys Glu Thr Pro Ser Gln Asp Ile 1 5 <210> 25 <211> 31 <212> PRT <213> Artificial sequence <220> <223> Peptide <400> 25 Asp Asp Ala Gln Glu Lys Leu Lys Val Glu Tyr Glu Glu Glu Glu Tyr 1 5 10 15 Glu Lys Glu Lys Ile Val Glu Lys Glu Thr Pro Ser Gln Asp Ile 20 25 30 <210> 26 <211> 24 <212> PRT <213> Artificial sequence <220> <223> Peptide <400> 26 Asp Asp Ala Gln Glu Lys Pro Lys Val Glu His Glu Glu Gly Asp Asp 1 5 10 15 Lys Glu Thr Pro Ser Gln Asp Ile 20

Claims

1. An isolated peptide, said isolated peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, and SEQ ID NO.8, and said isolated peptide having biostimulant and bioprotective activities against abiotic and biotic stresses in plants, said peptide optionally conjugated with a histidine tail at the amino terminus or carboxyl terminus.

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

3. An isolated nucleic acid sequence, said isolated nucleic acid sequence encoding the peptide according to claim 1 or 2.

4. An expression vector, said expression vector comprising the nucleic acid sequence according to claim 3.

5. A host cell, said host cell comprising the expression vector according to claim 4.

6. A method for preparing the peptide according to claim 1 or 2, said method comprising the step of culturing the host cell according to claim 5 under appropriate conditions for a time sufficient to express said peptide, and optionally comprising the step of recovering said peptide from the culture.

7. A biostimulant and bioprotective composition against abiotic and biotic stresses in plants, said biostimulant and bioprotective composition comprising at least one peptide according to claim 1 or 2 or any combination thereof, and at least one adjuvant.

8. The biostimulant and bioprotective composition according to claim 7, wherein, Said at least one peptide is present at a concentration of 0.01 picomolar (pM) to 100 pM.

9. The biostimulant and bioprotective composition according to claim 7 or 8, wherein, Said biostimulant and bioprotective composition further comprises microorganisms selected from the group consisting of mycorrhizal fungi, saprophytic fungi, plant growth-promoting bacteria, and any combination thereof.

10. The biostimulant and bioprotective composition according to claim 7 or 8, wherein, Said biostimulant and bioprotective composition is an aqueous liquid composition.

11. The biostimulant and bioprotective composition according to claim 7 or 8, wherein, Said biostimulant and bioprotective composition is in freeze-dried form.

12. A method for enhancing resistance to biotic and / or abiotic stresses in plants, said method comprising the step of applying the biostimulant and bioprotective composition according to any one of claims 7 to 11 to said plant or a part of said plant.

13. The method according to claim 12, wherein, Said biostimulant and bioprotective composition is applied by spraying, irrigation, or in a hydroponic solution.

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

15. Use of the isolated peptide according to claim 1 or 2, and / or the biostimulant and bioprotective composition according to any one of claims 7 to 11 in increasing resistance to abiotic and / or biotic stresses in plants.

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