Abstract
Isolated peptides having biostimulant and bioprotective activity against abiotic and biotic stress in plants are provided. Compositions comprising the peptides having biostimulant and bioprotective activity against abiotic and biotic stress in plants are also provided. Preferably, the peptides having biostimulant and bioprotective activity against abiotic and biotic stress in plants are from Solanum lycopersicum plants and are produced in a recombinant or synthetic manner. Methods for increasing resistance to biotic and/or abiotic stress in plants by applying to the plants the peptides having biostimulant and bioprotective activity and/or the compositions containing the peptides having biostimulant and bioprotective activity are further provided.
Claims
1. An isolated peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs. 1, 2, 3-9, 23-26 and having biostimulant and bioprotective activity against abiotic and biotic stress in plants, the isolated peptide being optionally conjugated with a histidine tail at the amino-terminal end or at the carboxy-terminal end.
2. (canceled)
3. The isolated peptide of claim 1, wherein the amino-terminal end is modified by acetylation and/or the carboxy-terminal end is modified by amidation.
4. An isolated nucleic acid sequence encoding the isolated peptide of claim 1.
5. An expression vector comprising the nucleic acid sequence of claim 4.
6. A host cell comprising the expression vector of claim 5.
7. A method for preparing an isolated peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs. 1, 2, 3-9, 23-26 and having biostimulant and bioprotective activity against abiotic and biotic stress in plants, the isolated peptide being optionally conjugated with a histidine tail at the amino-terminal end or at the carboxy-terminal end, the method comprising culturing a host cell according to claim 6 under suitable conditions and for a time sufficient for the expression of the peptide and, optionally, recovering the peptide from the culture.
8. A biostimulant and bioprotective composition against abiotic and biotic stress in plants, comprising at least one peptide according to claim 1, and at least one adjuvant, stabilizer and/or preservative.
9. The biostimulant and bioprotective composition of claim 8, wherein the at least one peptide is present in a concentration ranging from 0.01 picomolar (pM) to 100 pM.
10. The biostimulant and bioprotective composition of claim 8, further comprising a microorganism selected from the group consisting of mycorrhizal fungi, saprophytic fungi, plant growth promoting bacteria, Bacillus thuringiensis spores, and any combination thereof.
11. The biostimulant and bioprotective composition according to of claim 8, which wherein the biostimulant and bioprotective composition is a water-based liquid composition.
12. The biostimulant and bioprotective composition of claim 8, wherein the biostimulant and bioprotective composition is in lyophilized form.
13. A method for increasing resistance to biotic and/or abiotic stress in a plant, comprising applying a biostimulant and bioprotective composition according to claim 8 to the plant, parts of the plant, plant propagation material, and/or plant growth site.
14. The method of claim 13, wherein the biostimulant and bioprotective composition is applied by spraying, irrigation or is supplied in a hydroponic solution.
15. The method of claim 13, wherein the plant is a plant belonging to the Solanaceae, Vitaceae, Rosaceae or Oleaceae family.
16. A method for increasing resistance to abiotic and/or biotic stress in a plant, the method comprising applying to the plant the isolated peptide of claim 1.
Description
[0120] The experimental section that follows is provided for illustration purposes only and does not limit the scope of the invention as defined in the appended claims. In the experimental section, reference is made to the accompanying drawings, wherein:
[0121] FIG. 1 shows a schematic representation of the cloning vector pETM11 used by the present inventors for the recombinant production of the peptides of the invention.
[0122] FIG. 2 shows a schematic representation of the insert containing the nucleotide sequence encoding the 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 the peptide PS1-70 (SEQ ID NO. 10), correctly inserted in the cloning vector, is underlined in black; the sequence encoding for 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), placed downstream of the histidine tail to allow the removal of the latter, is highlighted in uppercase italics underlined in grey. The sequences of the forward primer P11F1 (SEQ ID NO. 17) and of the reverse primer P11R1 (SEQ ID NO. 18) used for amplifying and cloning the nucleotide sequence encoding the peptide PS1-70 are highlighted in light grey and dark grey, respectively. B) Amino acid sequence of the peptide PS1-70 (in bold, SEQ ID NO. 1), conjugated at the N-terminal position to the C-terminal end of a histidine tail (underlined sequence, SEQ ID NO. 15).
[0123] FIG. 3 shows a schematic representation of the insert containing the nucleotide sequence encoding the peptide PS1-120 after cloning into the vector pETM11. (A) Sequence of the recombinant insert obtained by Sanger sequencing (SEQ ID NO. 13). The nucleotide sequence encoding the peptide PS1-120 (SEQ ID NO. 11), correctly inserted in the cloning vector, is underlined in black; the sequence encoding for 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 allow the removal of the latter, is highlighted in uppercase italics underlined in grey. The sequences of the forward primer P11F1 (SEQ ID NO. 17) and of the reverse primer P11R3 (SEQ ID NO. 19) used for amplifying and cloning the nucleotide sequence encoding the peptide PS1-120 are highlighted in light grey and dark grey, respectively. (B) Amino acid sequence of the peptide PS1-120 (in bold, SEQ ID NO. 2), conjugated at the N-terminal position to the C-terminal end of a histidine tail (underlined sequence, SEQ ID NO. 15).
[0124] FIG. 4 is a table showing the nucleotide sequences and characteristics of the primers used for amplifying and cloning the nucleotide sequences encoding the peptides PS1-70 and PS1-120, respectively.
[0125] FIG. 5 shows the results of the affinity chromatography (IMAC), of the analysis of the eluted fractions by electrophoresis with 15% SDS-PAGE and of the Western blot analysis performed on the purified PS1-70 and PS1-120 peptides. (A1 and B1) Chromatographic profiles of the first step of purification of the peptides PS1-70 and PS1-120, which elute with 150 mM and 50 mM imidazole, respectively. (A2 and B2) Polyacrylamide gel analysis of the eluted fractions; M: molecular weight marker; black rectangle: eluted fractions containing the peptides PS1-70 and PS1-120. (A3 and B3) Identification of the peptides PS1-70 and PS1-120 by Western Blot analysis; M: molecular weight marker; black rectangle: PS1-70 and PS1-120 peptides.
[0126] FIG. 6 shows the results of the size exclusion chromatography (SEC) and of the analysis of the eluted fractions by electrophoresis with 15% SDS-PAGE. (A1 and B1) Chromatographic profiles of the second step of purification of the peptides PS1-70 and PS1-120 which have their elution peak at an elution volume of 12.16 ml and 10.92 ml, respectively. (A2 and B2) Polyacrylamide gel analysis of the eluted fractions; M: molecular weight marker; black rectangle: eluted fractions containing the peptides PS1-70 and PS1-120. (A3 and B3) Deconvoluted mass of the peptides PS1-70 and PS1-120.
[0127] FIG. 7 shows the profiles of the amino acid composition of the peptides PS1-70 (A) and PS1-120 (B). The graphs of the figure indicate that the amino acid sequences of said peptides both contain a significant representation of amino acids promoting structural disorder (dark grey) compared to amino acids promoting an ordered secondary structure (light grey).
[0128] In FIG. 8, graphs A and B illustrate the results of the Light Scattering experiments carried out by SEC-MALS-QELS on the peptides PS1-70 (A) and PS1-120 (B) at pH 8.0 as described in Examples 1 and 2. The peaks of the curves are representative of monomeric proteins in solution. FIG. 8 (C,D) shows the dichroic spectra of the purified PS1-70 (C) and PS1-120 (D) peptides recorded at the temperature of 20° C. using the peptides PS1-70 and PS1-120 at concentrations of 4.4 μM and 3.5 μM, respectively, in 10 mM phosphate buffer. The abscissa axis shows the wavelength (nm), the ordinate axis shows the mean residue molar ellipticity value.
[0129] FIG. 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. The analysis was carried out on the Lox C, AOS, Pin I and Pin II genes. The letters a, b, c indicate the statistical significance of the data (ANOVA), each letter representing a statistical group.
[0130] FIG. 10 illustrates the effects of the treatment of tomato plant leaves with the peptides PS1-70 and PS1-120 on S. littoralis lepidopteran larvae. The histograms (A,C) show the change in average weight, expressed in grams, of larvae fed with leaves treated with the peptides PS1-70 and PS1-120 at 100 pM and 100 fM, respectively, and the related controls, measured on several subsequent days. The letters a, b indicate the statistical significance of the data (ANOVA), each letter representing a statistical group. The graphs (B,D) show the mortality rate recorded each day for larvae fed with tomato plant leaves treated with the peptides of the invention and the related control (Log-Rank test; ****p<0.0001).
[0131] FIG. 11 illustrates the effects of the treatment of tomato plant leaves with the peptides PS1-70 and SEQ ID NO. 3, 5 and 8 on S. littoralis lepidopteran larvae. The histogram (A) shows the change in average weight, expressed in grams, of larvae fed with leaves treated with the above mentioned peptides and the related control, measured on days 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19. The letters a, b indicate the statistical significance of the data (ANOVA), each letter representing a statistical group. The graph (B) shows the daily mortality rate recorded for larvae fed with tomato plant leaves treated with the peptides of the invention and the related control (Log-Rank test; ****p<0.0001).
[0132] FIG. 12 shows the decrease in the necrosis areas generated by the necrotrophic B. cinerea fungus on tomato plant leaves (A,B), eggplant leaves (C) and vine plant leaves (D) after treatment with the PS1-70 and PS1-120 peptides, compared to untreated controls. The mean necrosis areas were measured 1, 3, 5 and 8 days after inoculation of the pathogen. The letters a, b, c, d indicate the statistical significance of the data (ANOVA), each letter representing a statistical group.
[0133] FIG. 13 shows the decrease in the necrotic areas generated by the necrotrophic B. cinerea fungus on tomato plant leaves after treatment with PS1-70, SEQ ID NO. 3, 5 and 8 compared to the untreated control. The mean necrosis areas were measured 1, 3, 5 and 8 days after inoculation of the pathogen. The letters a, b, c, d indicate the statistical significance of the data (ANOVA), each letter representing a statistical group.
[0134] FIG. 14 shows the decrease in the necrosis areas generated by the necrotrophic A. 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, 5 and 8 (B), compared to the untreated control. The mean necrosis areas were measured 1, 3, 5 and 8 days after inoculation of the pathogen. The letters a, b indicate the statistical significance of the data (ANOVA), each letter representing a statistical group.
[0135] FIG. 15 shows the effects of the treatment with the peptides PS1-70, PS1-120 and Systemin (Sys) in combination with Trichoderma harzianum T22 strain spores on 4-week plants born from seeds co-infected with Trichoderma T22 spores, on the survival of S. littoralis lepidopteran larvae (A1, A2 and A3), and on leaf colonization by necrotrophic B. cinerea (B1) and A. alternata (B2) fungi. Larval survival was measured daily (Log-Rank test; ****p<0.0001), each letter representing a statistical group. The mean necrosis areas were measured 1, 3, 5 and 8 days after inoculation of the pathogens. The letters a, b, c, d, e, f indicate the statistical significance of the data (ANOVA), each letter representing a statistical group.
[0136] FIG. 16 shows the decrease in the necrosis areas generated by the necrotrophic B. cinerea fungus on four-week-old tomato plant leaves born from seeds treated with a 100 fM suspension of the peptides PS1-70 and PS1-120, and the respective control. The mean necrosis areas were measured 1, 3 and 5 days after inoculation of the pathogen. The letters a, b indicate the statistical significance of the data (ANOVA), each letter representing a statistical group.
[0137] FIG. 17 shows the relative quantification of induced gene expression in tomato plants irrigated with the PS1-70 peptide at a concentration of 100 pM, in the absence of salt (A) (0 mM NaCl) and in the presence of salt (B) (80 mM NaCl). The analysis was carried out on the cat1, tft1, Sam, HSFA2, HSP70, HSP90, MPK1 and WRKY40 genes. The asterisks indicate the statistical significance of the data by Student's t-test (*p<0.05; **p<0.01; ***p<0.001).
[0138] FIG. 18 shows the relative quantification of induced gene expression in tomato plants irrigated with the peptides PS1-70, PS1-120 and SEQ ID NO.5 (100 fM) in the absence of salt (0 mM NaCl), in the presence of salt (150 mM NaCl), and related controls. The analysis was carried out on the CAT2(A), SAM (B) and APX2 (C) genes. The asterisks indicate the statistical significance of the data by Student's t-test (*p<0.05; **p<0.01; ***p<0.001).
[0139] FIG. 19 shows the average proline content in plant leaves irrigated with the peptides PS1-70, PS1-120 and SEQ ID NO. 5 (100 fM) in the absence of salt (0 mM NaCl), in the presence of salt (150 mM NaCl), and related controls. The letters a, b, c indicate the statistical significance of the data (ANOVA), each letter representing a statistical group.
[0140] FIG. 20 illustrates the effect of irrigation treatment with the peptides PS1-70, PS1-120 and SEQ ID NO.5 at a concentration of 100 fM on the biometric parameters of the tomato plant. The histogram (A) shows the root area, expressed in square centimetres, of the plants treated with the peptides object of the invention, and the related controls, in the absence of salt (0 mM NaCl) and in the presence of salt (150 mM NaCl), and related controls. The histogram (B) shows the change in fresh weight, expressed in grams, of the aerial part in plants treated with the peptides object of the invention, and the related controls, in the absence of salt (0 mM NaCl). The asterisks indicate the statistical significance of the data by Student's t-test (*p<0.05).
[0141] FIG. 21 shows a table with the results of the assessment of the direct toxic effect of the peptides of the invention assayed at increasing concentrations on S. littoralis larvae, as shown in Example 4. The survival rate was recorded up to the chrysalis stage for the larvae in which the peptides PS1-70 and PS1-120 were injected or applied to the epidermis.
[0142] FIG. 22 shows the assessment of the direct toxic effect of the peptides of the invention assayed at increasing concentrations when added to the growth medium of two different fungi: B. cinerea, (A) PS1-70 and PS1-120 and (B) PS1-70, SEQ ID NO. 3, 5 and 8, and Trichoderma T22, (C) PS1-70 and PS1-120. The growth of the fungus was measured 24 hours after the addition of the peptides of the invention as the turbidity level of the medium (absorbance at 600 nm). The letters a, b indicate the statistical significance of the data (ANOVA), each letter representing a statistical group.
Example 1: Production of the Peptides According to the Invention
[0143] Cloning, Expression and Purification
[0144] In order to isolate the nucleotide sequences encoding the peptides of the invention, PCR reactions were set up using the amplification primer pairs whose sequences are shown in the table of FIG. 4 and using the cDNA encoding for full-length Prosystemin as a template.
[0145] Amplicons were digested using NcoI and XhoI restriction enzymes, and subsequently cloned into the pETM11 vector previously digested with the same enzymes. pETM11 (courtesy of EMBL, Heidelberg) is a procaryotic expression vector, which is capable of adding a tail of six histidines (His-tag) at the amino(N-)terminal portion 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 (FIG. 1).
[0146] The integrity of the cloned fragments and the absence of possible mutations occurred during the amplification reaction were confirmed by sequencing the obtained constructs (FIGS. 2A and 3A). After an initial screening, large-scale expression of the peptides of the invention PS1-70 and PS1-120 was carried out in Escherichia coli BL21(DE3) strain for 16 hours at 22° C. in the presence of 2 mM IPTG in LB and 2-YT culture media. FIGS. 2B and 3B, 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, each conjugated at the N-terminal end to the C-terminal end of a histidine tail, highlighted with underscores.
[0147] Purification of peptides after expression was performed at room temperature on FPLC-ÄKTA (GE Healthcare) by affinity chromatography (IMAC) (FIG. 5) and size exclusion chromatography (SEC) (FIG. 6) obtaining yields of 2 mg/L cell culture.
[0148] Peptide Synthesis
[0149] The peptides of the invention having the sequences SEQ ID NO: 3, 5 and 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 procedure involved the use of a resin, which made it possible to obtain peptides modified by amidation at the carboxy-terminal end. At the end of the synthesis, the amino-terminal end of the peptides was also modified by acetylation. Purifications were carried out by reverse phase HPLC.
Example 2: Analysis of the Structure of the Peptides According to the Invention
[0150] During the procedure for the identification of the peptides of the invention, the present inventors encountered considerable difficulties due to the peculiar characteristics of the PS1-70 and PS1-120 peptides, primarily the significant presence in their primary sequence of amino acid residues promoting structural disorder (FIG. 7). Both peptides PS1-70 and PS1-120 exhibited aberrant migration when subjected to SDS-PAGE electrophoresis, migrating with an apparent molecular weight of 20-25 kDa relative to their actual weight (MW PS1-70=11 kDa; MW PS1-120=17 kDa) and again, mass spectrometry confirmed the exact molecular weight of the two recombinant peptides (FIGS. 6, A3 and B3).
[0151] Furthermore, during size exclusion chromatography (SEC), the peptides PS1-70 and PS1-120 showed a retention volume of 12.16 ml and 10.92 ml, respectively (FIG. 6), indicative of an oligomer or a poorly compact protein. Light Scattering experiments carried out by SEC-MALS-QELS showed that, regardless of the retention volume, the peptides of the invention are present in solution as monodispersed monomeric proteins having molecular weights of 9.36±0.6 kDa for PS1-70 and 19.98±1.5 kDa for PS1-120, respectively, consistent with the theoretical ones (FIG. 8A, 8B).
[0152] The secondary structure of the peptides PS1-70 and PS1-120 was then analysed by circular dichroism (CD). The Far-UV CD spectrum obtained showed negative molar ellipticity values at 198 and 190 nm for both tested peptides. However, the ellipticity values observed at 200 and 222 nm are indicative of some secondary structure (FIG. 8C, 8D). The above characteristics are typical of a disordered protein having large unstructured portions. This is probably related both to the high number of acidic residues (negatively charged at physiological pH) responsible for the intrinsic repulsion, and to the low content of hydrophobic residues which generally assist proteins in folding correctly.
Example 3: Induction of Defence Gene Expression in Plants by the Peptides According to the Invention
[0153] In order to test the biological activity of the peptides of the invention, the present inventors carried out studies to measure the expression of defence genes in tomato plants (Solanum lycopersicum) after application of the peptides PS1-70 and PS1-120 on these plants. Said peptides were assayed at picomolar (pM) and femtomolar (fM) concentrations in 1×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 comprising the peptides on several points on the upper side of expanded leaves of four-week-old tomato plants.
[0154] Leaf samples were taken 6 hours and 24 hours after application of the peptides of the invention to undergo RNA extraction and subsequent gene expression analysis. In particular, four genes known to be related to plant defence were selected and tested: two early-expression genes active in the octadecanoid biosynthesis pathway leading to the formation of Jasmonic Acid (JA), such as the Lipoxygenase C gene (Lox C) and the allene oxide synthase gene (AOS), and two late-expression genes such as the proteinase I inhibitor (Pin I) and proteinase II inhibitor (Pin II) genes.
[0155] All assayed genes were significantly over-expressed following exogenous application of both peptides of the invention at both concentrations tested (FIG. 9).
Example 4: The Peptides According to the Invention Promote Resistance to Biotic Stress in Plants
[0156] In order to demonstrate that the peptides of the invention are capable of promoting the resistance of plants against pathogenic organisms, the present inventors carried out studies for assessing the effects on herbivorous insects or phytopathogenic fungi resulting from the treatment of plants with peptides having the amino acid sequences SEQ ID NO.1 (PS1-70), 2 (PS1-120), 3, 5 and 8. More specifically, the present inventors monitored two different parameters, namely changes in weight gain and survival rate of larvae of Spodoptera littoralis, a lepidopteran that produces considerable damage to tomato plants, and the colonization of the plants by the phytopathogenic necrotrophic Botrytis cinerea fungus, an agent that causes tomato grey mould, and the parasitic Alternaria alternata fungus.
[0157] Experiments on Spodoptera littoralis Larvae
[0158] Briefly, S. littoralis larvae were grown in a climate chamber at 25° C., 70% relative humidity (RH), with a 16-hour light and 8-hour dark photoperiod and fed with an artificial diet until completion of the first moult. For the bioassays, 150 larvae for each thesis were grown on tomato leaves for the entire duration of the second instar to adapt them to the different diet. The newly moulted third-instar larvae up to the moult stage were fed with plants on which a composition comprising peptides PS1-70 and PS1-120 had been applied at 100 pM and 100 fM concentrations in 1×PBS buffer. Larvae fed with plants treated with 1×PBS buffer only were used as a control. Bioassays were carried out under the same environmental conditions, in 32-well plastic trays containing 1.5% (w/v) agar and 0.005% (w/v) methyl parahydroxybenzoate, which are useful for creating a humid environment for maintaining cell turgor of tomato leaves. Each experimental group consisted of 32 larvae. The survival of the larvae was monitored daily, and their weight every other day. Larvae fed with leaves treated with the peptides of the invention showed weight loss throughout the bioassay period compared to those fed with the control leaves, with a significant difference for both concentrations starting as early as the third day. In particular, on day 15 of the bioassay, for the 100 pM concentration, larvae fed with the control leaves had an average weight of 38 mg, whereas those fed with leaves treated with the peptides PS1-70 and PS1-120 had an average weight of 15 and 20 mg, respectively (FIG. 10A). In the bioassays carried out using 100 fM concentration of the peptides of the invention, on day 13 the larvae fed with the control leaves had an average weight of 44 mg, whereas those fed with leaves treated with the peptides PS1-70 and PS1-120 had an average weight of 11 and 12 mg, respectively (FIG. 10C). A significant decrease in the survival of larvae fed with leaves of plants treated with the peptides of the invention compared to those fed with leaves of the control plants was also observed for both assays (FIGS. 10B and 10D). In fact, on day 15 of the bioassay a survival rate of 96.87% was observed for larvae fed with the control leaves, and survival rates of 34.37% and 31.25%, respectively, were observed for those fed with leaves treated with the peptides of the invention at 100 pM concentrations (FIG. 10B). In the bioassay carried out at the 100 fM concentration, on day 13, a 100% survival rate was observed for larvae fed with the control leaves, and 0% and 21, 87% survival rates, respectively, were observed for those fed with leaves treated with the peptides of the invention (FIG. 10D).
[0159] The same assay scheme was carried out by the present inventors by feeding S. littoralis larvae with leaves treated with the peptide PS1-70 and with peptides having the sequences SEQ ID NO. 3, 5, and 8 at femtomolar concentration. Larvae fed with the treated leaves showed a significant reduction in their weight from day 3 compared to the control leaves (FIG. 11). In particular, on day 13 of the bioassay, larvae fed with the control leaves had an average weight of 59 mg, instead those treated with the peptides PS1-70, SEQ ID NO. 8, 3, and 5 had an average weight of 22 mg, 13 mg, 24 mg and 21 mg, respectively (FIG. 11A). A significant decrease was also observed in the survival of larvae fed with peptide-treated leaves compared to those fed with the control leaves (FIG. 11B). In fact, on day 13 of the bioassay a survival rate of 100% was observed for larvae fed with the control leaves, and survival rates of 31.25%, 15.62%, 43.75% and 15.62%, respectively, were observed for those fed with leaves treated with the peptides PS1-70, SEQ ID NO. 8, SEQ ID NO. 3, and SEQ ID NO. 5 (FIG. 11B).
[0160] Experiments on Botrytis cinerea and Alternaria alternata
[0161] For the performance of assays on the phytopathogenic Botrytis cinerea and Alternaria alternata fungi, the present inventors used spores of this microorganism obtained from cultures on solid PDA (Potato Dextrose Agar) sporulation substrate. The plates were inoculated with 20 μl of a conidial suspension at a concentration of 1×10.sup.6 spores/ml and incubated for 15 days at 25° C. in the presence of diffused light, so as to obtain complete sporulation. The spores were then collected in 5 ml of sterile water and, in order to remove the mycelium, were filtered through glass wool, washed with sterile distilled water and collected by centrifugation at room temperature. The concentration of spores suitable for inoculation (10.sup.5-10.sup.7 spores/ml) was determined by the serial dilution method using a Burker cell counting chamber for spore counting.
[0162] The assay was carried out on a detached leaf by taking a compound leaf for each treated tomato plant and for each control plant. Each leaflet of the compound leaf was marked with 3 markers in order to guide the subsequent application of the spores and detection of the pathogen developing necrotic areas.
[0163] The leaves were treated by applying 2 μl of a composition comprising the peptides PS1-70 and PS1-120 of the invention at a concentration of 100 pM, 100 fM or 1×PBS for the control leaves. After 6 hours, the time necessary for the perception of the peptides, the leaves were detached from the plant and inoculations with 10 μl of spore solution were carried out in the internerve spaces and near the previously marked points. Monitoring was carried out by measuring the necrosis areas (expressed in mm.sup.2) 1, 3, 5, 8 days after inoculation of the pathogen. The necrosis areas recorded on the control leaves were much larger than those detected on plants treated with the peptides of the invention. This difference increased as a function of the time elapsed since inoculation. The resistance inducing effect of the picomolar treatment is already statistically significant from the first day of inoculation of the pathogen up to the eighth day where values of 33 mm.sup.2 were reached on the control leaves, unlike the treated leaves where values not exceeding 18 mm.sup.2 were recorded for both peptides (FIG. 12A).
[0164] Similar results were observed following application of femtomolar concentrations of the peptides object of the invention. In particular, a strong reduction in the development of necrotic areas on the treated leaves compared to those on the control leaves was observed as early as day 1 of inoculation of the phytopathogenic fungus. This reduction was maintained until day 8 where values of 20 mm.sup.2 were reached on the control leaves, and values of 6.69 mm.sup.2 and 5.19 mm.sup.2 were reached on leaves treated with PS1-70 and PS1-120, respectively (FIG. 12B).
[0165] The present inventors also carried out tests aimed at determining the effects of exogenous application of the peptides of the invention, at picomolar and femtomolar concentrations, against the development of the necrotrophic fungus on Solanum melongena (eggplant) plants (FIG. 12C) and Vitis vinifera (vine) plants (FIG. 12D).
[0166] As shown in FIG. 12C, the tests performed showed a significant reduction in fungal colonization of the treated plants compared to the control plants, and the most marked effect was observed following treatment with the lowest concentration of the peptides of the invention. The positive effect of the treatment was statistically significant from day 3 of inoculation of the pathogen to day 8 where values of 11 mm.sup.2 were reached on the control leaves, unlike the treated leaves where values of 8.4 and 6.0 mm.sup.2 were recorded on leaves treated with the peptides PS1-70 and PS1-120 at a concentration of 100 pM, and values of 6.2 and 4.10 mm.sup.2 were recorded on leaves treated with said peptides at a concentration of 100 fM (FIG. 12C). Similar results were obtained on vine plants (FIG. 12D). The positive effect of the treatment was already statistically significant from day 1 of inoculation of the pathogen to day 8 where values of 43 mm.sup.2 were reached on the control leaves, and values of 16.7 and 13.6 mm.sup.2, respectively, were reached on leaves treated with the peptides PS1-70 and PS1-120 at a concentration of 100 pM, and values of 12 and 11.8 mm.sup.2 were reached on leaves treated with said peptides at a concentration of 100 fM (FIG. 12D). Similar results were obtained on olive trees.
[0167] The effect of application of femtomolar concentrations of the peptides having the sequences SEQ ID NO. 3, 5 and 8 on tomato leaves was also assessed against the development of the B. cinerea fungus.
[0168] As shown in FIG. 13, the necrosis areas recorded on the control leaves were much larger than those detected on plants treated with the peptides of the invention. The positive effect of the treatment was already statistically significant from day 1 of inoculation of the pathogen and continued until day 8 where values of 11.72 mm.sup.2 were reached on the control leaves, and values of 7.69 mm.sup.2, 7.67 mm.sup.2, 8.89 mm.sup.2 and 6.81 mm.sup.2 were reached on leaves treated with the peptides PS1-70, SEQ ID NO.8, SEQ ID NO.3, SEQ ID NO.5, respectively, at a concentration of 100 fM (FIG. 13).
[0169] The present inventors also performed assays aimed at determining the effects of exogenous application of the peptides of the invention on tomato plants against the development of the necrotrophic Alternaria alternata fungus (FIG. 14).
[0170] As shown in FIGS. 14A and 14B, the assays performed showed a significant reduction in fungal colonization of the treated plants compared to the control plants. The positive effect of the treatment was statistically significant from day 1 of inoculation of the pathogen to day 8 where values of 24 mm.sup.2 were reached on the control leaves, unlike the treated leaves where values of 8.4 and 8.0 mm.sup.2 were recorded on leaves treated with the peptides PS1-70 and PS1-120 at a concentration of 100 fM (FIG. 14A); and values of 17 mm.sup.2 were recorded on the control leaves and values of 8.3 mm.sup.2, 7.9 mm.sup.2, 8.9 mm.sup.2, and 7.8 mm.sup.2 were recorded on leaves treated with the peptides PS1-70, SEQ ID NO.8, SEQ ID NO.3 and SEQ ID NO.5, respectively, at a concentration of 100 fM (FIG. 14B).
[0171] Activity of the Peptides According to the Invention in Combination with Trichoderma T22 Spores
[0172] Further studies were carried out by the present inventors in order to determine the effects on tomato plants of treatment with the peptides of the invention, at femtomolar concentrations, in combination with Trichoderma T22 spores in reducing the survival rate of S. littoralis larvae (A1, A2, A3) and the development of the necrotrophic B. cinerea (B1) and A. alternata (B2) fungi (FIG. 15).
[0173] Tomato seeds were treated with a suspension of Trichoderma harzianum T22 strain spores (1×10.sup.7 spores/ml) or with water for the controls, allowed to dry and germinated in the dark on sterile adsorbent paper at 24° C. 4-week-old plant leaves were treated by applying 2 μl of a composition comprising the peptides Systemin (Sys), PS1-70 or PS1-120 at a concentration of 100 fM or 1×PBS on the control leaves. After 6 hours, the time necessary for the perception of the peptides, the leaves were detached from the plant to carry out both the assay with the S. littoralis larvae and the assay with the two necrotrophic fungi B. cinerea and A. alternata.
[0174] For the assay with the S. littoralis larvae, the survival of the larvae was monitored daily. To monitor the development of the two phytopathogenic fungi, on the other hand, measurements of the necrosis areas (expressed in mm.sup.2) were made 1, 3, 5, 8 days after the inoculation of 10 μl of spore solution (1×10.sup.6 spores/ml) on the previously treated tomato leaves.
[0175] FIG. 15 shows that treatment with the tested peptides in combination with Trichoderma T22 produces a surprising synergistic effect in counteracting the development and survival of the larvae and a significantly higher protection against the colonization by the two phytopathogenic fungi compared to treatments with Trichoderma T22 alone or with the peptides alone. The evidence of these effects increases as a function of time. Moreover, the effects produced by the peptides of the invention, used individually or in combination with the Trichoderma T22 spores, have been shown to be superior compared to the peptide systemin.
[0176] Seed Protective Effect of the Peptides According to the Invention
[0177] The present inventors also assessed the protective effect conferred by the treatment with the peptides object of the invention directly on the seed. Tomato seeds were treated with a composition comprising the peptides at a concentration of 100 fM or with 1×PBS for the controls, allowed to dry and germinated in the dark on sterile adsorbent paper at 24° C. 4-week-old plant leaves were detached from the plant and inoculated with 10 μl of spore solution in the internerve spaces and near the previously marked points. As shown in FIG. 16, the assay showed a significant reduction in fungal colonization on the leaves of plants born from seeds treated with PS1-70 and PS1-120 compared to control plants.
[0178] The positive effect of the treatment was statistically significant from day 1 of inoculation of the pathogen to day 8 where values of 11 mm.sup.2 were reached on the control leaves, and values of 7.1 and 7.4 mm.sup.2 were reached on leaves from plants born from seeds treated with the peptides PS1-70 and PS1-120 at a concentration of 100 fM (FIG. 16). Further studies were carried out by the present inventors in order to investigate the presence of any direct toxic effect of the peptides PS1-70 and PS1-120 on the tested pathogenic organisms. As can be seen from the data reported in the table in FIG. 21, the administration of the peptides of the invention orally or by injection in the epidermis of S. littoralis larvae had no effect on their survival and development.
[0179] Furthermore, as shown in FIGS. 22A and 22B, the growth of the B. cinerea fungus was not disrupted when the growth medium was added with the peptides PS1-70 and PS1-120 of the invention. Further studies were carried out on Trichoderma harzianum T22 strain and again, as shown in FIG. 22C, the addition of the peptides PS1-70 and PS1-120, at both the 100 pM and 100 fM concentrations, had no effect on the growth of the fungus. Instead, an increase in the growth of the T22 fungus was observed when the peptide PS1-70 was added to the growth medium, suggesting the possibility that the fungus uses this protein as a source of amino acids.
[0180] In summary, the experimental results described above show that the peptides object of the present invention are biologically active, and that exogenous application thereof promotes resistance to noxious insects and fungi in plants.
Example 5: The Peptides According to the Invention Promote Resistance to Abiotic Stress in Plants
[0181] During their studies, the present inventors have also set up experiments aimed at verifying the efficacy of the peptides of the invention in promoting the resistance of plants against various abiotic stresses.
[0182] Through the experiments carried out, the inventors first found that the application of the peptides of the invention on tomato plants causes an increase in their biomass and favours the production of larger berries with a greater number of seeds. The results of subsequent experiments, as shown in FIG. 17, showed that the application of the peptide PS1-70 protects the tomato plant from salt stress. Briefly, 48 hours after irrigation with the PS1-70 peptide at a concentration of 100 pM, the treated plants, unlike the control plants, were able to activate a series of genes and transcription factors responsive to salt stress, thereby demonstrating that the peptide of the invention is capable of stimulating an alert state in the plant, known as priming. Moreover, in the presence of moderate salt stress (80 mM NaCl), tomato plants treated with the peptide of the invention were more tolerant to salt than the untreated control plants, showing in particular a greater induction of genes responsive to this stress (FIG. 17B). An experiment was also set up to verify the efficiency of the peptides PS1-70, PS1-120 and SEQ ID NO.5, when administered at femtomolar concentrations, in promoting tolerance to a high salt stress condition. As shown in FIG. 18, 8 days after irrigation with the peptides PS1-70, PS1-120 and SEQ ID NO.5, the treated plants, unlike the control plants, were able to activate a series of salt stress-responsive genes, thus confirming that the peptides of the invention are capable of stimulating the priming defence state even at femtomolar concentrations (FIGS. 18A1, 18B1 and 18C1). Moreover, tomato plants treated with the above peptides and 24 hours later irrigated for 7 days at a high salt stress level (150 mM NaCl) were more salt-tolerant than the untreated control plants, showing in particular a greater induction of the same genes responsive to this stress (FIGS. 18A2, 18B2 and 18C2).
[0183] The average proline content was also assessed in these plants. Proline is an osmoprotector produced in the free form in the plant cell in response to salt stress and water deficiency. In plants subjected to salt stress, this amino acid, in fact, participates in the regulation of the osmotic potential, in the protection of membranes from free radicals, in the regulation of the cytoplasmic pH, and in the protection of enzymes from denaturation.
[0184] As shown in FIG. 19, plants treated with the peptides of the invention in the presence of high salt stress (150 mM NaCl) exhibit a lower average proline content than the salinized control plants, thus demonstrating their lower perception of the osmotic stress caused by the salt.
[0185] A few biometric parameters are shown in FIG. 20, measured after 14 days of continuous salt stress (150 mM) and 15 days after irrigation with the peptides PS1-70, PS1-120 and SEQ ID NO.5 at 100 fM concentrations, and the respective controls. FIG. 20A shows that peptide-treated plants exhibited greater tolerance to stress than untreated control plants, in particular with a greater root surface (FIG. 20A). Furthermore, in the absence of salt, the plants treated with the peptides object of the invention, unlike the control plants, are able to promote the growth of the aerial part of the plant, thus confirming that the peptides of the invention have a biostimulant effect even at femtomolar concentrations (FIG. 20B).
