WO2019116203A1

WO2019116203A1 - Peptides with fungicidal activity, their compositions and related uses in agronomic field

Info

Publication number: WO2019116203A1
Application number: PCT/IB2018/059834
Authority: WO
Inventors: Paolo PESARESI; Simona Masiero; Chiara MIZZOTTI; Luca TADINI; Sara PELLEGRINO; Monica Colombo; Silvia VEZZULLI; Michele PERAZZOLI; Riccardo Velasco
Original assignee: Universita' Degli Studi Di Milano; Fondazione Cassa Di Risparmio Delle Province Lombarde; Fondazione Edmund Mach
Priority date: 2017-12-11
Filing date: 2018-12-10
Publication date: 2019-06-20

Abstract

The present invention relates to new peptides with antimicrobial and fungicidal activity, the related phytopharmaceutical compositions and in particular their use for the control of Plasmopara viticola in viticulture.

Description

“Peptides with fungicidal activity, their compositions and related uses in agronomic field”

The present invention relates to new peptides with antimicrobial and fungicidal activity, the related phytopharmaceutical compositions and in particular their use for the control of Plasmopara viticola in viticulture.

Plant diseases caused by pathogenic agents such as viruses, bacteria and fungi are responsible for agricultural production losses quantifiable around 16% at the global level and can also influence the quality and safety of foods [1] In particular, fungicides constitute most of the phytosanitary products used in agriculture and viticulture is one of the leading sectors in terms of use of fungicides. It is estimated that, in Europe, 68,000 tonnes/year of fungicides are used to control grapevine diseases, accounting for 65% of the sum of the fungicides used in agriculture (Eurostat report, 2007).

The Oomycete Plasmopara viticola , obligate biotroph, is the causal agent of peronospora, one of the most severe grapevine diseases in the world [2].

Among the most common symptoms of the diseases are stem necrosis, grape withering and leaf discoloration. Severe attack generally determine the early fall of the leaves (phyloptosis). In the absence of treatments and in the presence of favourable meteorological conditions, peronospora can destroy up to 75% of the harvest in a single season and can weaken the new buds, causing loss of vigour and reduced production even in subsequent years, with consequent severe economic losses [3].

Currently, grapevine peronospora is controlled by frequent applications of fungicides, such as copper in the form of soluble salts (sulphate, Bordeaux mixture, etc.) or synthetic active ingredients.

However, the efficiency of these treatments, and in general of treatments with phytosanitary products, is very low; it is estimated that less than 0.1% of the active ingredient applied to crops is actually able to impact the designated pathogen [4]. The remaining part accumulates in the soil and from there, by washout of the contaminated grounds, can reach and pollute surface water and underground aquifers, becoming a hazard for fresh water organisms and human beings. Fungicides accumulated in soils can damage arthropods, earthworms, fungi, bacteria, protozoa and in general all the organisms that contribute to the function and to the structure of the soils. The chronic exposure to phytosanitary products of useful insects, like bees, and of wild birds can cause reductions in their reproductive capacity, with consequences at the level of species and ecosystem, while exposure to high concentrations can even cause the death of single individuals. Pets can also be influenced by exposure to phytosanitary products. Some active ingredients have poor degradability and hence they remain in the environment for a long time. For example, organochlorine insecticides, such as DDT, were identified in the surface water of the United States 20 years after their use had been forbidden. Moreover, the phytosanitary products that enter the food chain can experience the phenomenon of biomagnification. This term means the tendency of some chemical substances to become ever more concentrated the farther up one rises in the trophic chains. As a consequence, the concentrations accumulated in the tissues of organisms can be many times higher relative to the surrounding environment.

This is a partial list of the negative effects that agricultural technologies and practices can have on the ecosystem and that justify the need to develop new antimicrobial compounds that can be used in agriculture for control of vegetable diseases, characterized by law toxicity and a reduced environmental impact (Commission Regulation (EC) No 473/2002 of 15 March 2002).

The authors of the present invention have now identified a family of peptides characterized by a primary sequence of eight amino acids able to interact with the catalytic domain of cellulose synthase and to inhibit the activity of the enzyme. In particular, the inventors have verified that the peptides according to the invention are able to contrast the growth of Plasmopara viticola on grapevine, with improved properties of activity, specificity, biodegradability, toxicity and with relatively low production costs thanks to its small dimensions [5].

The peptides of the invention can advantageously be employed in the eradication and prevention of peronospora in different crops, in particular in grapevines, and they represent a valid alternative to copper-based preparations because: a) they are highly effective, as the minimum inhibitory concentration (MIC), in vitro , is around 20-50 mM;

b) they are not cytotoxic for human cells, as demonstrated by the assay for the determination of cell viability (MTT assay);

c) they are specific for Plasmopara viticola, while they are innocuous on non-target organisms, such as the soil bacteria Agrobacterium tumefaciens and Bacillus amyloliquefaciens and the Ascomycete Erysiphe necator (or Uncinula necator), causal agent of grapevine oidium;

d) they have a low cost of production, due to the small dimensions (eight amino acid residues) [5].

Moreover, the peptides according to the invention are sustainable fungicides that can sustain a reduction in use, or even the replacement, of traditional copper-based fungicides, as required by Commission Regulation (EC) no. 473/2002, thus promoting the transition towards green and sustainable agriculture.

Therefore, the present invention relates to a peptide characterized by a length sequence of 8 amino acids having the following sequence (I):

(X)„- R - (Y)_{m -} R -(Zi)_p (I)

wherein m is an integer number between 1-6;

n and p are each one an integer number between 0-5;

with m+n+p =6;

R is Arg;

wherein X, Y, Z are L-aminoacids selected from the group consisting of Leu (L), Thr (T), Ala (A), Cys (C), Gln (Q),

with the provision that the aminoacid Leu (L) is present twice and the aminoacids Thr (T), Ala (A), Cys (C), Gln (Q), are present once in the sequence of formula (I). According to a preferred embodiment of the invention the peptide of formula (I) is characterized by an amino acid sequence such that:

n= 0;

m= 5;

p = l; Yi = L; Y₂ = T; Y_{3 =} A; Y₄ = Q; Y₅ = C

Zi = L

or it is characterized by the amino acid sequence RLTAQCRL (SEQ ID NO:l). According to an alternative preferred embodiment of the invention the peptide of formula (I) is characterized by an amino acid sequence such that:

n= 2

m= 1

P = 3

X_{1 =} L; X_{2 =} C

Yi = L

Zi = A; Z₂ = T; Z₃= Q

or it is characterised by the amino acid sequence LCRLRATQ (SEQ ID NO:2).

The peptides according to the invention can be prepared by Fmoc solid-phase peptide synthesis or by recombinant expression. In particular, the nucleotide sequence for the expression of the octapeptide having SEQ ID No: 1 is as follows: 5'-CGTCTGACGGCGCAGTGTCGTCTT-3' (SEQ ID NO:6).

Instead for the peptide having SEQ ID NO:2 it is possible to use the nucleotide sequence 5'-CTG TGT CGT CTT CGT GCG ACG CAG-3' (SEQ ID NO:7).

The present invention also contemplates the peptidomimetic variants of said peptides, i.e. molecules that, while maintaining the structure and the bioactivity of the original molecule, exhibit greater stability (given by better resistance to proteolysis and/or to both physical and chemical degradation). These variants derive from the incorporation of amino acid residues not present in nature (D-amino acids) instead of L- amino acids and/or non-proteino genic amino acids.

By way of example, peptide fluorination is an effective strategy to improve the stability of the peptides of the invention with respect to enzymatic, chemical and thermal denaturation. Fluoroalkyl groups increase local hydrophobicity and facilitate membrane traversing. For practical uses in agriculture it is often preferable to use fungicide compositions containing the appropriately formulated active ingredients.

Therefore, further objects of the present invention are a phytopharmaceutical composition comprising at least one peptide according to the invention, a solid or liquid solvent and/or diluent, possibly adjuvants and/or co-formulants of various nature.

According to a preferred embodiment of the present invention, the aforesaid phytopharmaceutical composition can comprise one or more further active ingredients such as fungicides other than the peptide, selected from the group consisting of phytoregulators, antibiotics, herbicides, insecticides, fertilizers and/or mixtures thereof.

The aforesaid phytopharmaceutical compositions can be in solid form (such as granules, granules dispersible in water, dry powders etc.) of in liquid form (for example solutions, suspensions, emulsifiable concentrates, emulsions, microemulsions etc.): the selection of the type of composition will depend on the specific use.

The total concentration of the active peptide in the aforesaid compositions can vary within a broad range; in general, it varies from 1% to 90% by weight relative to the total weight of the composition, preferably from 5% to 50% by weight relative to the total weight of the composition.

The application of these phytopharmaceutical compositions can take place on each part of the plant, for example on leaves, stems, branches and roots, or on the seeds themselves before sowing, or on the soil in which the plant grows.

According to a preferred embodiment of the present invention, the compositions are in spray formulation.

A further object of the present invention, therefore, is the use of the peptides according to the invention or of the fungicidal compositions comprising at least one peptide of the invention for control of phytopathogenic fungi (Oomycetes class) in agricultural crops.

Thanks to their capability for specific interaction with the catalytic domain of the cellulose synthase enzyme the peptides according to the invention are able to perform a preventive fungicidal action and exhibit very low or zero toxicity on the treated crops.

Examples of phytopathogenic fungi that can be effectively treated and combated with the peptides according to the invention belong to the Oomycetes class and are selected from the group consisting of Plasmopara viticola, Peronospora spp, Phytophtora spp. (e.g. Phytophthora nicotianae, Phytophthora infestans, Phytophthora ramorum, Phytophthora sojae ), Pseudoperonospora cubensis and Bremia lactucae.

The main crops that can be protected with the compounds according to the present invention comprise fruit-bearing plants (e.g. grapevine), citrus trees (e.g. orange, lemon, tangerine, grapefruit), leguminous plants (e.g. bean, pea, alfalfa, clover, soy), vegetables (e.g. lettuce, onion, tomato, potatoes, eggplant, pepper), cucurbits (e.g. pumpkin, zucchini, cucumber, cantaloupe, watermelon), tobacco, coffee, tea, cocoa, sugar beet, sugar cane or cotton.

According to a particularly preferred embodiment of the present invention, the peptides described above have been found to be considerably effective in the control of Plasmopara viticola on grapevine.

The peptides of the invention can also be used in the control of Phytophtora infestans on tomato and potato; of Bremia lactucae on lettuce; of Phytophthora parasitica on pepper, eggplant, onion, citrus trees or Phytophthora nicotianae on tobacco or cotton; of Phytophthora sojae on leguminous plants; of Pseudoperonospora cubensis on cucurbits.

A further object of the present invention is a method for controlling phytopathogenic fungi in agricultural crops, which consists of applying on any part of the plants to be protected or on the soil effective, non-phytotoxic doses of compositions comprising the peptide according to the invention.

A further object of the present invention is then a method for controlling phytopathogenic fungi in agricultural crops, which consist of applying effective doses of the peptides according to the invention, used as such or formulated in fungicidal compositions as described above. The effective dose in the aforesaid compositions can vary within a broad range; in general, it varies from 1% to 90% by weight relative to the total weight of the composition, preferably from 5% to 50% by weight relative to the total weight of the composition.

The application of these compositions can take place on each part of the plant, for example on leaves, stems, branches and roots, or on the seeds themselves before sowing, or on the soil in which the plant grows.

The quantity of compound to be applied to obtain the desired effect can vary according to different factors such as, for example, the compounds used, the crop to be preserved, the type of pathogen, the degree of infection, the climatic conditions, the method of application, the adopted formulation.

Doses of compounds between 10 g and 5 kg per hectare of agriculture crop generally provide sufficient control.

The present invention will now be described, for non-limiting illustrative purposes, according to a preferred embodiment thereof, with particular reference to the attached figures, wherein:

- Figure 1 shows the amino acid sequence of cellulose synthase 2 of P. viticola (PvCesA2). The catalytic domain used in the yeast two-hybrid assay is underlined.

- Figure 2 shows the fungicidal activity of the peptide NoPvl co-inoculated together with P. viticola on grapevine leaves; dpi, days post infection.

- Figure 3 shows the images of grapevine foliar disks inoculated with P. viticola in the presence or absence of the peptide NoPvl. (a-c) As control, the sporangia of P. viticola were resuspended in water and used to infect the grapevine foliar disks (5 drops for each foliar disk). In these conditions, the sporulation of P. viticola is observable starting from 5 days from inoculation (b-d) The same sporangia of P. viticola were mixed with the peptide NoPvl (200 mM) and then used to infect the grapevine foliar disks. In this case, the presence of NoPvl was able to inhibit completely the growth of P. viticola without causing any damage to foliar tissues. The images refer to 7 days post infection.

- Figure 4 shows the fungicidal activity of the peptide NoPvl administered before infection with P. viticola (pre-inoculation).

- Figure 5 shows the comparison between the fungicidal activity of the peptide NoPvl with two fungicides currently on the market: Kocide 2000^®, based on Copper and Pergado^®. The fungicides were administered with the Potter spray Tower before the inoculation of P. viticola. The data refer to 5 and 7 days post infection.

- Figure 6 shows the fungicidal activity of the peptide NoPvl on greenhouse- cultivated grapevine plants. The peptide was administered to the leaves by spray a day before infection with P. viticola and the severity of the infection was assessed 7 days from inoculation. The data clearly indicate that NoPvl is also effective in greenhouse conditions.

- Figure 7 shows the bactericidal/bacteriostatic activity of NoPvl verified on Agrobacterium tumefaciens and Bacillus amyloliquefaciens. NoPvl was used to verify its impact on the growth of soil bacteria such as Agrobacterium tumefaciens (a) and Bacillus amyloliquefaciens (b). Bacterial growth was analysed for 5 hours in the presence of NoPvl (100 mM and 200 Mm) and in its absence (Control). The optical density measurements at 600 nm were carried out every hour. The charts clearly show that NoPvl have no inhibitory effect on bacterial growth.

- Figure 8 shows the biological activity of NoPvl on Phytophthora infestans and Erysiphe necator. (a) Alignment of the amino acid sequence of the PvCesA2 domain, used in the yeast two-hybrid assay, with the homologue of P. infestans (PiCES2). (b) The ability of NoPvl of inhibiting the mycelial growth of P. infestans at 18 °C was verified in vitro through the addition of NoPvl to the culture medium and the measurement of the diameter of the colony of the oomycete at 4, 5, 7 and 11 days post inoculation. The images refer to 7 days post inoculation (c) Young grapevine leaves were treated with NoPvl (200 and 400 pM) and inoculated with oidium spores. The high level of infection, observed 14 days post infection, indicates that NoPvl does not alter the growth of Erysiphe necator , even at high concentrations. The images refer to 14 dpi.

- Figure 9 shows the MTT assay to assess the potential cytotoxicity of NoPvl. Immortalised human cells (HKC8), cultivated in the medium DMEM-F12 at different densities (1000, 3000 and 6000 per 100 pl), were grown in the presence of 400 pM NoPvl and in its absence (Control) for 24 and 48 hours. The results obtain demonstrate the absence of any effect of NoPvl on the growth and on the vitality of the cells in the culture.

- Figure 10 shows the fungicidal activity of the“scramble” peptide of NoPvl (NoPvl sc) on P. viticola. (a) the activity of the peptide NoPvl sc solubilised in water and inoculated together with P. viticola on grapevine foliar disks, in the form of droplets, was analysed 5 and 7 dpi. (b) Photos of grapevine foliar disks with P. viticola in the presence of NoPvl sc (20, 50 and 100 mM) and in its absence (Control) (c) Fungicidal activity of the peptide NoPvl sc, solubilised in water and administered with the Potter spray tower before infection with P. viticola. The data refer to 5 and 7 dpi.

- Figure 11 shows the fungicidal activity of mutated versions of the peptide NoPvl analysed on co-inoculated grapevine foliar disks. The activity of the peptides NoPvl R1A, where the Arg in position 1 was replaced with Ala, NoPvl R7A, where the Arg in position 7 was replaced with Ala, and NoPvl R1A-R7A, where both Args were replaced with Ala, was analysed at 5 and 7 dpi. The charts highlight the importance of the two Args for the fungicidal activity of NoPvl.

The better to illustrate the invention, the following examples are provided below to illustrate the invention without limiting it.

EXAMPLE 1: Identification of the peptide according to the invention NoPvl (SEQ ID NO:l )

The peptide NoPvl having amino acid sequence RLTAQCRL (SEQ ID NO: 1) was selected by a yeast two-hybrid assay in which small peptides had to be identified, able to interact with the catalytic domain of the cellulose synthase 2 of P. viticola , PvCesA2 (Figure 1), to inhibit the activity of the enzyme.

It has been demonstrated in Phytophthora infestans, a pathogenic Oomycete close to Plasmopara viticola from the phylogenetic viewpoint, that the cellulose synthases play a fundamental role in the establishment of the infection [6]. The peptide library was generated following the protocol shown in [7], which provides the use of a modified yeast expression vector, in which the gene of thioredoxin A of Escherichia coli (TrxA), encoding for the scaffold protein, was fused in frame with the transcriptional activation domain (Activation Domain, AD) of the yeast transcription factor GAL4 (TrxA-GAL4AD). The fragment of random nucleotide sequences of 24 base pairs (bp), encoding for the 8 amino acid long peptide library, was then inserted in the vector containing the fusion protein TrxA-GAL4AD, more precisely it was positioned in the catalytic centre of the protein TrxA. The insertion of random peptide sequences in this position makes the enzyme TrxA inactive, eliminates the potential interactions with its specific natural interactors and exposes on the surface the random sequence of 8 amino acids. The library thus built was used to transform a yeast strain (AH109) bearing the vector pGBKT7 in which the catalytic domain of the gene PvCesA2 was fused with the DNA Binding Domain (BD) of the yeast transcription factor GAL4 (PvCesA2-GAL4BD). The peptides able to interact with the catalytic domain of PvCesA2 were selected on the basis of the capability of the positive yeast clones to grow on a selective medium. The plasmid DNA was then purified and sequenced. The sequence of the fragment of 24 nucleotides allowed to deduct the primary structure of the peptide NoPvl.

Properties of the peptide NoPyl

The peptide NoPv 1 is a peptide consisting of eight L-amino acids, NH₂-Arg - Leu - Thr - Ala - Gln - Cys - Arg - Leu-COOH (SEQ ID NO:l) with a molecular weight of 960.16 Da, an isoelectric point at pH 10.43, a net charge of 1.9 at pH 7, and good solubility in water. It was hypothesised that peptides with a net positive charge, between +2 and +9, are excellent antimicrobial peptides because their charge promotes the initial electrostatic interaction with the negatively charged phospholipid membranes of bacteria, fungi and other micro-organisms ([8][9][l0][l l]). However, the positive charge of NoPvl is not sufficient to explain its specificity for P. viticola and the absence of toxicity on human cells.

To verify the importance of the primary sequence of the peptide NoPvl for its biological activity, a scramble peptide was generated, with the name of NoPvl sc (NH₂-Leu - Cys - Arg - Leu - Arg - Ala - Thr - Gln-COOH, SEQ ID NO:2).

The peptide NoPvl sc demonstrated its ability, both in co-inoculation assays and in pre-inoculation assays, to inhibit the infection from P. viticola in a similar manner to NoPvl, suggesting that both the primary sequence of the peptide, and its biochemical properties are important and both contribute to determine its biological activity (Figure 10).

Lastly, a test was conducted of the importance for the biological activity of NoPvl of the two arginine residues, which provide the peptide with the positive charge, by the generation of three new peptides in which one or both arginine residues were replaced by alanine resides (Ala-scan) (Figure 11).

All the peptides thus generated, NoPvl R1A (NFL -Ala - Leu - Thr - Ala - Gln - Cys - Arg - Leu-COOH, SEQ ID NO: 3), NoPvl R7A (NH₂-Arg - Leu - Thr - Ala - Gln - Cys - Ala - Leu-COOH, SEQ ID NO:4), and NoPvl R1A-R7A (NH₂-Ala - Leu - Thr - Ala - Gln - Cys - Ala - Leu-COOH, SEQ ID NO:5) proved to be unable to prevent the infection of Plasmopara viticola at the levels of NoPvl, underlining the fundamental role played by the positive charge in the biological activity of the peptide NoPvl.

EXAMPLE 2: Synthesis of the peptide and purification

The peptides were prepared by Fmoc solid-phase peptide synthesis [12].

The peptides were prepared using the Wang resin with a loading of 0.4 mmol g ¹ and on 0.2 mM scale.

0.2 M of solutions of 9-Fluorenylmethyloxycarbonyl (Fmoc) amino acids were used in N,N-dimethylformamide (DMF) or l-Methyl-2-pyrrolidinone (NMP). As coupling reagent, a mixture of l-hydroxybenzotriazole/O-benzotriazol-N,N,N0,N0- tetramethyluronium hexafluoro-phosphate (HOBT/HBTU) 0.45M in DMF and as diisopropylethylamine (DIPEA) 2 M in NMP.

The coupling reaction was conducted for 5 min at 40 W with a maximum temperature of 75°C. The deprotection is carried out with 2 cycles of 5 min and 10 min respectively (75°C, 40 W).

For the deprotection reaction of the Fmoc group, a 20% piperidine solution in DMF was used.

The detachment of the resin was carried out using the reagent K (trifluoroacetic acid/phenol/thioanisole/triisopropylsilane/water, 82.5:5:5:5:2.5 v/v) for 3 hours. The peptide was precipitated from cold ethyl ether and purified by RP-HPLC with a 5- 70% gradient of the solvent B (solvent A:water/acetonitrile/TFA 95/5/0.1; solvent B: water/acetonitrile/TFA 5/95/0.1) in 20 min with a flow of 20 ml/min. The peptides were lyophilised and preserved at 0°C. EXAMPLE 3: Study on the biological activity of the peptide NoPvl

To assess the effects of the peptide NoPvl on the infection by P. viticola, different types of experiments were carried out both in vitro, in controlled conditions in a laboratory by inoculation on foliar disks obtained from susceptible plants of Vitis vinifera cultivar (cv) Pinot noir, and in vivo on greenhouse-grown plants.

Co-inoculation assays

The fungicidal activity of the NoPvl, dissolved in water at different concentrations (20, 50, 100, 150, 200, 400 mM) and inoculated together with P. viticola on grapevine leaves in the form of small drops, was assessed at 5 and 7 days from inoculation (dpi), in accordance with the protocol described in [13] and [14].

Each foliar disk was inoculated positioning on it 5 drops (of 10 pl each) of a suspension of sporangia of P. viticola (at the concentration of 1 x 10⁵ sporangia/ml) as shown in Figure 2.

For each concentration to be tested, 5 replications were carried out, each consisting of 5 foliar disks. The disks, placed in Petri dishes with a plastic film, were incubated in the dark in a growth chamber at the temperature of 22 ± 1 °C for one night to allow the penetration of P. viticola inside the foliar tissues. The following day, the drops were dried with filtering paper and the disks were incubated again in the growth chamber, at the same temperature and a photoperiod of 16 hours of light and 8 ours of darkness, for a period of seven days. The severity of the disease was assessed at five and seven days after inoculation as the percentage of surface area of the foliar disk covered by sporulation. This percentage was calculated as the sum of the severity of the disease in each of the five drops positioned on the disk. To each drop was assigned a value variable from 0% (absence of sporulation at the drop) to 20% (abundant sporulation). NoPvl showed a weak antimicrobial activity at the concentration of 20 mM, but it was able to block almost completely the infection of P. viticola at 100 mM (Figures 2 and 3).

Pre-inoculation assays:

The effects of the peptide NoPvl against P. viticola were assessed also using the Potter spray Tower (Burkard Scientific, UK). The Petri dishes containing the foliar disks were sprayed with 1.67 ml of a 400 mM solution of NoPvl (corresponding to a standard dosage of 10 hl/ha in a vineyard with Pergola Trentina cultivation system) at the pressure of 55 kPa.

The peptide NoPvl was applied at different times (from seven days to two hours) before the inoculation of the pathogen. After each application, the foliar disks were left to dry in a chemical fume hood and the preserved in the growth chamber. The foliar disks were then sprayed with a fresh suspension of sporangia of P. viticola (0.6 ml of suspension for each Petri dish, containing 5 foliar disks, at a concentration of 1 x 10⁵ sporangia/ml) and incubated for one night in the dark in a growth chamber at the temperature of 22 ± l°C. The following day, the dishes were made to dry in a laminar flow hood and then maintained in the growth chamber of a period of seven days. At 7 dpi, the percentage of surface area of the foliar disk covered by sporulation was assessed visually.

The peptide NoPvl showed a significant capability of inhibiting the Plasmopara viticola , even when applied seven days before infection indicating that NoPvl can be used as a fungicide with preventive action. The peptide (400 mM) was supplied to foliar disks by means of the Potter spray Tower at different times before the infection with Plasmopara viticola (g, days; O, hours). The corresponding controls (Co), without peptide NoPvl, are shown in the chart. The chart also shows the co inoculation assay (0, O). The data show that NoPvl is effective even when it is administered before the infection, hence in pre-inoculation. The values of severity of the infection shown in the chart refer to 7 days from the inoculation of Plasmopara viticola (7 dpi) (see Figure 4).

Even more interesting is the fact that NoPvl showed a very similar effectiveness to copper sulphate, currently the best fungicidal compound used in agriculture, in preventing the attack of peronospora and it also showed a far higher effectiveness than Pergado SC, because the strains of P. viticola that attack vineyards in Trentino have become resistant to this fungicide (see Figure 5).

Assays on plants in greenhouse

The ability of NoPvl to prevent the Plasmopara viticola infection was also analysed in greenhouse conditions, in this case the solution of NoPvl was sprayed on the plants to be tested one day before inoculation with Plasmopara viticola (Figure 6). In detail, the plants of V. vinifera cv Pinot Noir were cultivated in a greenhouse (20°C, 70% ± 10% RH), so that each plant had two buds bearing 10-15 leaves each. The solution of the peptide NoPvl, at the concentration of 400 of 800 mM, was applied on the lower page of all leaves by a spray. For each concentration to be tested, five plants were used. To each plant were applied 15-20 ml of solution of the peptide NoPvl on the basis of the number of leaves present. As a control, five plants were sprayed with water (untreated control). For the inoculation of Plasmopara viticola , the plants were sprayed with a fresh suspension of sporangia (1.8 x 10⁵ sporangia / ml) and incubated in the dark in a greenhouse for one night. The percentage of foliar surface area covered by sporulation was assessed visually at 7 dpi. In this case, too, NoPvl confirmed its protective action, since it demonstrated its ability to reduce the P. viticola infection in greenhouse conditions.

EXAMPLE 4: Properties of the peptide NoPvl

Specificity of NoPyl

The micro-organisms present in the ground represent an essential component of the soil ecosystem, as well as of the agricultural system. They perform a fundamental role in the biogeochemical cycles of the nutrients and each of them, indicated as PGPR bacteria (plant growth-promoting rhizobacteria), are able to establish more or less close relations with the superior plants, promoting their growth [15]. Among PGPR bacteria we find micro-organisms belonging to the genera Agrobacterium, Arthrobacter, Azotobacter, Azospirillum, Bacillus, Burkholderia, Caulobacter, Chromobacterium, Erwinia, Flavobacterium, Micrococcous, Pseudomonas and Serratia. Therefore, it was verified whether the peptide NoPvl could somehow influence the growth of these bacteria. For this purpose, the peptide NoPvl was added to a concentration of 200 mM in liquid cultures of Agrobacterium tumefaciens and Bacillus amyloliquefacien . In both cases, the addition of the peptide NoPvl to the culture medium caused no alteration in bacterial growth (Figure 7), suggesting that NoPvl can be used in vineyards without the occurrence of undesired bacteriostatic or bactericidal effects on the soil ecosystem. The biological activity of NoPvl was also tested on the Oomycete fungus P. infestans, closely correlated to P. viticola, and on the Ascomycete Erysiphe necator, causal agent of grapevine oidium (Figure 8).

The catalytic domain of the gene PvCesA2 of P. viticola used in the yeast two- hybrid assay is extremely similar to the corresponding gene of P. infestans (PiCES2), having an identity equal to 97% (Figure 8a); consequently, NoPvl proved to be able to influence the growth of P. infestans in vitro (Figure 8b) when added to the growth medium. On the contrary, NoPvl does not block the growth of Erysiphe necator (Figure 8c), an Ascomycete that has a cell wall constituted mainly by chitin, and not by cellulose as in Oomycetes. These experimental results strongly support the hypothesis that the activity of NoPvl is specific and hence that the peptide acts inhibiting the activity of the cellulose synthase.

On the basis of these results it is also possible to hypothesise that the peptide NoPvl can be active against any organism having an enzyme for the cellulose synthase that is very similar to PvCesA2. Analyses conducted with the TBLASTIN software allowed us to identify numerous organisms, all Oomycetes and plant pathogens, whose cellulose synthases show an amino acid sequence homology above 90% with PvCesA2.

The list of oomycetes and of the host vegetable species whose cellulose synthase has an amino acid sequence homology above 90% with PvCesA2 is shown in Table 1 below.

The homology analysis was conducted using the TBLASTN software (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Table 1

Potential cytotoxicity of the peptide NoPyl

The peptide NoPvl never showed phytotoxicity symptoms, even when used in relatively high concentrations.

No damage was observed either on the foliar disks, or on the young or adult leaves of the greenhouse plants treated with the peptide.

The potential cytotoxicity of the peptide NoPvl on human cells was tested in vitro by the cell viability test, or MTT assay.

It is a standard colorimetric assay that allows to measure the activity of the succinate dehydrogenase mitochondrial enzyme, active only in living cells, and able to reduce MTT (bromide of 3-[4,5-dimethylthiazol-2-il]-2,5-diphenyltetrazole, yellow coloured) to formazan, a blue/purple coloured substance, whose formation can be followed measuring absorption at 570 nm. Therefore, formation and absorbance levels at 570 nm are directly proportional to the quantity of vital cells present in the sample. Cultures of immortalised human cells (HKC8) at different densities (1000, 3000 and 6000 per 100 pl) were grown in DMEM-F12 medium in the presence of 400 mM NoPvl and their vitality was measured after 24 and 48 hours. No significant differences were observed between control and treated, indicating that NoPvl is not toxic for human cells (see Figure 9).

REFERENCES

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Claims

1. Peptide characterized by a length sequence of 8 amino acids having the following sequence (I):

(X)_{n -} R - (Y)_{m -} R -(Zi)_p (I)

wherein m is an integer number between 1-6;

n and p are each one an integer number between 0-5;

with m+n+p =6;

R is Arg

with the provision that the aminoacid Leu (L) is present twice and the aminoacids Thr (T), Ala (A), Cys (C), Gln (Q), are present once in the sequence of formula (I).

2. Peptide according to claim 1, wherein:

n= 0;

m= 5;

p = l;

Y_{1 =} L; Y_{2 =} T; Y_{3 =} A; Y₄ = Q; Y_{5 =} C

Zi = L

3. Peptide according to claim 1, wherein:

n= 2

m= 1

P = 3

Xi = L; X₂ = C

Yi = L

Zi = A; Z₂ = T; Z₃= Q

4. Phytopharmaceutical composition comprising at least one peptide according to any one of the claims 1-3 as active principle, and optionally a solid or liquid solvent and /or diluent, at least an adjuvant and/or coformulating agent.

5. Phytopharmaceutical composition according to claim 4, comprising one or more further active ingredients such as a fungicide other than the peptide according to any one of the claims 1-3, selected from the groups consisting of phytoregulators, antibiotics, herbicides, insecticides, fertilizers and/or mixtures thereof.

6. Use of the peptide according to any one of the claims 1-3 or a phytopharmaceutical composition according to anyone of the claims 4-5, as antimicrobial or fungicide agent in agronomic field.

7. Use of the peptide according to claim 6, for treating or preventing the infections mediated by Oomycetes.

8. Use of the peptide according to claim 7, wherein said Oomycetes are selected from the group consisting of Plasmopara viticola, Peronospora spp, Phytophtora spp. , Pseudoperonospora cubensis and Bremia lactucae.

9. Use of the peptide according to claim 7, for treating or preventing peronospora caused by Plasmopara viticola on vines.

10. Method for controlling phytopathogenic fungi in agricultural crops, which consists in applying, on any part of the plants to be protected or on the ground, effective and non-phytotoxic doses of a peptide according to any of the claims 1-3, used as such or formulated in phytopharmaceutical composition according to one or more of claims 4-5.

11. Method for controlling phytopathogenic fungi according to claim 10, wherein said phytopathogenic fungi belong to Oomycetes class and are selected from the group consisting of Plasmopara viticola, Peronospora spp, Phytophtora spp. , Pseudoperonospora cubensis and Bremia lactucae.

12. Method for controlling phytopathogenic fungi according to any one of the claims 10-11, wherein said agricultural crops comprising fruit bearing crops, preferably vine, citrus, legume, horticultural crops, cucurbitaceous crops, tobacco, coffee, the, cacao, sugar beet or cotton.