Method for producing a combination of phages for controlling phytopathogenic bacteria

Abstract

The present invention discloses the development of bacteriophage technology for controlling bacterial diseases caused by phytopathogens. In the development to demonstrate the effectiveness of the method, three bacteriophages from natural sources were selected and their ability to destroy different strains of Pseudomonas syringae was demonstrated. In vitro efficacy tests showed that the bacteriophages eliminated 99% of the bacterial population within an hour. During effectiveness tests in plants, the treatment with bacteriophages showed a preventive and healing effect against bacterial cancer in cherry trees.

Description

[0001]TITLE: Method for producing a combination of phages for the control of bacterial phytopathogens TECHNICAL FIELD The invention falls within the technical agricultural field because it seeks to control a pest and within the field of biotechnology by the proposed method. BACKGROUND AND STATE OF THE ART. There are multiple phytopathogenic pests of different origins that cause diseases to plants, trees, vegetables, whether of bacterial, fungal or other origin. The invention addresses the problem of controlling and / or treating diseases generated by infections, avoiding the generation of resistance of phytopathogens to treatment. Throughout this document, phytopathogens will be considered to exemplify the invention; however, this does not represent a limitation on the scope of protection.One of the phytopathogens that serve to exemplify the invention is Pseudomonas syringae which affects different crops causing, for example, bacterial canker in cherry trees, blossom burst in pears, bacterial speck in tomatoes and bacterial leaf blight in blueberries, in addition to affecting other crops such as peaches and apricots. Other important phytopathogens are Xanthomonas (X. arboricola pv. Juglandis, X. erboricola pv. Corylina, X. arboricola pv. Pruni, X. campestris pv. Campestris, X. vesicatoria pv. Vesicatoria, X. avellanae), Pseudomonas, P. syringae pv. tomato, P. viridiflava), Clavibacter michiganensis, Ralstonia solanacearum, Pectobacterium carotovorum, Erwinia amylovora, Agrobacterium tumefaciens. P. syringae survives winters in infected plant tissues, which then remain in the leaves during the spring or summer seasons, where the infection will continue without generating the disease.The bacteria then penetrate the leaves to eventually cause disease. Evidence of the disease is reflected in necrotic spots on leaves or woody tissues, where polysaccharide production allows the bacteria to adhere and form biofilms that make them difficult to control. Finally, the plant pathogen explodes in its next cycle, causing damage by generating abnormal growths. Some of the common problems associated with controlling Pseudomonas syringae infections include: 1. Antibiotic resistance: Bacteria can develop resistance to antibiotics, limiting the effectiveness of traditional treatment methods. 2. Environmental persistence: Pseudomonas syringae can survive in diverse environmental conditions, making it difficult to eradicate infected areas. 3.Host range: The pathogen has a wide host range, infecting numerous plant species, complicating control efforts. 4. Disease spread: The bacteria can be spread through irrigation water, rain, and wind, causing widespread infections in agricultural settings. Regarding the above, currently available solutions for controlling Pseudomonas syringae infections include cultural practices, where proper sanitation, crop rotation, and pruning can help reduce disease incidence; biological control, where some beneficial microorganisms can suppress the growth of Pseudomonas syringae, and using them as biopesticides can be an environmentally friendly approach; and chemical control, where certain biological and copper-based pesticides are used to control Pseudomonas syringae infections.However, this approach must be carefully regulated due to environmental concerns; genetic resistance, in which the development and planting of genetically resistant plant varieties can be an effective long-term strategy to combat the disease. Integrated pest management (IPM), with the combination of multiple control methods, including cultural practices, biological control, and selective pesticide use, can improve overall disease management. It is essential to note that the availability and efficacy of these solutions can vary depending on specific Pseudomonas syringae strains and regional factors. Therefore, ongoing research and adaptation strategies are crucial to stay ahead of the effective control of this plant pathogen.The following is a summary of the prior art related to the present invention: WO2011014693A2 describes modified bacteriophages that express specific peptides with antibacterial activity. Specifically, the document mentions P. syringae as one of the bacteria that can be combated with the bacteriophages described in the publication. It should be noted that this document is very broad in its description and includes medical applications, such as for the treatment of Alzheimer's, and for the control of infections in plants. WO02086072A2 describes specific polypeptides with antimicrobial activity, specifically against phytopathogens. More specifically, it indicates that the polypeptides are isolated from the hemolymph of insect larvae that have been infected with plant-pathogenic fungi.The mention of bacteriophages is due to their classification as one of the alternative vectors for transforming bacteria and producing the peptide with antimicrobial activity. In addition, seven related documents are published, focusing on the different polypeptides in each document. US2020354689A1 describes modified bacteriophages that can be specific to certain bacteria, although the treatment of plant infections is not specifically mentioned. US2020254035A1 describes modified phages to combat bacterial infections. Among the pathogenic bacteria mentioned, P. syringae is indicated. The document is also very general, as it mentions that humans, animals, or plants can be chosen as treatment subjects. US2015050717A1 describes bacteriophages for the control of bacterial infections in plants.It is specified that bacteriophages are selected based on the antibacterial peptides expressed (indolicidin, CecropinP1, Dermaseptin, Ponericin W1, Ponericin W3, Ponericin W4, Ponericin W5, Ponericin W6). US2010322903A1 describes bacteriophages that express antimicrobial agents. The document is written in a general way, so humans, animals and plants appear as application subjects. Specifically, a section mentions P. syringae as one of the bacteria to be targeted. Microorganisms 2019, 7(9), 286 describes in vitro evidence of the effects of phage phi6 against P. syringae. Applied Microbiology and Biotechnology volume 104, pages 1319-1330 (2020) describes in vitro tests using phage phi6 to inactivate P. syringae. Specifically, tests are being carried out on kiwifruit leaves. PHAGE. Dec 2020.245-250 describes three isolated phage strains targeting various P. syringae strains.Specifically, the strains were isolated from organic waste obtained from a treatment plant. Applied Biotechnology International, Volume 13, Number 5, Thematic Issue on Agricultural Biotechnology, September 2020, pages 1428-1445 describes the isolation of more than 70 phages from natural environments for use in the control of P. syringae in cherry trees. As can be seen, the use of bacteriophages for the control of phytopathogens has been explored; however, the problem of generating resistance to bacteriophages persists. This invention proposes as a solution a method for the selection of specific phytopathogenic bacteriophages that do not affect other beneficial bacteria for crops, while ensuring that the development of long-term resistance is avoided. BRIEF DESCRIPTION OF THE FIGURES Figure 1: The three selected bacteriophages have a clear or transparent lysis halo, demonstrating lytic activity on P. syringae pv. syringes (Pss).Furthermore, phages RQ1 (RQ1, deposited with the International Depositary Authority of Canada, IDAC, with accession number 281124-03), C1 (IDAC accession number 281124-01), and D3 (IDAC accession number 281124-02) have enzymatic activity in addition to cell lytic activity. This image shows synergy between bacteriophages for the control of bacteria, since some bacteriophages, due to the presence of specific enzymes, can facilitate the work of other bacteriophages that do not have them. Figure 2: Multiple sequence alignment for lytic enzymes in selected phages. Figure 3: Lifespan of formulated and unformulated bacteriophages (formulated squares, unformulated circles, x-axis days). Figure 4: The whole genome phylogenetic tree was made. Phages were compared with the top 10 blast hits per phage (30 phages total). Figure 5: Bacterial resistance to bacteriophage combinations.Figure 6: When there is a variation in the multiplicity of infection (MOI), a bacteriophage significantly decreases its lytic activity against Pss in an in vitro culture. However, the fact that this bacteriophage is mixed with 1 or 2 different bacteriophages and the presence of other enzymes / proteins reverses its effect. This represents the synergy between the different phages, since the combination of the different enzymes or other metabolites produced by each of them allows a combination that is not possible to obtain with each of them separately. Figure 7: Synergy of Pss bacteriophages in vivo. Time in hours. SUMMARY OF THE INVENTION The invention includes the development of a bacteriophage technology for the control of bacterial diseases caused by phytopathogens.In the development to demonstrate the effectiveness of the method, three bacteriophages from natural sources were selected and their ability to destroy different strains of Pseudomonas syringae was demonstrated. In vitro efficacy tests showed that the bacteriophages eliminate 99% of the bacterial population within one hour. In plant efficacy tests, treatment with bacteriophages showed a preventative and curative effect against bacterial canker in cherry trees. DETAILED DESCRIPTION OF THE INVENTION Firstly, the invention corresponds to a method of selecting bacteriophages for biological control by preventative and curative treatment in phytopathogen infections of plants, trees or vegetables where the selection of bacteriophages is carried out seeking the synergistic effect of their combination and consequently avoiding the appearance of resistance to the proposed treatments.In a second aspect, the bacteriophage strains selected according to the bacteriophage selection method of the present invention are considered. In a third aspect, a preventive and curative treatment method against phytopathogen infections using the phages selected with the method of the invention is contemplated. In a particular embodiment and as a first aspect, the bacteriophage selection method includes the following steps: ● Determine a geographic area with water sources close to the crops of interest; ● Obtain samples of the soil, water sources and / or plant tissue identified in the previous step; ● Generate the first round of bacteriophage amplification using samples, previously treated with an organic solvent (e.g., chloroform, ethanol, isopropanol), using host bacteria (virulent strain of Pseudomonas syringae pathovar syringae (Pseudomonas syringae Pss) IDAC accession number 281124-04, Pseudomonas pv.morsprunorum, Pseudomonas syringe pv. actinidae, Pseudomonas pv. tomato, Pseudomonas gramini, Xanthomonas (X. arboricola pv. Juglandis, X. erboricola pv. Corylina, X. arboricola pv. Pruni, tomato, P. viridiflava), Clavibacter michiganensis, Ralstonia solanacearum, Pectobacterium carotovorum, Erwinia amylovora, Agrobacterium tumefaciens or similar) and grown in a microbiological nutrient culture medium (based on tryptones, peptones and / or yeast extracts, for example, Luria Bertani or LB (2x) for a maximum of 24 hours at 25°C and 150 rpm shaking. ● Generate a second round of bacteriophage amplification using a filtration of the pre-culture (0.22 μm) with a host strain known to allow bacteriophage multiplication, of your choice and under the same operating conditions described above.● Obtain individual lysis plaques using the double-layer agar technique, discarding those with cloudy lysis plaques, as these will be considered preliminary as lysogenic phages. ● Select bacteriophage strains that have lytic activity against phytopathogens, with or without enzymatic activity against this bacteria, and that do not have lytic activity against beneficial bacteria. • Determine the resistance or cross-synergy of the mixture of bacteriophage strains. ● Select at least 2, and preferably 3 or more bacteriophages that have lytic and / or enzymatic activity against phytopathogens and that do not have lytic activity against beneficial bacteria, and where at least 2, or preferably 3, selected bacteriophages present resistance or cross-synergy.As regards the second aspect of the invention, the strains selected using the above method have been deposited with the International Depository Authority of Canada (IDAC): RQ1, accession number IDAC 281124-03, C1 accession number IDAC 281124-01, D3 accession number IDAC 281124-02. Finally, in the third aspect of the invention, the protocol for preventive or curative treatment against phytopathogens includes the following steps: 1. Dilution of the product depending on the crops to be treated with a concentration of 10. 5 to 10 9PFU / ml. 2. Foliar, root or substrate application, by spraying, irrigation and / or immersion, during flowering and / or other phenological stage of the plant that involves a high bacterial load. 3. Apply a minimum of 2 times, preferably 3 or more times with intervals of 7-15 days. EXAMPLES (A) Detection of differentiating parameters of bacteriophages: 1) Host range: Methodology: Through a spot test using the double layer agar technique, the ability of previously isolated bacteriophages to destroy different representatives of bacterial phytopathogens used as indicators was evaluated. The bacteriophages selected according to the method of invention were called RQ1, C1 and D3. These bacteriophages have specific activity against the Pseudomonas syringae group, without causing death by lysis of other bacteria such as Xanthomonas, Curtobacterium, Erwinia and Pectobacterium, included in the test.Furthermore, using the same methodology, the mixture of the three indicated bacteriophages has the ability to produce bacterial lysis of different pathogenic representatives of the Ps group, both in Pseudomonas syringae pv. syringae (Pss), Pseudomonas syringae pv. morsprunorum (Psm) and Pseudomonas syringae pv. actinidiae (Psa). 2) Presence of lytic activity and action of another lysis enzyme / protein. Methodology: To determine the lytic activity of the selected bacteriophages from the previous test, the double-layer agar technique was developed using a Pss isolate as a strain indicator. In this case, isolated lysis plaques were photographed to determine the clarity of the halo, demonstrating lytic activity and ruling out unwanted lysogenic activity (preliminary analysis). From this photographic analysis, the presence of other enzymes / protein activity was determined, forming a second halo around the central lysis halo.Result: The three selected bacteriophages have a clear or transparent lysis halo, demonstrating lytic activity on Pss. In addition, phages RQ1 and C1 have second enzyme activity in addition to lytic activity. Result: All three selected bacteriophages presented a clear or transparent lysis halo, suggesting the presence of lytic activity on Pss. Subsequent genomic analyses generated with PhageAI software confirmed the absence of genes related to lysogeny, antibiotic resistance, and virulence, thus confirming the lytic nature of the phages. Furthermore, phages RQ1 and C1 show hydrolytic activity in Pss culture (indicated by a red arrow), which is imperceptible with bacteriophage D3. The selected bacteriophages do not have genes encoding depolymerases, however, all three phages have genes encoding endolysins (N-acetylmuramoyl-L-alanine amidase; Pfam PF01510.25).The amino acid sequences of these endolysins were aligned using MUSCLE 3.8 (https: / / www.ebi.ac.uk / jdispatcher / msa / muscle). As a result, it was shown that the amino acid sequences of C1 and RQ1 are identical, whereas the D3 sequence differs from these with a low identity (82.19) (Figure 2). 3) Lifespan Stability: Methodology: To determine the lifespan of bacteriophages, assays were performed using a 1X10 stock. 5PFU / mL of a phage cocktail between RQ1, C1 and D3 and the addition of coformulants based on i) 1-3% thickening agents (celluloses, xanthan gum, alginate or other polysaccharides; ii) 1-5% dispersants (celluloses and polyvinylpyrrolidone), iii) 3-8% humectants (glycerol, polyethylene glycol, ethylene glycol and iv) UV protectors (zinc oxide, titanium oxide). These phages, in triplicate, were placed in test tubes at 25°C for 5 months. The variation in PFU / mL was occasionally evaluated compared to a bacteriophage stock in the absence of coformulants. The addition of coformulant to a stock of bacteriophages allows the shelf life of a product based on these bacteriophages to be maintained for at least 5 months, which projects a minimum period of 12 months of final shelf life.English: Results: As shown in the attached graph (Figure 3), the addition of coformulant to a stock of bacteriophages allows to maintain the shelf life of a product based on these bacteriophages for at least 5 months, which projects a minimum period of 12 months of final shelf life. 4) Genomic analysis of bacteriophages: Methodology: To demonstrate that the selected bacteriophages group into different phylogenetic groups, comparative genomic studies were carried out. To this end, the genetic material of each selected bacteriophage was initially obtained and sequenced using the Ilumina platform by the company MicrobesNG (United Kingdom). The information obtained was processed with the bioinformatics software Geneious Prime to annotate structural genes and enzymes that are associated with the lytic processes of each virus.To study the intergenomic similarity of bacteriophages, the VIRIDIC platform (Virus Intergenomic Distance Calculator; https: / / www.viral.org / en / taxonomy / prediction / 2018 / 01 / 04 / virology-2018-01-04) was used for taxonomic prediction with PhageAI. Using the VICTOR tool (Virus Classification and Tree Building Online Resource; https: / / ggdc.dsmz.de / victor.php), a genome-wide phylogenetic tree was created. Phages were compared to the top 10 blast hits per phage (30 phages in total) (Figure 4). These tools were used because they are developed for the analysis of viral genomes. Results: The results of the PhageAI tool suggest that the phages belong to the order Caudoviricetes, family Autographiviridae, and genus Ghunavirus. On the other hand, the analyzed bacteriophages showed intergenomic similarities that can be observed in Table 2. According to the International Committee on Taxonomy of Viruses (ICTV) standards, the phages belong to three different species. Phage RQ1 was found close to CHF phages previously isolated in Chile as a potential biocontrol against Pseudomonas syringae pv. actinidiae (Flores et al. 2020), with similarities ranging from 96,553 to 99,112. According to ICTV recommendations, RQ1 would belong to a variant of this same species. On the other hand, bacteriophage C1 is close to phages KNP (NC_047827.1; similarity 95,006) and WRT (NC_047826.1; similarity 93,944), isolated phages that infect Pseudomonas fluorescens (Nowicki et al. 2017). Bacteriophage D3 has a similarity of 92,593 to phage P413 (OM282085.1), which infects Pseudomonas savastanoi pv. glycine (Psg), the causative agent of bacterial spot in soybean (Tarakanov et al. 2022). Phage D3 also has a similarity of 87,617 to phage MR1 (MT104465.1), which was previously used in phage biological control to combat the disease-causing pathogen Pseudomonas syringae in cherry plants (Rabiey et al.2020). Therefore, the phylogenetic tree generated by VICTOR allows us to conclude that the three selected bacteriophages are from different species, which justifies their placement in different groups. B) Selection of the bacteriophage cocktail: 1) Cross-resistance (cross-synergy) Methodology: In order to understand the emergence of bacteriophage-resistant Pss variants, two assays were carried out to determine the synergistic effect of bacteriophages in reducing the in vitro appearance of bacteriophage-insensitive mutants (BIMs). To do this, initially the individually selected bacteriophages as a mixture were added to flasks up to an MOI of 100 and left in coculture for 20 minutes.After this time, serial dilutions were made and the colonies resistant to Pss bacteriophages were quantified (CFU / mL) on microbiological agar plates, which contained individual or mixed bacteriophages, as appropriate for the treatment, to ensure resistance. All assays were performed in triplicate and compared with Pss bacterial cultures without the addition of bacteriophages. Subsequently, and to confirm the results of the synergy, colonies were randomly isolated from the BIM assays and lysis assays were performed on plates with these bacteria, evaluating the ability of other bacteriophages to lyse them in vitro (double layer of agar), thus determining a range of lytic action against Pss populations (intraspecific host range). Furthermore, to confirm the synergistic effect of these bacteriophages, cross-lysis assays were performed.As shown in the accompanying table, it is possible to determine which bacteriophage-resistant bacteria can be lysed by other bacteriophages included in the study or cocktail. For example, RQ1. R1 isolated from Pss resistant to phage Rq1 can be lysed by phages C1 and / or D3 (Figure 5). ✔ : with lytic effect X: without lytic effect C) In vitro effectiveness model (synergy) Methodology: The objective of this methodology is to demonstrate a synergistic control of Pss in in vitro cultures between the selected bacteriophages by observing the decrease in the optical density of the culture. In this sense, the time taken for total lysis of Pss in the presence of individual or mixed phages was determined. To do this, three Phage Multiplicity of Infection (MOI: 10, 1 and 0.1) values ​​were applied in liquid cultures of Pss at flask scale. The kinetics of bacterial lysis with individual or mixed phage treatments (RQ1, RQ1+C1, RQ1+D3, C1+D3, RQ1+C1+D3) was compared with Pss cultures in the absence of viruses. Results: The graphs obtained show that bacteriophages RQ1 and C1 individually or in mixture present a similar pattern in the kinetics of bacterial decomposition of Pss regardless of the applied MOI.On the contrary, as the MOI of application specifically to the lytic bacteriophage D3 decreases, its ability to eliminate the bacterial population of Pss during the evaluated period decreases proportionally. However, when this bacteriophage is mixed with RQ1 and C1, the behavior is reversed, drastically reducing the growth of Pss in vitro. Methodology: The objective of this methodology is to demonstrate synergistic control of Pss in in vitro cultures between the selected bacteriophages by observing the decrease in the optical density of the culture. In this sense, the time required for total lysis of Pss in the presence of individual phages or in a mixture was determined. To this end, three Phage Multiplicity of Infection values ​​(MOI: 10, 1 and 0.1) were applied in liquid cultures of Pss at the flask scale.The kinetics of bacterial lysis with individual or mixed phage treatments (RQ1, RQ1+C1, RQ1+D3, C1+D3, RQ1+C1+D3) were compared with Pss cultures in the absence of viruses. Results: The graphs obtained show that bacteriophages RQ1 and C1 individually or in mixtures present a similar pattern in the kinetics of bacterial decomposition of Pss regardless of the applied MOI. On the contrary, as the MOI of application specifically to the lytic bacteriophage D3 decreases, its ability to eliminate the bacterial population of Pss during the evaluated period decreases proportionally. However, when this bacteriophage is mixed with RQ1 and C1, the behavior is reversed, drastically reducing the growth of Pss in vitro.(Figure 6) E) Effectiveness in the plant model (synergy) Methodology: The objective of this methodology is to demonstrate Pss control in in vivo models in tomato plants, to demonstrate the synergistic effect of the selected bacteriophages. In this sense, the effectiveness of bacteriophages in reducing the Pss load (CFU / mL) was determined, applied individually and in mixtures. For this purpose, bacteriophages will be applied at a final MOI of 0.1, on plants previously inoculated by spraying with Pss (1x10. 6CFU / mL). For the purposes of this study, control tomato plants inoculated with Pss and absolute humid controls were included. All results were analyzed in triplicate, in a climate chamber under constant control of temperature (20°C), relative humidity (80%), and photoperiod (16 hours of light / 8 hours of darkness). The results were analyzed using the one-way ANOVA test. Result: As expected, the groups inoculated with Pss (control) have counts that exceed 10 6CFU / mL of Pss for up to 48 hours of exposure. Individual bacteriophages were able to reduce the bacterial load by almost 2 orders of magnitude at the end of 48 hours of analysis. Similar results were obtained with phages mixed in pairs. However, the addition of all three bacteriophages together achieved a reduction of Pss of almost three orders of magnitude, confirming the synergy of the bacteriophage mixture in reducing the bacterial load of Pss in plant models (Figure 7). Identification of deposited bacteriophages of the invention RQ1 (accession number IDAC 281124-03); C1 access number IDAC 281124-01, and D3 access number IDAC 281124-02, and host bacteria for selection is Pseudomonas syringae pathovar syringae (Pseudomonas syringae Pss) access number IDAC 281124-04. INDUSTRIAL APPLICATION The present invention has industrial, agricultural and biotechnological applications.

Claims

CLAIMS 1. Method for selecting bacteriophages for biological control by means of preventive and curative treatment of infections in plants, trees or plant phytopathogens in which the selected bacteriophages have a synergistic effect, thus preventing the appearance of resistance to the proposed treatments, CHARACTERIZED because the method comprises the following stages: a) Determine a geographical area with water sources close to the crops of interest; b) Obtain samples from the soil or from the water sources identified in the previous step; c) Generate a first round of bacteriophage amplification using samples, previously treated with an organic solvent (chloroform, ethanol, isopropanol), using host bacteria and grown in a suitable microbiological nutrient culture medium (based on tryptones, peptones and / or yeast extracts); d) Generate a second round of bacteriophage amplification by filtering the previous culture;e) Obtain individual lysis plaques using the double-layer agar technique, discarding those with turbidity-lysis plaques, as these will be considered preliminary as lysogenic phages; f) Select bacteriophage strains that have lytic activity against phytopathogens, with or without enzymatic activity against this bacteria, and that do not have lytic activity against beneficial bacteria; g) Determine the resistance or cross-synergy of the mixture of bacteriophage strains;h) Selecting at least 2 or preferably 3 or more bacteriophages that have lytic and / or enzymatic activity against phytopathogens and that do not have lytic activity against beneficial bacteria and in which at least 2 or preferably 3 selected bacteriophages present resistance or cross-synergy.

2. The bacteriophage selection method according to claim 1, CHARACTERIZED in that the host bacteria are virulent strains of Pseudomonas syringae pv. syringae, Pseudomonas syringe pv. morsprunorum, Pseudomonas syringe pv. actinidiae, Pseudomonas syringe pv. tomato, Pseudomonas gramini, Xanthomonas (X. arboricola pv. Juglandis, X. erboricola pv. Corylina, X. arboricola pv. Pruni, solanacearum, Pectobacterium carotovorum, Erwinia amylovora, Agrobacterium tumefaciens or other bacterial phytopathogen. 1; 3. The bacteriophage selection method according to claim 1, CHARACTERIZED in that the organic solvent is chloroform.

4. The bacteriophage selection method according to claim 1, CHARACTERIZED in that a microbiological nutrient culture medium (based on tryptones, peptones and / or yeast extracts) is required and is maintained for a maximum of 24 hours at 25°C and 150 rpm of agitation.

5. The bacteriophage selection method according to claim 1, CHARACTERIZED in that the filtrate is carried out with a 0.22 μm in LB medium with a host strain known to allow the multiplication of bacteriophages, of choice and under the same operating conditions described above. 6.Preventive and curative treatment method against phytopathogen infections using the phages selected with the method of claims 1 to 5, CHARACTERIZED in that the preventive and curative method comprises the following steps: a) dilution of the product according to the crops to be treated with a concentration of 10. 5 to 10 9PFU / ml; b) providing foliar, root or substrate application, by spraying or immersion, during flowering and / or other phenological stage of the plant that involves a high bacterial load; c) Applying the foliar application a minimum of 2 times, preferably 3 or more times with intervals of 7-15 days.

7. Consortium of bacteriophages obtained using the method of claims 1 to 5, CHARACTERIZED in that the access numbers of the bacteriophages before the International Depositary Authority of Canada (IDAC) are: RQ1 access number IDAC 281124-03; C1 access number IDAC 281124-01, and D3 access number IDAC 281124-02.

8. Consortium of bacteriophages obtained using the method of claims 1 to 5, CHARACTERIZED in that the host bacterium for the selection of bacteriophages according to the method of claims 1 to 5 is Pseudomonas syringae pathovar 2 syringae (Pseudomonas syringae Pss) IDAC accession number 281124-04. 3

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