Bacteriophages, bactericidal position comprising same and method for the biocontrol of pseudomonas syringae

Abstract

The present invention relates to new isolated and purified bacteriophages, a bactericidal composition comprising same, and a method for the biocontrol of diseases caused by strains of the species Pseudomonas syringae that effect plant crops, using the bacteriophages and / or composition comprising same.

Description

[0001] BACTERIOPHAGES, BACTERICIDAL COMPOSITION CONTAINING THEM AND METHOD FOR THE BIOLOGICAL CONTROL OF PSEUDOMONAS SYRINGAE

[0002] TECHNICAL FIELD

[0003] The present invention relates to the technical field of agroindustry, particularly to the biological control of phytopathogens. Specifically, the present invention relates to novel bacteriophages, a bactericidal composition comprising them, and a method for the biological control of diseases caused by strains of the species Pseudomonas syríngae that affect plant crops, using said bacteriophages and / or a composition comprising them.

[0004] BACKGROUND OF THE INVENTION

[0005] Phytopathogenic bacteria of the species Pseudomonas syríngae affect a wide variety of economically important vegetable crops, such as kiwi, cherries, plums, tomatoes, beans, among others. Among the most important diseases caused by this bacterial species are bacterial canker of kiwi, bacterial canker of cherry, halo blight of beans, and bacterial spot of tomatoes. Diseases caused by P. syríngae are associated with significant losses in the agricultural sector, as the infection causes everything from decreased plant growth to total crop failure.

[0006] An important characteristic of this bacterial species is that, depending on the host plant, it is subclassified into more than 50 different pathovars. For example, bacterial spot disease in tomatoes (Solanum lycopersicum) is caused by the strain Pseudomonas syríngae pv. tomato. This disease produces dark lesions on the foliage and fruit of these plants, resulting in leaf death and a subsequent reduction in their photosynthetic capacity. Another example is halo blight disease affecting beans (Phaseolus vulgaris), which is caused by infection with the strain Pseudomonas syríngae pv. phaseolicola. This disease produces irregular necrotic spots surrounded by a yellow halo on the leaves, and reddish spots on the pods.

[0007] Currently, treatments to control infections caused by P. syringae include antibiotics such as streptomycin and oxytetracycline, or copper-derived chemicals such as hydroxides, oxides, sulfates, and oxychlorides. However, prolonged use of any of these compounds promotes the selection of resistant bacterial strains, resulting in ineffective treatments against the various diseases caused by these plant pathogenic bacteria. Furthermore, the uncontrolled use of inorganic copper compounds can alter the native (beneficial) microbiota of plants and accumulate in water and agricultural soils, contaminating the environment and decreasing crop productivity by eliminating microorganisms from the rhizosphere.

[0008] For the reasons explained above, the use of chemicals in the agricultural sector is gradually being abandoned, giving way to the use of biological control agents that have a neutral or positive environmental impact. For several years, various biological control strategies have been developed using bacteria, viruses, and organic compounds, such as terpenes, chitosan, and antimicrobial peptides, which act as bactericides or bacteriostatics. However, one of the most promising strategies that has proven effective for controlling phytopathogens is the use of lytic bacteriophages.

[0009] There are some bacteriophage-based formulations in the state of the art to combat or prevent plant diseases caused by strains of the species P. syringae. For example, Rombouts S. et al., discloses a mixture of bacteriophages for the treatment of halo blight in leeks caused by P. syringae pv. porri (Rombouts S. et al. Characterization of Novel Bacteriophages for Biocontrol of Bacterial Blight in Leek Caused by Pseudomonas syringae pv. porri. Front Microbiol. 2016. 15; 7:279). Frampton RA. et al., discloses bacteriophages for the control of bacterial canker in kiwifruit produced by P. syringae pv. actinidiae (Frampton RA. et al. Identification of Bacteriophages for Biocontrol of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae. Appl Environ Microbiol. 2014. 80(7):2216-2228). Patent documents KR101768778B1 and KR101786221 B1 refer to lytic bacteriophages that infect P. syringae pv.actinidiae, whose deposit numbers are KFCC1 1635P and KFCC1 1634P, respectively. Patent document US4828999A discloses bacteriophages that infect P. syringae strains to prevent damage caused by this bacterial species by acting as a nucleus for ice formation in the plants.

[0010] However, despite the existence of these formulations, the host range of a bacteriophage is known to be highly variable. Many bacteriophages are specific to a single bacterial species, and are usually specific to only some strains of that species, demonstrating the high degree of specialization that bacteriophages have for certain strains. There is also evidence that bacteriophages can adapt locally to specific strains of a bacterial species present in a particular region (Koskella B & Meaden S. Understanding Bacteriophage Specificity in Natural Microbial Communities. Viruses. 2013. 5:806-823). Therefore, it is common for formulations containing bacteriophages isolated from one country to be ineffective against plant pathogens present in crops from other countries.

[0011] Consequently, it is necessary to develop alternative control strategies for this important phytopathogenic bacterium that are effective, environmentally friendly, and minimize the potential emergence of resistant strains.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention relates to new lytic bacteriophages capable of infecting strains of the phytopathogenic species Pseudomonas syringae, a bactericidal composition containing them, and a method for the biological control of diseases caused by said phytopathogen comprising the use of said bacteriophages and / or a composition containing them.

[0014] A first object of the present invention is an isolated and purified lytic bacteriophage that infects strains of the species Pseudomonas syringae, which is selected from the group consisting of the bacteriophage deposited with the International Depositary Authority of Canada, with the access code 280823-01 (bacteriophage E), the bacteriophage deposited with the International Depositary Authority of Canada, with the access code 280823-02 (bacteriophage 21 1 1), the bacteriophage deposited with the International Depositary Authority of Canada, with the access code 280823-03 (bacteriophage EGF) and the host bacterial strain of the bacteriophages deposited with the International Depositary Authority of Canada, with the access code 280823-04 (Pseudomonas syringae pv. tomato DC3000).

[0015] A second object of the present invention is a bactericidal composition for the control of diseases caused by strains of the species Pseudomonas syríngae in plant crops, which comprises an effective amount of at least one bacteriophage that infects strains of the species Pseudomonas syríngae and an agronomically acceptable additive. Said bacteriophage is selected from the group consisting of the bacteriophage deposited with the International Depositary Authority of Canada, with access code 280823-01 (bacteriophage E); or the bacteriophage deposited with the International Depositary Authority of Canada, with access code 280823-02 (bacteriophage 211 1); or the bacteriophage deposited with the International Depositary Authority of Canada, with access code 280823-03 (bacteriophage EGF); or a mixture of at least two of the bacteriophages 280823-01 (bacteriophage E), 280823-02 (bactehophage 211 1) and 280823-03 (bacteriophage EGF).

[0016] In a preferred embodiment, the composition has an effective amount between 1 x10 7 and 1 x10 9 PFU of the bacteriophage per mL of composition, preferably 1 x10 9 PFU of the bacteriophage per mL of composition.

[0017] In another preferred embodiment, the additive present in the composition is selected from the group consisting of a carrier, an adhering agent, an osmoprotectant, an anti-drift adjuvant, and a UV radiation protective agent; where the carrier is preferably bentonite in a concentration between 0.05% and 0.1% (w / v); the adhering agent is preferably magnesium sulfate in a concentration between 0.1% and 0.15% (w / v); the osmoprotectant is preferably glycerol in a concentration between 0.1% and 0.15% (w / v); the anti-drift adjuvant is preferably sucrose in a concentration between 0.2% and 0.3% (w / v); and the UV radiation protective agent is preferably soy milk in a concentration between 0.5% and 0.75% (w / v).

[0018] A third object of the present invention is a method for the biological control of diseases caused by strains of the species Pseudomonas syringae in plant crops, which comprises the steps of providing a bactericidal composition comprising an effective amount of at least one bacteriophage that infects strains of the species Pseudomonas syringae, and applying said composition to the plant crop.Said bacteriophage is selected from the group consisting of the bacteriophage deposited with the International Depositary Authority of Canada, under accession code 280823-01 (bacteriophage E); or the bacteriophage deposited with the International Depositary Authority of Canada, under accession code 280823-02 (bacteriophage 211 1 ); or the bacteriophage deposited with the International Depositary Authority of Canada, under accession code 280823-03 (bacteriophage EGF ); or a mixture of at least two of bacteriophages 280823-01 (bacteriophage E), 280823-02 (bacteriophage 21 1 1 ) and 280823-03 (bacteriophage EGF).

[0019] In a preferred embodiment of the method of the present invention, the composition has an effective amount between 1x10 7 and 1 x10 9 PFU of the bacteriophage per mL of composition, more preferably between 1 x10 8 and 1 x10 9 PFU of the bacteriophage per mL of composition.

[0020] In another preferred embodiment of the method of the present invention, the composition is applied to a plant crop or crop, to parts of said plant, or to the soil where said crop or plant grows. Within these parts of the plant, the composition may be applied to the seeds, stems, leaves, flowers, roots, bark, branches, trunk, and fruits.

[0021] Preferably, the plant crops are selected from the group consisting of plants of the genera Solanum, Phaseolus, Prunus, Malus and Pyrus, even more preferably from the species Solanum lycopersicum and Phaseolus vulgaris, Prunus cerasus, Prunus armeniaca, Prunus domestica, Malus domestica and Pyrus communis L.

[0022] In a preferred embodiment of the method of the present invention, between 150 and 15,000 L / ha of the composition is applied to the plant crop, more preferably between 1,000 and 2,000 L / ha of the composition to said plant crop.

[0023] In another embodiment of the present invention, the composition is applied to the plant crop by a technique selected from the group consisting of direct injection into the soil, drip irrigation, sprinkling, spraying and manual watering.

[0024] BRIEF DESCRIPTION OF THE FIGURES

[0025] FIG. 1 shows transmission electron microscopy photographs showing the structure of the three bacteriophages. FIGS. 1 A, 1 B, and 1 C show the structure of bacteriophage 21 1 1 , E, and EGF, respectively.

[0026] FIG. 2 shows transmission electron microscopy photographs showing the structure of the three bacteriophages infecting P. syringae pv. tomato DC3000. FIGs. 2A, 2B, and 2C show the adsorption of bacteriophage 2111, E, and EGF to the cell surface of P. syringae pv. tomato DC3000, respectively.

[0027] FIG. 3 shows photographs of Petri dishes showing the results of infection of the bacterial strain Pseudomonas syringae pv. tomato DC3000 by the three bacteriophages. FIGs. 3A, 3B, and 3C show the results of infection by bacteriophage 21 1 1 , E, and EGF, respectively.

[0028] FIG. 4 shows graphs with the adsorption curve of bacteriophage 21 1 1 on the surface of the bacterial strain Pseudomonas syringae pv. tomato DC3000. FIG. 4A shows a graph with the result of the one-step adsorption assay. FIG. 4B shows the linearization of the first part of the curve of the graph shown in FIG. 4A to calculate the adsorption rate of bacteriophage 211 1 . The letter “A” in the graph corresponds to the adsorption time and the letter “L” is the latent period.

[0029] FIG. 5 shows graphs of the adsorption curve of bacteriophage E on the surface of the bacterial strain Pseudomonas syringae pv. tomato DC3000. FIG. 5A shows a graph of the result of a one-step adsorption assay. FIG. 5B shows the linearization of the first part of the curve in the graph shown in FIG. 5A to calculate the adsorption rate of bacteriophage E. The letter “A” in the graph corresponds to the adsorption time, and the letter “L” is the latent period. FIG. 6 shows graphs of the adsorption curve of bacteriophage EGF on the surface of the bacterial strain Pseudomonas syringae pv. tomato DC3000. FIG. 6A shows a graph of the result of a one-step adsorption assay. FIG. 6B shows the linearization of the first part of the curve in the graph shown in FIG. 6A to calculate the adsorption rate of bacteriophage EGF.The letter “A” in the graph corresponds to the adsorption time and the letter “L” is the latent period.

[0030] FIG. 7 shows a graph of the results of a one-stage viral multiplication assay for bacteriophages 21 1 1 , E, and EGF. The data correspond to the plaque-forming units (PFU) at different times, divided by the initial number of PFU. The graphed values ​​correspond to the average of experiments performed in triplicate and are shown with their corresponding standard deviations (SD).

[0031] FIG. 8 shows graphs with the results of the lytic activity of bacteriophage E. FIGs. 8A and 8B show the estimated time to kill (TOD) of Pseudomonas syringae pv. tomato DC3000 and Pseudomonas syringae pv. phaseolicola PSP at different MOIs (multiplicity of infection), respectively. Bacterial cultures were evaluated in the presence of bacteriophages over time, from 0 to 24 h. The MOIs evaluated were 0.1, 0.01, and 0.001. The data correspond to the average of experiments performed in triplicate and are shown with their corresponding standard deviations (SD).

[0032] FIG. 9 shows graphs with the results of the lytic activity of bacteriophage EGF. FIGs. 9A and 9B show the estimated time to kill (TOD) of Pseudomonas syringae pv. tomato DC3000 and Pseudomonas syringae pv. phaseolicola PSP at different MOIs (multiplicity of infection), respectively. Bacterial cultures were evaluated in the presence of bacteriophages over time, from 0 to 24 h. The MOIs evaluated were 0.1, 0.01, and 0.001. The data correspond to the average of experiments performed in triplicate and are shown with their corresponding standard deviations (SD).

[0033] FIG. 10 shows graphs with the results of the lytic activity of bacteriophage 21 1 1 . FIGs. 10A and 10B show the estimated time to kill (TOD) of Pseudomonas syringae pv. tomato DC3000 and Pseudomonas syringae pv. phaseolicola PSP at different MOIs (multiplicity of infection), respectively. Bacterial cultures were evaluated in the presence of bacteriophages over time, from 0 to 24 h. The MOIs evaluated were 0.1 , 0.01 and 0.001. The data correspond to the average of experiments performed in triplicate and are shown with their corresponding standard deviations (SD).

[0034] FIG. 11 shows graphs with the results of the lytic activity of the mixture of bacteriophages E, EGF and 211 1. FIGs. 1 1 A and 1 1 B show the estimated time to kill (TOD) of Pseudomonas syringae pv. tomato DC3000 and Pseudomonas syringae pv. phaseolicola PSP at different MOIs (multiplicity of infection), respectively. Bacterial cultures were evaluated in the presence of bacteriophages over time, from 0 to 24 h. The MOIs evaluated were 0.1, 0.01 and 0.001. The data correspond to the average of experiments performed in triplicate and are shown with their corresponding standard deviations (SD).

[0035] FIG. 12 shows graphs of the viability results over time for bacteriophage E mixed with different additives. FIGs. 12A and 12B show the plaque-forming units per mL (PFU / mL) at different times over 14 days for bacteriophage stored at 4°C and 25°C, respectively. The data correspond to the average of experiments performed in triplicate and are shown with their corresponding standard deviations (SD).

[0036] FIG. 13 shows graphs of viability over time for bacteriophage 21 1 1 mixed with different additives. FIGs. 13A and 13B show plaque-forming units per mL (PFU / mL) at different times over 14 days for bacteriophage stored at 4°C and 25°C, respectively. The data correspond to the average of experiments performed in triplicate and are shown with their corresponding standard deviations (SD).

[0037] FIG. 14 shows graphs with the viability results over time of bacteriophages E, EGF and 211 1 on cherry tomato plants (Solanum lycopersicum var. cerasiforme). FIG. 14A shows the plaque-forming units per gram (PFU / g) of bacteriophages E, EGF and 211 1 at a 1 x 10 suspension. 6 PFU / mL (F2, F5, and F4, respectively). FIG. 14B shows the plaque-forming units per gram (PFU / g) of bacteriophages E, EGF, and 21 at a 1 x 10 suspension. 8 PFU / mL (F3, F7, and F6, respectively). Data correspond to the average of experiments performed in triplicate and are shown with their corresponding standard deviations (SD).

[0038] FIG. 15 shows a graph with the viability results over time of a combination of bacteriophages 21 11 and EGF on cherry tomato plants (Solarium lycopersicum var. cerasiforme). The graph shows the plaque-forming units per gram (PFU / g) of a combination of bacteriophage 21 1 1 at a 1 x 10 suspension. 6 UFP / mL together with the bacteriophage EGF at a suspension of 1 x10 8 UFP / mL (F9), and a combination of bacteriophage 21 1 1 at a 1 x10 suspension 8 UFP / mL together with the bacteriophage EGF at a suspension of 1 x10 6 PFU / mL (F8). Data correspond to the average of experiments performed in triplicate and are shown with their corresponding standard deviations (SD).

[0039] FIG. 16 shows a graph of the viability results over time of a combination of bacteriophages EGF and E on cherry tomato plants (Solarium lycopersicum var. cerasiforme). The graph shows the plaque-forming units per gram (PFU / g) of a combination of bacteriophage EGF at a 1x10 suspension. 6 PFU / mL together with bacteriophage E to a suspension of 1 x10 8 UFP / mL (F1 1 ), and a combination of bacteriophage EGF at a suspension of 1 x10 8 PFU / mL together with bacteriophage E to a suspension of 1 x10 6 PFU / mL (F10). Data correspond to the average of experiments performed in triplicate and are shown with their corresponding standard deviations (SD).

[0040] FIG. 17 shows a graph with the viability results over time of a combination of bacteriophages E and 21 11 on cherry tomato plants (Solarium lycopersicum var. cerasiforme). The graph shows the plaque-forming units per gram (PFU / g) of a combination of bacteriophage E at a 1 x 10 suspension. 6 PFU / mL together with bacteriophage 21 11 to a suspension of 1 x10 8 PFU / mL (F13), and a combination of bacteriophage E to a 1 x10 suspension 8 PFU / mL together with bacteriophage 21 11 to a suspension of 1 x10 6PFU / mL (F12). Data correspond to the average of experiments performed in triplicate and are shown with their corresponding standard deviations (SD). FIG. 18 shows a graph with the viability results over time of a combination of bacteriophages E, EGF and 211 1 on cherry tomato plants (Solarium lycopersicum var. cerasiforme). The graph shows the plaque-forming units per gram (PFU / g) of a combination of bacteriophages E, EGF and 211 1 at a suspension of 1 x 10 8 UFP / mL (F15) and a combination of bacteriophages E, EGF and 21 1 1 at a suspension of 1 x10 6 PFU / mL (F14). Data correspond to the average of experiments performed in triplicate and are shown with their corresponding standard deviations (SD).

[0041] FIG. 19 shows a graph with the results of the in vivo assay of protection against infection of the P. syringae pv. tomato DC3000 strain. The data correspond to the colony forming units per gram of plant tissue (CFU / g) of tomato leaves infected with the DC3000 strain and controlled with 150 pg / mL streptomycin, 0.075% copper sulfate and with bacteriophage 2111 in a 1 x 10 suspension. 9 PFU / mL (MOI 0.01). Data correspond to the average of experiments performed in triplicate and are shown with their corresponding standard deviations (SD). Letters indicate significant differences (p<0.05) according to Tukey's test, two-way ANOVA.

[0042] FIG. 20 shows a graph with the results of the in vivo protection assay with the composition comprising the mixture of bacteriophages E, EGF and 21 11 against infection by P. syringae pv. tomato DC3000. The colony forming units per gram of plant tissue (CFU / g) over time are shown. The data correspond to the average obtained from quadruplicate of tomato leaves infected with the DC3000 strain at a suspension of 1 x 10 7 CFU / mL with the bacteriophage composition with 1 x10 8 UFP / mL (MOI 0.01 ) (Plant 1 and 2 c / mix E- EGF-21 11 ) and without the composition (Plant 3 and 4 DC3000 control). The data correspond to the average of experiments performed in triplicate and are shown with their corresponding standard deviations (SD). The letters correspond to the significant differences p < 0.05, according to the Tukey test, two-way ANOVA. DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention relates to novel lytic bacteriophages capable of infecting strains of the phytopathogenic species Pseudomonas syringae, which is capable of causing serious diseases in various plant species. These novel bacteriophages provide an efficient alternative for the biological control of various P. syringae pathovars, without harming other beneficial bacterial species found in plant crops.

[0044] The present invention also includes a novel bactericidal composition suitable for use in agriculture and a method of applying it to plant crops to prevent, treat, and / or cure diseases caused by strains of the P. syringae species. The novel composition comprises said novel isolated and purified bacteriophages and agronomically acceptable additives that provide stability and viability for use in the field.

[0045] All technical and scientific terms used to describe this invention have the same meaning as understood by a person with basic knowledge in the technical field concerned. However, to more clearly define the scope of the invention, a list of the terminology used in this description and its meaning is included below.

[0046] The term “isolated and purified” refers to any bacteriophage that has been removed and purified from its natural environment and is propagated and preserved under controlled artificial conditions.

[0047] The term “plant crop” should be understood as any plant or plant species and / or the environment in which said plant grows and develops.

[0048] The terms “bacteriophage” and “phage” will be used interchangeably in this description to refer to viruses that infect prokaryotic cells.

[0049] A first object of the present invention is an isolated and purified lytic bacteriophage that infects strains of the species Pseudomonas syringae, which is selected from the group consisting of the bacteriophage deposited with the International Depositary Authority of Canada, with the access code 280823-01 (called phage E), the bacteriophage deposited with the International Depositary Authority of Canada, with the access code 280823-02 (called phage 21 1 1), and the bacteriophage deposited with the International Depositary Authority of Canada, with the access code 280823-03 (called phage EGF).

[0050] All three bacteriophages mentioned have icosahedral and hexagonal capsids approximately 50 nm in diameter and short rigid tails, suggesting that they belong to the Podoviridae family, which is part of the Caudovirales order. Furthermore, all three bacteriophages possess double-stranded DNA genomes with approximate molecular sizes shown in Table 1.

[0051] Table 1. Molecular sizes of the bacteriophage genome

[0052] In a preferred embodiment, the bacteriophages preferentially infect the pathovars P. syringae pv. tomato, P. syringae pv. phaseolicola and P. syringae pv. syringae but it is not excluded that they are capable of infecting and lysing other pathovars. In an even more preferred embodiment, these bacteriophages are capable of infecting and lysing the strains P. syringae pv. tomato 500, P. syringae pv. tomato 792, P. syringae pv. tomato 856, P. syringae pv. tomato DC3000, P. syringae pv. phaseolicola PSP and P. syringae pv. syringae PC3500

[0053] A second object of the present invention is a bactericidal composition for the biological control of diseases caused by strains of the species Pseudomonas syríngae in plant crops. Preferably, this bactericidal composition is useful for the prevention and / or treatment of bacterial spot or speck in tomatoes (Solanum lycopersicum), which is caused by the strain Pseudomonas syríngae pv. tomato, for the prevention and / or treatment of halo blight disease affecting beans (Phaseolus vulgaris), which is caused by infection with the strain Pseudomonas syríngae pv. phaseolicola and for bacterial canker (cancer) in cherry trees (Prunus cerasus), a disease caused by Pseudomonas syríngae pv. syríngae. The bactericidal composition of the present invention comprises an effective amount of a bacteriophage that infects strains of the species Pseudomonas syringae and an agronomically acceptable additive.Said bacteriophage may be any of the three deposited with the International Depositary Authority of Canada, under accession code 280823-01 (phage E), 280823-02 (phage 21 1 1); or 280823-03 (phage EGF); or it may be a mixture of at least two of these bacteriophages, preferably a mixture of all three.

[0054] In a preferred embodiment, for the composition to be effective against strains of the species Pseudomonas syringae, it must contain an amount of at least 1 x10 6 PFU (plaque forming units) of the bacteriophage per mL of composition, preferably between 1 x10 7 and 1x10 9 PFU of bacteriophage per mL of composition, even more preferably 1x10 8 PFU of the bacteriophage per mL of composition.

[0055] In one embodiment of the present invention, the bactericidal composition contains at least one agronomically acceptable additive that can provide stability to the bacteriophages, or maintain their viability, or even increase their efficacy. The additive present in the composition can be a carrier or vehicle, an adhesion promoter, an osmoprotectant, an anti-drift adjuvant, a UV radiation protection agent, or any other compound that enhances the action of the bacteriophages and / or improves the physical characteristics of the composition.

[0056] The carrier or vehicle is preferably bentonite in a concentration between 0.05% and 0.1% (w / v); the adhesive agent is preferably magnesium sulfate in a concentration between 0.1% and 0.15% (w / v); the osmoprotectant is preferably glycerol in a concentration between 0.1% and 0.15% (w / v); the anti-drift adjuvant is preferably sucrose in a concentration between 0.2% and 0.3% (w / v); and the UV protection agent is preferably soy milk in a concentration between 0.5% and 0.75% (w / v).

[0057] A third object of the present invention is a method for the biological control of diseases caused by strains of the species Pseudomonas syringae in parts of plants or plant crops, comprising the steps of providing a bactericidal composition comprising an effective amount of a bacteriophage that infects strains of the species Pseudomonas syringae, and applying said composition to parts of plants or plant crops. Said bacteriophage is selected from the group consisting of the bacteriophage deposited with the International Depositary Authority of Canada, with access code 280823-01 (called phage E), the bacteriophage deposited with the International Depositary Authority of Canada, with access code 280823-02 (called phage21 11), and the bacteriophage deposited with the International Depositary Authority of Canada, with access code 280823-03 (called phageEGF); or a mixture of at least two of the bacteriophages called E, 21, 11 and EGF.

[0058] In a preferred embodiment of the method of the present invention, the composition has an effective amount between 1x10 7 and 1 x10 9 PFU of the bacteriophage per mL of composition, more preferably between 1 x10 8 and 1x10 9 PFU of the bacteriophage per mL of composition.

[0059] In another preferred embodiment of the method of the present invention, the composition is applied to parts of the plant or plant crop, which are selected from the group consisting of seeds, stems, leaves, flowers, roots, fruits, bark, branches, trunk and soil.

[0060] Preferably, the plant crops are selected from the group consisting of plants of the genera Solanum and Phaseolus, Prunus, Malus and Pyrus, even more preferably from the species Solanum lycopersicum, Phaseolus vulgaris, Prunus cerasus, Prunus armeniaca, Prunus domestica, Malus domestica and Pyrus communis L.

[0061] In a preferred embodiment of the method of the present invention, between 150 and 15,000 L / ha of the composition is applied to the plant crop, more preferably between 1,000 and 2,000 L / ha of the composition to said plant crop.

[0062] In another embodiment of the present invention, the composition is applied to the plant crop using a technique selected from the group consisting of direct soil injection, drip irrigation, sprinkling by wetting the entire plant, spraying, and manual watering. The following are examples of embodiments of the invention, which have been included for the purpose of illustrating the invention, its preferred embodiments, and comparative examples, but in no case should they be considered to restrict the scope of the patent application, which is only delimited by the content of the claims attached hereto.

[0063] EXAMPLES OF IMPLEMENTATION

[0064] Example 1. Isolation and characterization of Utic bacteriophages that infect P. syringae pathovars

[0065] Leaf samples were obtained from kiwi (Actinidia deliciosa) plants at the Viconto fruit farm (Lat., Long.: -33.74715594404317; -70.7892222822868) and irrigation ditch water samples from the Caperana estate, Terramater (Lat., Long.: -33.736361588969, -70.858237612128) in the Metropolitan Region, Chile. Both samplings were conducted in April. Leaf samples were taken randomly and stored in Falcon tubes, which were then stored in an insulated box to maintain a constant temperature (20°C). In the laboratory, solid samples were incubated overnight with shaking at 25°C in phosphate-buffered saline (PBS), while water samples were stored at 4°C. The following day, the samples were concentrated on Amicon columns (50 kDa filter), 500 pL of these samples were added to cultures belonging to different strains of Pseudomonas syringae and incubated overnight.The following day, each culture was centrifuged and the obtained supernatant was added to fresh cultures. This procedure, called enrichment technique (Van Twest R., Kropinski AM (2009) Bacteriophage Enrichment from Water and Soil. In: Clokie MR, Kropinski AM (Ed.s) Bacteriophages (pp. 15-21 )), was carried out for 4 days in order to increase the analyzed samples with possible viral particles. To isolate the phages, a double agar layer assay was performed (Abedon ST, Yin J. (2009) Bacteriophage plaques: theory and analysis. In: Clokie MR, Kropinski AM (Eds.) Bacteriophages (pp. 161 -174)), a lysis plaque was chopped with a sterile toothpick and resuspended in 1 mL of PBS. Using this assay, three phages were isolated, which were named E, EGF and 21 1 1.The viral structure of the isolated phages was determined by transmission electron microscopy (TEM), visualizing icosahedral and hexagonal capsids of approximately 50 nm in diameter and with small rigid tails, suggesting that they belong to the Podoviridae family, which is within the order Caudovirales (FIGs. 1 A, 1 B and 1 C). FIGs. 2A, 2B and 2C show the adsorption of phages 211 1 , E and EGF, respectively, on the surface of P. syringae pv. tomato DC3000 cells. This morphological characterization plus the genome sizes of these three bacteriophages (Table 1 ) allows us to assume that they correspond to members of the Podoviridae family, group C1 .

[0066] Bioinformatic analysis of the complete genome DNA sequence of the three bacteriophages revealed that no genes were found whose products participate in the integration of the viral genome into the bacterial genome. Nor were probable recombination sites between the two genomes (bacterial and phage). These results demonstrate and confirm that the phages do not follow a lysogenic cycle and are 100% lytic, permanently producing productive infections that guarantee their effectiveness in controlling Pseudomonas syringae. The three bacteriophages were shown to exhibit lytic activity against P. syringae pv. tomato strains DC3000, 792, and 500, P. syringae pv. phaseolicola strain PSP, and P. syringae pv. syringae PC3500, producing clear lysis plaques on solid medium (Figs. 3A, 3B, and 3C).

[0067] Example 2. Determination of the host range of bacteriophages and optimal multiplicity of infection (MOI)

[0068] In order to determine the host range of the three bacteriophages object of the invention, different strains were grown in liquid King B medium overnight with shaking at 30 ° C. Signal dilutions were prepared from an aliquot of viral suspension and analyzed by double agar layer assays to determine the infection susceptibility against the phages (Di Lallo, G., Evangelist!, M., Mancuso, F., Ferrante, P., Marcelletti, S., Tinari, A., & Scortichini, M. (2014). Isolation and partial characterization of bacteriophages infecting Pseudomonas syringae pv. actinidiae, causal agent of kiwifruit bacterial canker. Journal of Basic Microbiology, 54 (1 1 ), 1210-1221 ). Pseudomonas fluorescens was used as a control bacterial strain. Table 2 presents the results of the test carried out with different strains of Pseudomonas syringae corresponding to the pathovars tomato, actinidae, syringae and phaseolicola.

[0069] Table 2. Lytic activity of bacteriophages

[0070] Additionally, the adsorption rate of each virus was determined. To do this, a culture of P. syringae pv. tomato DC3000 was grown to exponential phase and infected with bacteriophages at an MOI of 0.01. Subsequently, 1 mL aliquots were taken every 2 min for 20 min, filtered, centrifuged for 2 min at 4°C, and serial dilutions were made from an aliquot of the resulting supernatant at each time point. The result was visualized by a double-layer agar assay. Adsorption was expressed as a percentage, as the decrease in free phage in the medium from time 0 of viral infection. The virus adsorption rate constant (k) was calculated from the equation

[0071] 2.3 Po where B is the initial bacterial count and t is the time it takes for free phage to decrease from an initial maximum (Po) to half (P). The time interval in which this decrease occurs was determined by linear regression from the graph obtained (Barry, GT, & Goebel, WF (1951 ). The effect of chemical and physical agents on the phage receptor of Phase II Shigella sonnet. Journal of Experimental Medicine, 94(5), 387-400).

[0072] The adsorption rates of phages 21 1 1 , E, and EGF were 21.5%, 14.4%, and 15.1% adsorbed bacteriophages / min, respectively (Figs. 4B, 5B, and 6B). The latency times of phages 21 1 1 , E, and EGF were 2, 6, and 4 min, respectively (Figs. 4A, 5A, and 6A).

[0073] In addition to the above, the number of viral particles released per infected cell (burst size) was determined. To this end, a culture of P. syringae pv. tomato DC3000 was grown to exponential phase and infected with bacteriophages at an MOI of 0.01. It was incubated for 20 min at 100 rpm, 25°C, to adsorb the phages to the bacteria. It was then centrifuged and the obtained pellet was resuspended in the same volume. Aliquots of the medium were taken every 20 min and the viral titer was immediately determined by a double agar layer assay. The burst size was calculated as the ratio between the number of viral particles released and the initial number of viruses with which the bacterial cells were infected (Adams, MH (1959). Bacteriophages. Interscience Publishers, 450-456).

[0074] The magnitude of viral multiplication was 29.3, 1 1 ,7 and 604 for 21 1 1 , E and EGF, respectively (FIG. 7). In turn, the optimal multiplicity of infection (MOI) was determined to be 0.01 , since with this the best lytic efficiency and the shortest bacterial death time (2 hours) were obtained, with each virus separately (FIGs. 8A, 8B, 9A, 9B, 10A and 10B).

[0075] In accordance with the above, when the combination of the three bacteriophages was used at different MOIs (0.1, 0.01, 0.001), it was observed that the lytic efficiency at an MOI of 0.01 was similar to that observed with each virus separately, obtaining a bacterial death time at 2 hours, while when using an MOI of 0.1, the bacterial death time decreased to 1 hour (FIGs. 11 A and 11 B). Although a shorter bacterial death time is obtained with an MOI of 0.1, this can be limiting, since when spreading viruses, rapid bacterial lysis is obtained and also a reduction in the amount of bacteria to infect, thus reducing the final yield (Fortier, LC, & Moineau, S. (2009). Phage production and maintenance of stocks, including expected stock lifetimes. (Eds) Bacteriophages, pp. 203-219).With these data it was determined that the optimal propagation conditions for the composition are an MOI of 0.01, orbital shaking at 130 rpm for 4 hours at 25°C. Example 3. Evaluation of the compatibility of bacteriophages with different additives.

[0076] To determine the importance of additives in the composition, bacteriophage viability was evaluated over time with each component separately, in order to determine whether these components affected their infectivity. For this purpose, different molecules categorized according to their characteristics and function in the composition were used (Table 3). For the evaluation, each additive was added to the bacteriophage suspension separately at the concentration indicated in said table, and the mixtures were stored at 4°C and 25°C for 14 days. Bacteriophage viability was assessed every 3 days using a double-layer agar infection assay.

[0077] Table 3. Additives that maintain the viability of the active ingredient

[0078] On average, at the start of the test, the number of bacteriophages that were quantified varied between 5x10 9 and 1x10 10 PFU / mL. A decrease in the number of viable bacteriophages was observed at both 4°C and 25°C after 14 days of storage (Figs. 12A, 12B, 13A, and 13B). In the case of phage E, this decrease was 20% in samples stored at 4°C (Fig. 12A) and 5% in those stored at 25°C (Fig. 12B). For phage 21 1 1, the decrease was 3% at 4°C (Fig. 13A) and 6% when stored at 25°C (Fig. 13B). In both cases, no differences in bacteriophage viability were observed between those maintained with the additives and the control without them. Example 4. Determination of bacteriophage viability

[0079] Once the compatibility of the bacteriophages with the additives was determined, the viability over time of bacteriophages E, 21, 11 and EGF on cherry tomato plants (Solarium lycopersicum var. cerasiforme) was evaluated in a greenhouse. For this purpose, 15 treatments were designed with each bacteriophage (contained in phosphate-buffered saline) separately and their mixture in pairs and trios (Table 4). The viability of the bacteriophages was determined by a double-layer agar assay with P. syríngae pv. tomato DC3000. Samples were obtained from tomato leaves biocontrolled with the respective treatments. Table 4. Bacteriophage treatments on cherry tomato plants.

[0080] By applying the bacteriophages individually to a 1 x 10 suspension 6 UFP / mL (FIG. 14A), it was observed that the active ingredient drops to zero after one week after application, while when applied to a suspension of 1 x10 8PFU / mL (FIG. 14B), it was observed that the bacteriophages remained viable for two weeks, and then dropped to zero as observed after the fourth application. In the case of using the bacteriophage pairs (FIGs. 15, 16 and 17), the suspensions were generated so that one of the bacteriophages was at a dilution of 1 x 10 6 UFP / mL and the other at a dilution of 1 x10 8 PFU / mL. All 6 treatments (F8, F9, F10, F11, F12, and F13) showed the ability to remain viable for 14 days, regardless of the combination used. This was similar to what was observed with the individual application of phages to a 1 x 10 suspension. 8 PFU / mL. Regarding the treatments prepared with the three bacteriophages together (FIG. 18), a similar behavior to that described above was observed, since when using the three bacteriophages at a suspension of 1x10 6PFU / mL, viability drops to zero after 7 days after application, however, by maintaining the phages at a suspension of 1 x10 8 PFU / mL viability remained stable for 14 days. The set of results obtained from the application of the compositions in plants show that even when the titer drops by 3 to 4 orders of magnitude, the composition containing at least one of the phages in a suspension of 1 x 10 8 UFP / mL, maintains viability for 14 days, regardless of which of the three bacteriophages are added to the composition.

[0081] Example 5. Plant protection trials of cherry tomatoes against infection with P. syringae pv. tomato DC3000

[0082] In vivo protection tests against infection with the P. syringae pv. tomato DC3000 strain were performed on cherry tomato plants. First, in vivo protection was evaluated with different bactericides, applying separately the compounds copper sulfate at 0.075% w / v, streptomycin at 150 pg / mL, and a phage 21 1 1 suspension of 1 x 10 9 PFU / mL (MOI 0.01), 24 h before infection with the bacteria. The plants were incubated at 20°C with a 12-h photoperiod. To quantify the bacteria, leaves were randomly collected from each treatment, ground with a mortar and pestle, and incubated with phosphate-buffered saline for 2 h. Colony-forming units (CFU) were then quantified by microdroplet assay.

[0083] A decrease in the quantified amount of bacteria was observed as the days went by (FIG. 19). In turn, better control of the infection was observed with the phage suspension 21 1 1 , where the amount of bacteria was maintained two orders of magnitude (8.2x10 4 CFU / g) below control (1.6x10 6 CFU / g). Therefore, greater protection was determined with phage 21 1 1 after 7 days post-infection, compared to the other treatments used.

[0084] An in vivo protection test was also performed with the prototype of the composition containing the combination of the three bacteriophages as the active ingredient. The formulation was applied by spraying to a final suspension of 1 x 10 8 PFU / mL, 24 hours prior to infection with the bacteria. The following day, the bacteria was sprayed to a final suspension of 1 x 10 7. Plants were incubated with a 12-h photoperiod at 24°C. To quantify the bacteria, leaves were randomly collected from each treatment, ground with a mortar and pestle, and incubated with phosphate-buffered saline for 2 h. Colony-forming units (CFU) were then quantified using a microdroplet assay.

[0085] A decrease in the bacterial population was observed after two weeks (FIG. 20). In plant 1, a drop of one order of magnitude was observed, from 2.1 x 10 6 at 2.9x10 5 CFU / g between day 4 and day 14. Likewise, on plant 2, a similar behavior was observed, with the amount of bacteria decreasing from 2.5x10 6 at 2.1 x10 5 CFU / g. In contrast, an increase in the bacterial population of 2.0x10 was observed in untreated plants. 6 at 5.0x10 7 CFU / g in plant 3 and an increase of 2.3 x10 6 at 5.9 x10 7CFU / g in plant 4. The results revealed effective control of the composition, preventing the spread of the bacteria in the plant.

Claims

CLAIMS 1. An isolated and purified lytic bacteriophage that infects strains of the species Pseudomonas syríngae, CHARACTERIZED in that it is selected from the group consisting of the bacteriophage deposited with the International Depositary Authority of Canada, under access code 280823-01 (bacteriophage E), the bacteriophage deposited with the International Depositary Authority of Canada, under access code 280823-02 (bactehóphage21 1 1), and the bacteriophage deposited with the International Depositary Authority of Canada, under access code 280823-03 (bactehóphageEGF).

2. A bactericidal composition for the control of diseases caused by strains of the species Pseudomonas syríngae in plant crops, CHARACTERIZED in that it comprises an effective amount of a bacteriophage that infects strains of the species Pseudomonas syríngae that is selected from the group consisting of: - a bacteriophage deposited with the International Depositary Authority of Canada, under accession code 280823-01 (bacteriophage E); - a bacteriophage deposited with the International Depositary Authority of Canada, under accession code 280823-02 (bacteriophage21 1 1 ); - a bacteriophage deposited with the International Depositary Authority of Canada, under accession code 280823-03 (bactehophageEGF); - a mixture of at least two of the bacteriophages E, EGF and 21 1 1; and an agronomically acceptable additive.

3. The composition according to claim 2, CHARACTERIZED in that said effective amount includes between 1 x10 7 and 1 x10 9 PFU of the bacteriophage per mL of composition.

4. The composition according to claim 2, CHARACTERIZED in that the additive is selected from the group consisting of a carrier, an adhesive agent, an osmoprotectant, an anti-drift adjuvant and a UV radiation protective agent.

5. The composition according to claim 4, CHARACTERIZED in that the carrier is bentonite in a concentration between 0.05% and 0.1% (w / v).

6. The composition according to claim 4, CHARACTERIZED in that the adhesive agent is magnesium sulfate in a concentration between 0.1% and 0.15% (w / v).

7. The composition according to claim 4, CHARACTERIZED in that the osmoprotector is glycerol in a concentration between 0.1% and 0.15% (w / v) 8. The composition according to claim 4, CHARACTERIZED in that the anti-drift adjuvant is sucrose in a concentration between 0.2% and 0.3% (w / v).

9. The composition according to claim 4, CHARACTERIZED in that the UV radiation protective agent is soy milk in a concentration between 0.5% and 0.75% (w / v).

10. A method for the biological control of diseases caused by strains of the species Pseudomonas syringae in plant crops, CHARACTERIZED because it includes the stages of: - providing a bactericidal composition comprising an effective amount of a bacteriophage that infects strains of the species Pseudomonas syringae that is selected from the group consisting of: - a bacteriophage deposited with the International Depositary Authority of Canada, under accession code 280823-01 (bacteriophage E); - a bacteriophage deposited with the International Depositary Authority of Canada, under accession code 280823-02 (bacteriophage2111); - a bacteriophage deposited with the International Depositary Authority of Canada, under accession code 280823-03 (bacteriophageEGF); - a mixture of at least two of the bacteriophages E, EGF, and 2111; and applying said composition to parts of the plant or plant crop. 1 1. The method according to claim 10, CHARACTERIZED in that said effective amount includes between 1 x10 7 and 1 x10 9 PFU of the bacteriophage per mL of composition.

12. The method according to claim 10, CHARACTERIZED in that it comprises applying the composition to parts of the plant or plant crop, which are selected from the group consisting of seeds, stems, leaves, flowers, roots, fruits, and soil.

13. The method according to claim 10, CHARACTERIZED in that said plant crop is selected from the group consisting of crops of the genera Solanum, Phaseolus, Prunus, Malus and Pyrus.

14. The method according to claim 12, CHARACTERIZED in that it comprises applying between 150 and 15,000 L / ha of the composition to the plants or trees of the vegetable crop.

15. The method according to claim 12, CHARACTERIZED in that it comprises applying the composition by a technique selected from the group consisting of direct injection into the soil, drip irrigation, sprinkling by completely wetting the plant, spraying and manual watering.

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