Bacteriophage capable of infecting xanthomonas arboricola pv. juglandis bacteria and its use

Abstract

The invention relates to an isolated bacteriophage deposited under the accession number DSM 34666 at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ); Inhoffenstraße 7B, 38124 Braunschweig, Science Campus Braunschweig-Süd, Germany) on May 25, 2023; or a derivative, variant or mutant thereof, the genomic sequence of which is at least 95% identical to the sequence of SEQ ID NO: 1. Preferably, the bacteriophage is capable of killing the bacterium Xanthomonas arboricola pv. juglandis.. The invention also relates to a composition comprising the bacteriophage. The invention also relates to the use of the bacteriophage or the composition comprising the bacteriophage on a plant for the prevention or treatment of a bacterial infection caused by Xanthomonas arboricola pv. juglandis.

Description

[0001] Bacteriophage capable of infecting Xanthomonas arboricola pv. juglandis bacteria and its use

[0002] FIELD OF THE INVENTION

[0003] The invention relates to an isolated bacteriophage capable of infecting Xanthomonas arboricola pv. juglandis (Xaj) bacteria. The invention also relates to compositions comprising the bacteriophage and a method for producing the bacteriophage, as well as the composition. The invention also relates to the use of the bacteriophage or the composition comprising the bacteriophage for the prevention or treatment of a bacterial infection caused by Xanthomonas arboricola pv . juglandis (Xaj). Furthermore, a method for the prevention or treatment of an infection wi Xanthomonas arboricola pv . juglandis (Xaj).

[0004] TECHNICAL BACKGROUND

[0005] Plants are constantly exposed to numerous abiotic pressure and biotic jeopardy. One major biothreat is bacterial pathogens causing various diseases, critically diminishing crop yields, and threatening worldwide food security. The necessity of developing sustainable biocontrol agents is markedly rising.

[0006] The Xanthomonas genus contains more than forty bacterial species causing diseases in over 400 plant species [Stefani et al., 2021], One of these Gram -negative bacterium species is Xanthomonas arboricola pv . juglandis (Xaj), a member of Gammaproteobacteria, the causative agent of the bacterial blight of walnut [Frutos, 2010], Xaj causes leaf and fruit spot of walnuts, and according to some authors, Xaj is the most significant above-ground bacterial pathogen infecting walnuts [Assis et al., 2021],

[0007] The disease can be controlled with copper or antibiotics.

[0008] The disadvantage of antibiotic treatment is that prolonged antibiotic treatment in a given area - for years or even decades - leads to the development of resistance.

[0009] The other common method of protection against Xaj infection is the use of copper-containing compounds. However, copper treatment can also induce resistance [Assis etal., 2021], which can easily spread between Xaj strains [Kaluzna etal., 2021], The likelihood of copper resistance spreading between Xaj strains is increased by copper treatments (through selection pressure), and in fact, the presence of these genes can be detected, even on plasmids. Carrying them on a plasmid further increases the mobility of these genes.

[0010] In addition to the emergence of resistance to copper or antibiotic treatments, these treatments also have a serious environmental impact.

[0011] The current control methods are not effective and / or burden the environment and / or are illegal (like the usage of streptomycin in most countries) [Pereira et al., 2015; Giovanardi et al., 2016; Stefani et al., 2021], There are limited tools available for organic production. Recently, a novel biopesticide containing the Bacillus subtilis QST 713 strain has been registered against Xaj (Bayer), which has a broad antibacterial spectrum.

[0012] The emergence of antibiotic and copper resistance has prompted researchers to find alternative, yet effective solutions against bacterial infections in orchards. One such alternative method is bacteriophage therapy, which is an environmentally friendly and side -effect-free control option in plant protection.

[0013] The reemerging phage therapy approach to control plant-pathogenic bacteria is promising: cost- effective, eco-friendly, extremely specific, and safe for the host plant.

[0014] However, given the narrow spectrum of phages, it is widely believed [Okabe and Goto, 1963] that phages are not suitable for meaningful use in plant protection. To overcome the obstacles, phage cocktails containing several phage strains began to be used, and lysogenic phages were excluded from use.

[0015] The development of phage resistance, which initially caused problems in treatments, has become a manageable problem partly by using so-called h-mutant phages (phages with extended host specificity, see patent US4828999), and partly by using several phages with broad host specificity simultaneously. Another problem was caused by the phenomenon of transduction. Transduction is when a phage transfers DNA from the host bacterium. Two types are known: specialized and generalized transduction. The prerequisite for specialized transduction is that the phage DNA is integrated into the chromosome or plasmid of the host bacterium, and then exits from there, taking with it a piece of host DNA adjacent to the site of integration. Generalized transduction is characterized by the fact that the phage packages any DNA that is similar in size to the phage DNA. The prerequisite for this is that the phage DNA is packaged into the capsid by the so-called headful mechanism, and the point where the DNA is attached to the capsid is loosely defined. (For more on transduction, see Waldor and Friedman, 2005). Fortunately, not all phages are capable of generalized or specialized transduction. The use of phages capable of transduction should be avoided in phage therapy. Their screening can be achieved in a simple experiment: the phages are grown on a host bacterium that is resistant to an antibiotic, and then the sterile-filtered phage population is combined with a host bacterium that is sensitive to said antibiotic. If, after co-cultivation with the phages, the survivors of this bacterium are spread out on a medium comprising said antibiotic and colonies are obtained with a relatively high frequency, then the phage is capable of transduction. (The type of transduction can be determined by further studies, but this is beyond the scope of phage therapy experiments.)

[0016] Bacteriophages (or phages for short), viruses infecting bacteria exclusively, are good candidates for active ingredients of biopesticides. A recent review summarizes the feasibility of applications of bacteriophages as agents for the biological control of Xanthomonads [Stefani etal., 2021], Phages have significant advantages over traditional chemical pesticides, including specificity (minimalizing the intervention in the natural microflora), efficacy, and decomposition after treatment [Greer, 2005; Jones etal., 2007; Hathaway etal., 2017; Abrahamian etal., 2019; Liu etal., 2020], UV sensitivity of phages, resistance and immunity emerging in the host bacteria during treatment, and narrow host range are important limitations of the bacteriophage therapy against phytopathogenic bacteria [Jones et al., 2005; Obradovic et al. , 2020; Stefani et al. , 2021], The application of uncharacterized phages may strengthen the pathogenicity of the host bacterium, which feature is mainly caused by the horizontal gene transfer mediated by temperate phages [Kovacs et al., 2021; Stefani et al., 2021], Therefore, a thorough characterization of bacteriophages to be applied for biocontrol is a must. This should include the determination of the complete genomic sequence of the phages, besides the morphologic characterization, host range, and efficacy investigations.

[0017] The first isolation of bacteriophages against Xaj was reported from soil samples in New Zealand [McNeil et al., 2001], This study contained a deep characterization of the isolated phages, albeit no complete genomic sequences (only sequence fragments) were reported. Twenty-six Podoviridae and Siphoviridae Xaj bacteriophages were isolated 11 years later [Romero-Suarez et al., 2012], The first complete genomes of two Xaj phages were reported in 2016 [Domotor et al., 2016], Also, the genomic sequences of three Xaj bacteriophages were published in the same year [Retamales et al., 2016],

[0018] Bacteriophages against Xanthomonas arboricola pv. juglandis have been isolated and characterized by several research groups (comprehensive review: Stefani et al., 2021).

[0019] Romero-Suarez et al. (2012) isolated bacteriophages infecting Xanthomonas arboricola pv. juglandis in New Zealand. A total of 26 X. arboricola phages were identified and characterized. Some of the phages belonged to the Podoviridae family, while others belonged to the Siphoviridae family. They hypothesized that these phages might be suitable for use in biocontrol, and in this regard, they examined how long the phages remained viable under different storage conditions. According to their results, the phages were cold-sensitive, media (chloroform)-sensitive, and they were also affected by storage time, which led them to conclude that although there is a possibility that a group of phages could be used in the control of Xanthomonas arboricola, phage efficacy may depend on different environmental conditions, and therefore further studies are necessary.

[0020] Domotor et al. (2016) identified 24 bacteriophages from soil and walnut aerial tissues infected with Xanthomonas arboricola pv. juglandis (Xaj). Of the isolated bacteriophages, two polyvalent bacteriophages were characterized in detail, belonging to the Siphoviridae and Podoviridae families, respectively. Both phages demonstrated lytic effect on Xaj bacteria under laboratory conditions. The complete genome sequences of the phages were also determined, which consisted of a total of 49214 and 44861 nucleotides encoding 80 and 53 genes, respectively. The sequences are available in the GenBank database under accession numbers KU197014 and KU197013.

[0021] The first field trial results were reported just recently [Retamales et al., 2022], In this study, a cocktail of three bacteriophages was applied in different doses on Chandler variety walnut trees in Chile. A significant reduction in disease severity was observed. Retamales etal. (2022) described specific lytic bacteriophages ( / 20-Xaj, / 29-Xaj and / 30-Xaj) against Xanthomonas arboricola pv. juglandis strains isolated in Chile and France. The phages belong to the Pradovirus genus. The application of a mixture of these bacteriophages showed similar effectivity to treatment with copper-containing compounds (e.g. C11SO4). and moreover, the bacterial load of Xanthomonas arboricola pv. juglandis was significantly reduced in the presence of the bacteriophages. The morphology of the three phages described is typical of Podovirus (head with a diameter of about 50 nm and short stubby tail). The authors also reported the complete genomes of two of the three phages, / 20-Xaj and / 30-Xaj (GenBank accession numbers of KU595432 and KU595433, respectively). The genome of / 20-Xaj is about 43.9 kbp, while the genome of / 30-Xaj is about 44.3 kbp. Based on the genomes, the phages were classified into the Pradovirus genus (Podoviridae family) (in accordance with the morphology). When compared with the NCBI Blastn algorithm, the complete genomes of / 20-Xaj and / 30-Xaj show 98.46% identity with each other.

[0022] WO2017 / 113029A1 discloses a bacteriophage-based composition forthe prevention ortreatment of infection caused by Xanthomonas arboricolci pv . juglandis . The composition comprises at least one phage selected from bacteriophages with the following deposit numbers: PCM F / 00087, PCM F / 00088, PCM F / 00089, PCM F / 00090, PCM F / 00092, PCM F / 00091 and PCM F / 00093. The composition can be used for the prevention and / or treatment of infection by copper-resistant Xanthomonas arhoricola pv. juglandis in a plant (part). The plant is preferably a walnut. Of the 7 bacteriophages disclosed, 6 phages are of the Podovirus type (family Podoviridae), while 1 phage is of the Sifovirus type (family Siphoviridae). The complete genome sequences of the phages were not determined, instead they were distinguished and identified by the RFLP (restriction fragment length profile) method. The genome size of the phages is about 38 to 42 kbp (there were no data for two phages).

[0023] W02023191071A1 discloses a novel bacteriophage for the prevention of plant diseases caused by bacteria of the genus Xanthomonas, and a composition containing the same. The disclosed bacteriophage contains a gene encoding a tail fiber protein in its genomic DNA, which protein has target bacterium recognition activity.

[0024] Due to the relatively narrow host specificity of bacteriophages and the presence of different Xaj strains, there is still a need to isolate bacteriophages that are capable of killing different strains of X. arhoricola pv . juglandis .

[0025] The aim of the solution according to the invention was therefore to isolate novel bacteriophages effective against Xaj bacterium and to produce compositions comprising them.

[0026] BRIEF DESCRIPTION OF THE INVENTION

[0027] The invention relates to a bacteriophage deposited under the accession number DSM 34666 at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ); InhoffenstraBe 7B, 38124 Braunschweig, Science Campus Braunschweig-Siid, Germany) on May 25, 2023, the genomic sequence of which is optionally the sequence of SEQ ID NO: 1.

[0028] Furthermore, a bacteriophage deposited under the accession number DSM 34666 at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ); InhoffenstraBe 7B, 38124 Braunschweig, Science Campus Braunschweig-Siid, Germany) on May 25, 2023; or a derivative, variant or mutant thereof, the genomic sequence of which is at least 80% identical to the sequence of SEQ ID NO: 1. Preferably, the genomic sequence of the bacteriophage or derivative, variant or mutant thereof is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the sequence of SEQ ID NO: 1.

[0029] Preferably, the genomic sequence of the bacteriophage or derivative, variant or mutant thereof is at least 95% identical to the sequence of SEQ ID NO: 1.

[0030] Preferably, the genomic sequence of the bacteriophage is the sequence of SEQ ID NO: 1 or a sequence at least 95% identical thereto.

[0031] Preferably, the genomic sequence of the bacteriophage or derivative, variant or mutant thereof does not contain an integrase gene.

[0032] Preferably, the bacteriophage is capable of infecting, damaging or killing the bacterium Xanthomonas arboricolci pv . juglandis or is capable of inhibiting its growth.

[0033] Preferably, the bacteriophage is capable of killing Xanthomonas arhoricola pv. juglandis bacteria. Optionally, the Xanthomonas arhoricola pv. juglandis is a Xanthomonas arhoricola pv. juglandis bacterium or bacterial strain resistant to an antibiotic and / or to a copper-containing compound.

[0034] Preferably, the bacteriophage is capable of killing Xanthomonas arhoricola pv. juglandis, optionally resistant to an antibiotic and / or to a copper-containing compound, wherein preferably the bacteriophage capable of killing Xanthomonas arhoricola pv. juglandis forms plaque in a plaque assay, wherein optionally the plaque assay comprises the following steps: i) preparing a multi-step, for example five-step, decimal dilution from a stock solution comprising the bacteriophage, ii) adding a drop, for example about 0. 1 mL, of the dilutions to a Xanthomonas arhoricola pv. juglandis bacterial lawn, wherein optionally the bacterium is resistant to an antibiotic and / or a copper-containing compound, iii) covering the bacterial lawn with a gel-forming substance, thereby forming a gel, iv) examining the gel after a given time to determine whether plaques have formed, v) if plaques have formed, the bacteriophage is capable of killing Xanthomonas arhoricola pv. juglandis, optionally resistant to an antibiotic and / or to a copper-containing compound, if plaques have not formed, the bacteriophage is not capable of killing Xanthomonas arhoricola pv. juglandis, optionally resistant to an antibiotic and / or to a copper-containing compound.

[0035] Preferably, the bacteriophage is isolated or produced by another technical process.

[0036] The invention also relates to a composition comprising any of the bacteriophages as defined above.

[0037] In an embodiment, the composition comprises the bacteriophage deposited under the accession number DSM 34666 at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ); InhoffenstraBe 7B, 38124 Braunschweig, Science Campus Braunschweig-Siid, Germany) on May 25, 2023.

[0038] Furthermore, a composition comprising any of the bacteriophages as defined above and additional bacteriophage(s), preferably additional bacteriophage(s) against Xanthomonas arboricola juglandis. Preferably, the composition comprises at least two bacteriophages. Preferably, the composition comprises at least three bacteriophages. Preferably, the composition comprises at least four bacteriophages. Preferably, the composition comprises at least five bacteriophages. Preferably, the composition comprises at least six bacteriophages. In an embodiment, one of the bacteriophages in the composition is a bacteriophage deposited under the accession number DSM 34666 at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ); InhoffenstraBe 7B, 38124 Braunschweig, Science Campus Braunschweig-Siid, Germany) on May 25, 2023.

[0039] In an embodiment, the composition is in the form of a suspension comprising a diluted lysate of the bacteriophage(s). In a preferred embodiment, the composition comprises or consists of a suspension of a bacteriophage deposited under the accession number DSM 34666 at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ); InhoffenstraBe 7B, 38124 Braunschweig, Science Campus Braunschweig-Siid, Germany) on May 25, 2023.

[0040] In an embodiment, the composition is in the form of a lyophilisate, which further comprises an excipient, wherein optionally the excipient is a cryoprotectant, preferably sucrose. In an embodiment, the lyophilized composition comprises a bacteriophage deposited under the accession number DSM 34666 at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ); InhoffenstraBe 7B, 38124 Braunschweig, Science Campus Braunschweig-Siid, Germany) on May 25, 2023, and comprises a cryoprotectant, preferably sucrose.

[0041] A kit comprising any of the bacteriophages as defined above or any of the compositions as defined above and comprising a UV-protective agent. Preferably, the UV-protective agent is Na-alginate.

[0042] The invention also relates to the use of any of the bacteriophages or any of the compositions as defined above on a plant for the prevention or treatment of a bacterial infection caused by Xanthomonas arboricola pv . juglandis .

[0043] Preferably, in the use, the plant is a walnut tree (Juglans regia) or a European hazelnut (Corylus avellana). more preferably a walnut tree.

[0044] Preferably, in the use, the Xanthomonas arboricola pv. juglandis is a bacterium or bacterial strain resistant to an antibiotic and / or to a copper-containing compound.

[0045] In an embodiment, a bacteriophage deposited under the accession number DSM 34666 at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ); InhoffenstraBe 7B, 38124 Braunschweig, Science Campus Braunschweig- Slid, Germany) on May 25, 2023 is used on a plant for the prevention or treatment of a bacterial infection caused by Xanthomonas arboricolci pv . juglandis . Preferably, the plant is a walnut tree (Juglans regid) or a European hazelnut (Corylus ave Ilana), more preferably a walnut tree. Preferably, in the use, the Xanthomonas arhoricola pv . juglandis is a bacterium or bacterial strain resistant to an antibiotic and / or to a copper-containing compound.

[0046] In an embodiment, a composition comprising a bacteriophage deposited under the accession number DSM 34666 at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ); InhoffenstraBe 7B, 38124 Braunschweig, Science Campus Braunschweig-Siid, Germany) on May 25, 2023 is used on a plant for the prevention or treatment of a bacterial infection caused by Xanthomonas arhoricola pv . juglandis . Preferably, the plant is a walnut tree (Juglans regia) or a European hazelnut (Corylus avellana), more preferably a walnut tree. Preferably, the Xanthomonas arhoricola pv. juglandis is a bacterium or bacterial strain resistant to an antibiotic and / or to a copper-containing compound. Preferably, the composition is a suspension or a reconstituted lyophilized composition.

[0047] The invention also relates to a method for preventing or treating a bacterial infection of a plant caused by Xanthomonas arhoricola pv. juglandis, wherein the method comprises using any of the bacteriophages as defined above or any of the compositions as defined above on the plant or on a part of the plant or on a location of the plant.

[0048] Preferably, the part of the plant is the crown, leaf, trunk, stem, root or shoot of the plant. Preferably, the part of the plant is the foliage of the plant.

[0049] Preferably, the plant is a walnut tree (Juglans regia) or a European hazelnut (Corylus avellana), more preferably a walnut tree.

[0050] Preferably, the Xanthomonas arhoricola pv . juglandis is a bacterium or bacterial strain resistant to an antibiotic and / or to a copper-containing compound.

[0051] In an embodiment, the method comprises using a bacteriophage deposited under the accession number DSM 34666 at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ); InhoffenstraBe 7B, 38124 Braunschweig, Science Campus Braunschweig-Siid, Germany) on May 25, 2023 on the plant or on a part of the plant or on a location of the plant. Preferably, the part of the plant is the crown, leaf, trunk, stem, root or shoot of the plant. Preferably, the part of the plant is the foliage of the plant. Preferably, the plant is a walnut tree (Juglans regia) or a European hazelnut (Corylus avellana), more preferably a walnut tree. Preferably, the Xanthomonas arhoricola pv. juglandis is a bacterium or bacterial strain resistant to an antibiotic and / or to a copper-containing compound.

[0052] Preferably, the bacteriophage is isolated or produced by another technical process.

[0053] In a further embodiment, the method comprises using a composition comprising a bacteriophage deposited under the accession number DSM 34666 at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ); InhoffenstraBe 7B, 38124 Braunschweig, Science Campus Braunschweig-Siid, Germany) on May 25, 2023 on the plant or on a part of the plant or on a location of the plant. Preferably, the part of the plant is the crown, leaf, trunk, stem, root or shoot of the plant. Preferably, the part of the plant is the foliage of the plant. Preferably, the plant is a walnut tree (Juglans regid) or a European hazelnut (Corylus avelland), more preferably a walnut tree. Preferably, the Xanthomonas arboricolci pv. juglandis is a bacterium or bacterial strain resistant to an antibiotic and / or to a copper-containing compound. Preferably, the composition is a suspension or a reconstituted lyophilized composition.

[0054] The invention also relates a method for producing a bacteriophage, which comprises propagating the bacteriophage deposited under the accession number DSM 34666 at the German Collection of Microorganisms and Cell Cultures (DSMZ) on May 25, 2023 in a suitable host cell.

[0055] Preferably, the suitable host cell is the bacterium Xanthomonas arhoricola pv . juglandis . In an embodiment, the host cell is strain KFB 302; 235; 1105; 1155; 1317; XajHUl; XajHU2; DSMZ 1049 or NCAIM 02489 of the bacterium Xanthomonas arhoricola pv . juglandis .

[0056] In an embodiment, the method for producing a bacteriophage comprises:

[0057] (i) culturing Xanthomonas arhoricola pv. juglandis bacteria in a culture medium,

[0058] (ii) adding a bacteriophage deposited under the accession number DSM 34666 at the German Collection of Microorganisms and Cell Cultures (DSMZ) on May 25, 2023 to the incubated bacterial culture of (i),

[0059] (iii) incubating the culture obtained in step (ii), thereby producing the bacteriophage.

[0060] A method for producing a suspension comprising a bacteriophage deposited under the accession number DSM 34666 at the German Collection of Microorganisms and Cell Cultures (DSMZ) on May 25, 2023, comprising the following:

[0061] (i) growing the bacteriophage on Xanthomonas arhoricola pv. juglandis host bacterium, thereby producing a lysate,

[0062] (ii) centrifuging the lysate and filtering it to sterile,

[0063] (iii) diluting the lysate, thereby producing a suspension.

[0064] A method for producing a lyophilized composition comprising a bacteriophage deposited under the accession number DSM 34666 at the German Collection of Microorganisms and Cell Cultures (DSMZ) on May 25, 2023, comprising the following:

[0065] (i) growing the bacteriophage on Xanthomonas arhoricola pv. juglandis host bacterium, thereby producing a lysate,

[0066] (ii) centrifuging the lysate and filtering it to sterile,

[0067] (iii) diluting the lysate, thereby producing a suspension, (iv) lyophilizing the suspension, thereby producing a lyophilized composition.

[0068] Preferably, the lyophilization according to (iv) is carried out with the addition of a cryoprotectant, preferably sucrose.

[0069] Preferably, the concentration of the suspension is 1010to 1012PFU / mL.

[0070] DEFINITIONS

[0071] A “bacteriophage” or “phage” for short is a virus that can infect bacteria. Its two main types are lytic phage and lysogenic phage.

[0072] A “lytic phage” is a bacteriophage that reproduces according to a lytic cycle. In the lytic cycle, during the reproduction of viruses, the viral DNA replicates separately from the DNA of the host cell. In the lytic cycle, the infected bacterial cell is broken open and destroyed (lysis occurs). Phages that use the lytic cycle exclusively are also called virulent phages.

[0073] A “lysogenic” phage or “temperate” phage is a bacteriophage that reproduces according to a lysogenic cycle. In the lysogenic cycle, the viral DNA integrates into the DNA of the host cell and replicates with it. The lysogenic cycle does not result in the immediate destroying of the bacterial cell, since the viral DNA (prophage) incorporated into the host cell DNA is in a “dormant” state and is only activated when conditions in the bacterial cell deteriorate (e.g., decreasing nutrients). Only the reproductive cycle initiated by activation causes the lysis of the host cell. For this reason, lysogenic phages are not suitable for phage therapy treatment.

[0074] The bacteriophage “XajPHUl” refers to a bacteriophage deposited under the accession number DSM 34666 at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ for short; InhoffenstraBe 7B, 38124 Braunschweig, Science Campus Braunschweig-Siid, Germany) on May 25, 2023.

[0075] A “derivative” of a bacteriophage is a bacteriophage progeny produced by culturing, propagating or growing a given bacteriophage. The derivative is preferably genetically identical or substantially identical to the parent bacteriophage.

[0076] A “variant” refers to a subtype of a given bacteriophage that is genetically different from the given bacteriophage, but not so distinct in its characteristics as to be considered a separate bacteriophage.

[0077] A “mutant” bacteriophage refers to a bacteriophage that comprises one or more mutations in its genome compared to the genome of the parent bacteriophage. A mutant generally loses or gains one or more features or characteristics compared to the parent bacteriophage.

[0078] ABBREVIATIONS

[0079] DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures) pv. pathovar

[0080] Xaj Xanthomonas arboricolci pv . juglandis BRIEF DESCRIPTION OF THE FIGURES

[0081] Figure 1. Laboratory efficacy trials. The experiments were conducted in a multimode reader, as described in the “Materials and Methods” part of Example 2. Results of three independent tests are demonstrated. Error bars mark the standard deviation. A: Comparison of the growth of bacterial strain XAJ 1155 in the presence of XAJ2-like bacteriophages. B: Growth of bacterial strain XAJ 1155 in the presence of bacteriophages (Xaj2, Xaj5, Xaj6, Xaj7, Xaj8) of the Xaj Phage cocktail. The efficacy of five of the six phages in the composition is presented in this figure. For better clarity of the curves, the results are presented in two diagrams for both Figures A and B.

[0082] Figure 2. Results of the efficacy trial of the isolated bacteriophage XajPHU 1 (accession number: DSM 34666). The figure shows the growth of bacterial strain Xaj 1049 alone, without phage (“control”), or in the presence of phage Xaj PHU 1. The experiments were conducted in a multimode reader, as described in the “Materials and Methods” part of Example 2. Results of three independent tests are shown. Error bars indicate the standard deviation.

[0083] Figure 3. Results of two field trials (see Example 5). A: cultivar Roxana, 2021; B: cultivar Jupanesti, 2021; C: cultivar Roxana, 2022; D: cultivar Jupanesti, 2022. In the figures, for each treatment group, from left to right, the first column is DD (%) for shoots, the second column is DD (%) for leaves and the third column is DD (%) for fruits. XAJPHAGE XI, XAJPHAGE X2 and XAJPHAGE X3 represent the results of experimental groups treated with the XAJPHAGE composition once, twice and three times, respectively. Cuproxat Flowable (ST) x3 represents the results of the samples sprayed with the Cuproxate Flowable product (three times).

[0084] Figure 4. In the field trials (see Example 5), the product efficacy (EAbbott%) values calculated according to the Abbott formula for the different organs assessed. A: cultivar Roxana, 2021; B: cultivar Jupanesti, 2021; C: cultivar Roxana, 2022; D: cultivar Jupanesti, 2022. In the figures, for each treatment group, from left to right, the first column is the EAbbott (%) value for shoots, the second column is the EAbbott (%) value for leaves and the third column is the EAbbott (%) value for fruits. XAJPHAGE* IX, XAJPHAGE* 2X and XAJPHAGE* 3X represent the results of experimental groups treated with the XAJPHAGE composition once, twice and three times, respectively. Cuproxat Flowable (ST) *3X represents the results of the group sprayed with the standard Cuproxate Flowable preparation (three times).

[0085] DETAILED DESCRIPTION OF THE INVENTION

[0086] Our goal was to isolate and characterize a bacteriophage that is suitable as an active ingredient for the destruction of Xanthomonas arboricolci pv . juglandis .

[0087] The bacteriophage we isolated was genetically characterized and tested for host specificity. It is important to mention that this phage is also suitable for use in combination with other phages in a biopesticide composition. The main advantages of using phages over antibiotics are the following: phages are self-replicating and self-limiting: they reproduce only as long as the host bacterium is present, and are rapidly degraded in its absence; phages are natural components of the biosphere; phages are non-toxic to eukaryotic cells [Greer, 2005] ; phages are highly species-specific, so they have minimal environmental impact, and even with well-designed human therapy, they do not destroy normal intestinal flora bacteria, so the side effects typical of most antibiotics due to this do not occur; phage compositions can be manufactured quite easily, and can be stored for a long time at 4 °C [Greer, 2005]; furthermore, the effect of phages is not affected by most agrochemicals [Zaccardelli etal., 1992],

[0088] We have isolated a bacteriophage which has a unique genome sequence, and have confirmed the efficacy of this phage in laboratory experiments.

[0089] In the examples, we describe the properties and efficacy of the bacteriophage XajPHUl isolated and characterized by us against the bacterium X. arboricola pv . jugandis .

[0090] If phages are used instead of antibiotics, even if the bacteria become resistant to the composition used, the phages “ensure” that new phages capable of attacking resistant bacteria are created, which can be isolated in a short time and at low cost (compared to the development of antibiotics), and a new, effective composition can be produced.

[0091] Several bacteriophages have already been isolated against Xanthomonas arboricola \Y\ . juglandis [Stefani et al., 2021], and in several cases, development has been or is underway to create biopesticides comprising bacteriophages [Stefani et al., 2021],

[0092] The isolated XajPHUl bacteriophage can be suitable for use as an active ingredient in a bacteriophage -based pesticide, due to its broad host specificity and the fact that its genome does not encode an integrase gene. Bacteriophage-based biopesticides usually contain several different bacteriophages [Stefani etal., 2021], As described in Example 1, the genome of bacteriophage XajPHUl is completely unique, i.e., it does not show any significant similarity to the genome of any other bacteriophage. (For example, neither to the genome of / 20-Xaj nor to the genome of / 30-Xaj described by Retamales et al. (2022), which was confirmed by a search in the NCBI Blast nucleotide collection (nr / nt) database, as well as a direct comparison using the NCBI Blastn algorithm.) The unique genome sequence is surprising because, out of the isolated phages, 7 other phages had genome sequences that were nearly identical (99.0% to 99.9% identity) to each other (see Example 1). The unique genome sequence results in the following advantageous properties with regard to the use as a biopesticide active ingredient:

[0093] • If a host bacterium is infected by a lysogenic bacteriophage, infection with bacteriophages with a similar genome may be unsuccessful (protection against superinfection, see Labrie et al., 2010). Since the genome of XajPHUl is unique, it can be infectious even in cases where a lysogenic bacteriophage infects the host bacterium.

[0094] • Due to the CRISPR / Cas system, bacteria can develop immunity against bacteriophages [Labrie et al., 2010], As one element of this, the bacterium stores in its genome a genetic “memory trace” characteristic of a given bacteriophage (“proto -spacer” - Labrie et al., 2010), and if the given spacer is present in the genome of the bacteriophage infecting the host bacterium, the bacterial cell cleaves the phage genome introduced into the cell, thus the infection will be unsuccessful. If the given spacer is present in the genome of several different bacteriophages, it follows that the genome of all such phages will be cleaved by the bacterial enzyme. Since the genome of the XajPHUl bacteriophage is unique, the chance that the CRISPR / Cas system of the host bacterium would contain a spacer from it, i.e., that the bacterial cell would be immune to the XAJPHU1 bacteriophage, is small, thus the infection is more likely to be successful.

[0095] In addition to phage XajPHUl with the unique genome, we found 11 other bacteriophages infecting Xanthomonas arboricola pv. juglandis (Xaj) and characterized them morphologically and genetically according to a unique protocol. Their efficacy was also analyzed, and basic life cycle parameters (burst time and burst size) were determined in vitro in laboratory trials. See Examples 1 to 2.

[0096] The bacteriophage XajPHUl can be used not only alone, but also in combination with other bacteriophages infecting Xanthomonas arboricola pv . juglandis (Xaj).

[0097] To demonstrate this, based on the characterization of the 11 other isolated bacteriophages, we formulated a cocktail containing six bacteriophages and performed field trials with it for two consecutive years in Pitesti, Romania. The applications were implemented through foliar spraying the suspension containing bacteriophages on different Juglans regia varieties. The phage cocktail treatment protected walnut trees against Xaj effectively, albeit efficiency discrepancies were observed depending on the variety treated. Biosafety and ecotoxicity tests were also performed to evaluate the bacteriophages’ effect on the ecosystem. The results showed that the use of the cocktail was safe and has neutral effect on the environment. The presented results aim to assess the potential of bacteriophages as biocontrol agents and further emphasize their biopesticide activity against Xanthomonas spp.

[0098] In summary, twelve novel bacteriophages were isolated and characterized in our work. The characterization of these phages was aimed at the safety of their field application. Interestingly, bacteriophages with high genetic similarity showed distinct biological features. A bacteriophage cocktail composed of six different bacteriophages (XajPHAGE composition, see Example 4) provided significant protection for Juglans regia cultivars (Jupanesti and Roxana) when applied twice or thrice during both consecutive years (see Example 5). This effect was better than the three-times application of a copper-based pesticide. Disease damage reduction by the Xaj Phage composition was cultivardependent. To determine the minimum applicable effective dose and to optimize the parameters of the treatment desires, more field trials are needed in the future. Investigations of the molecular interactions of bacteriophage-host bacterium-plant triads could serve as data for even more successful applications of bacteriophage-based biopesticides. Nevertheless, the presented results confirm that the XajPHUl phage and bacteriophage-based biopesticides can be an effective and green tool for the protection against Xaj, including organic production.

[0099] EXAMPLES

[0100] EXAMPLE 1: Isolation and characterization of phages

[0101] A) Materials and methods

[0102] 1. Bacterial strains and culture conditions

[0103] Xaj strains used in our work are listed in Table 1. Bacteria were cultivated in Nutrient Broth (NB). The NB liquid medium comprised 0.5% (w / v) peptone, 0.3% (w / v) yeast extract, 0.5% (w / v) sodium chloride and had a pH of 6.8 to 7.2. The NB solid medium comprised 0.5% (w / v) peptone, 0.3% (w / v) yeast extract, 0.5% (w / v) sodium chloride and 1.5% (w / v) agar and had a pH of 6.8 to 7.2. The bacteria were cultured as previously described [Dbmbtbr et al., 2016], Briefly, the bacteria (Xanthomonas arboricola pathovars) were incubated in NB liquid medium at 28 °C with constant (160 rpm) shaking on an orbital shaker for 36 to 48 hours.

[0104] Table 1. Xaj bacterium strains used in the study.

[0105] 2, Isolation of bacteriophages

[0106] Aerial tissue (shoots and leaves), soil, and effluent water samples were collected from walnut fields in Hungary (Lengyeltoti and Celldbmblk) and Romania (Targu Mures, Transylvania) in 2021- 2022. Bacteriophage isolation was performed as described earlier [Dbmbtbr et al., 2016], Briefly: samples were collected from infected walnut fields. Bacteriophages were isolated from aerial tissues of walnut plants (bark, leaves and fruits) and the surrounding soil. In order to facilitate the isolation of a large number of diverse phages with broad host ranges, multiple host enrichment systems described by Gill et al. (2003) were applied with some modifications. Plant and soil filtrates were prepared by macerating 10 g of chopped plant tissue (bark, fruits, leaves) and 40 g of soil in 50 mL of NB in 150 mb Erlenmeyer flasks, respectively. The samples were incubated for 24 hours at 28 °C in a rotary shaker at 160 rpm, then sieved through sterilized gauze, and centrifuged at 2600 x g for 40 minutes at 4 °C. Supernatant was removed and filtered using 0.2 pm syringe filters. 5 mL of the resulting filtrates were added to 5 mL of NB supplemented with 1 mM MgCU in test tubes. Then, each tube was inoculated with 250 pL overnight cultures of six host bacterial strains (cell density of 108CPU mL1) and incubated for 24 h at 28 °C with shaking at 160 rpm. The 18 propagation hosts were grouped into three host cocktails; therefore, three tubes were prepared for each sample. Phages were obtained from enriched samples by centrifugation and subsequent filtration through a 0.2 pm syringe filter. The presence of lytic phages was tested by spotting 20 pL of filtrate onto a lawn of Xaj grown on Nutrient agar (NA) at 28 °C. All phage isolates were purified by three successive single-plaque isolations on their original hosts. Phage stocks used in this study were prepared by infecting the host strain (108CFU mL1) in NB at a Multiplicity of Infection (MOI) of 1. After 24 h incubation at 28 °C with shaking at 160 rpm, phages were recovered as described above.

[0107] The bacteriophage later named XajPHUl was isolated from leaves of walnut tree infected with Xanthomonas arboricola pv . juglandis in Celldomolk in 2022. Five flasks containing 60 mL of liquid medium each were inoculated with 200 pL of overnight cultures of six strains of Xanthomonas arboricola pv . juglandis . 40 g of leaf sample, 1.2 mL of sterile glycerol, 60 pL of 1 M MgCL x 6 FLO were added to the flasks and incubated for 24 hours at 28 °C in a shaker incubator. The samples were filtered through sterile gauze, and then centrifuged at 2600 x g for 40 minutes at 4 °C. After removing the supernatant, another filtration followed, on microfilter with a pore size of 0.20 pm. The phages were purified by three passages using the plaque isolation technique.

[0108] 3, Characterization of bacteriophages: examination of phage morphology by transmission electron microscopy

[0109] Electron microscopy investigations were conducted according to the method of Domotor et al. [Domotor et al., 2016], The morphology of phages was examined by transmission electron microscopy following a negative staining protocol. 10 mL of bacteriophage suspension (109PFU mL1) was centrifuged at 16 000 x g for 1 h at 4 °C. Supernatant was discarded and the phage pellet was resuspended in 100 pL of sterile distilled water. Phage suspension was spotted on a 200 mesh formvar- coated copper grid, stained with 3% (w / v) sodium phosphotungstate (Merck, Germany). The solution was aspirated, and the grid was air-dried for 40 s. Phages were observed at various magnifications by JEOL JEM-1200EX II transmission electron microscope operating at an acceleration voltage of 64 kV. At least five virions of each phage were analyzed to calculate average phage dimensions (head diameter and tail length). Based on their morphologies, phages were classified into their respective family according to the guidelines of the International Committee of Taxonomy of Viruses [King et al., 2011],

[0110] 4, Host-range analysis

[0111] Host-range experiments were conducted according to a previously described procedure [Domotor et al., 2016], with the difference that medium WALNUT was used as the medium and the culturing temperature was 28 °C. WALNUT medium comprised the following: 5 g casein peptone (digested with pancreatic enzymes), 5 g NaCl, 3 g yeast extract, made up to 1 liter with water, adjusted to pH 6.8 to 7.2. For a solid medium, 15 g agar was added.

[0112] Briefly, the procedure used in the host-range experiments: the host range of high titer (109PFU mL1) phages was assessed on a range of Xaj strains and two different genera using the drop-on-lawn technique [Adams, 1959] . Phages were suspended in NB, and control drops of NB containing no phages were dropped in each case in order to ensure that the observed lysis was caused by phages. Plaque morphology was assessed using the soft agar overlay method [Adams, 1959],

[0113] The bacterial strains listed in Table 1 were used for the host-range analysis.

[0114] 5 , Genome sequencing and bioinformatics analysis

[0115] Genome sequencing and bioinformatics analysis were performed as described earlier [Kovacs et al., 2019],

[0116] DNA extraction was performed as follows. 1.5 mL of culture was centrifuged at 10500 x g for 10 min at 4 °C. 1.2 mL of the supernatant was aspirated, and then subjected to DNasel and RNase treatment for 30 min at room temperature. 100 pL of 0.5 M EDTA (pH 8) was added. Incubation was performed at 75 °C for 10 min, followed by the addition of 10 pL of ProteinaseK (20 mg / mL) and incubation for 1 h at 65 °C. Subsequently, isolation was performed using the High Pure Viral Nucleic Acid Kit manufactured by Roche Diagnostics GmbH (Germany) according to the manufacturer's instructions.

[0117] DNA library was prepared using the Nextera XT kit (Illumina Inc., USA). DNA sequencing was done with an Illumina MiSeq (Illumina Inc., USA) platform using an Illumina V2 (500 cycles) or Illumina V3 (600 cycles) sequencing kit (Illumina Inc., USA) according to the manufacturer's instructions.

[0118] Paired-end reads were quality trimmed (cut-off value 30) with Trim Galore! (Babraham Bioinformatics, version 0.4.4 with paired mode) and Trimmomatic (version 0.36 with paired mode and using the CROP: 150 MINLEN: 150 parameters) programs [Bolger et al., 2014], The trimmed and quality-filtered reads were assembled using the MyPro pipeline [Liao et al., 2015],

[0119] The genome was assembled using Geneious Prime software (version 2023.0.4). Machine annotation was done using the RAST server (https: / / rast.nmpdr.org / ).

[0120] B) Results - features of the isolated phages

[0121] 1. The isolated bacteriophages and their characterization (electron microscopy results, plaque morphology, life cycle parameters) Four bacteriophages were isolated from aerial tissues, six from soil samples, and one from effluent water (Table 2).

[0122] During the project, a further, 12th phage was also isolated, which we named XajPHUl (Table 2). This phage was also deposited under the accession number DSM 34666 on May 25, 2023 at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ); InhoffenstraBe 7B, 38124 Braunschweig, Science Campus Braunschweig -Slid, Germany).

[0123] The plaque morphology, electron microscopy results, and the life cycle parameters (determined by the one-step growth curve trials) are summarized in Table 2. Table 2. Plaque morphology, electron microscopy and life cycle data of the isolated Xaj bacteriophages, n.d.: not determined. Based on the data presented in Table 2, Xaj l had a short, non -contractile tail, XajPHUl had a long, non-contractile tail, and the other Xaj phages had long, flexible tails. The isolated Xaj bacteriophages had a latent period of 4-90 min, and a relatively small burst size of 4-33. Phage XajPHU 1 had a burst size of 94, and a burst time of 165 min. 2, Results of host-range experiments

[0124] The host range of the isolated bacteriophages listed in Table 2 was determined (Table 3). The bacteriophage Xaj 6 presented the broadest host range, infecting ten of the fourteen investigated Xaj strains (see Table 1). Xaj3 and XajPTR2 could infect 9-9 strains, and further three phages (Xaj4, Xaj7 and XajPTRl) could lyse 8-8 host strains. Phages Xaj5 and Xaj 8 had the narrowest host range (infecting 5-5 strains). Significant differences could be observed when investigating the susceptibility of Xaj strains against bacteriophages. The strain Xaj 2490 could not be lysed by any of the applied phages. The strains Xaj234 and XajTR2 could be infected with only 2-2 phages. Contrarily, the strains Xaj 1105, Xaj 1317, XajHU 1 and XajHU2 were demonstrated to be susceptible to 10-10 bacteriophages.

[0125] Table 3. Host range results of 11 isolated Xaj phages. Column 1 lists the bacterial strains, and row 1 lists the isolated phages. + indicates host; - indicates not host.

[0126] The host range results of the bacteriophage XajPHUl are given in Table 4.

[0127] Table 4. Host range results of the isolated bacteriophage named XajPHUl (accession number:

[0128] DSM 34666)

[0129] According to the host-range experiments, the bacteriophage XajPHUl can be considered broad- ranged, as it was able to infect more than 50% (in our case 57%) of the investigated bacterial strains, with the remark that at least 10 different bacterial strains should be included in such experiments (our experiments were conducted using 14 bacterial strains).

[0130] We also note that the host-range experiments were performed on solid media, while the laboratory use studies (see Example 2) were performed in shaking culture, in suspension. The results measured in the two systems do not always overlap, since the gene expression patterns of the bacteria in the attached state and in suspension differ. It is conceivable that in one case the bacterium expresses the protein that is the receptor of the bacteriophage, while in the other case it does not. For example, in the case of phage XajPHUl, according to the results in Table 4, the DSMZ 1049 strain is not a host for the phage, but in the laboratory studies, phage XajPHUl effectively inhibited the growth of bacterial strain 1049 (see Example 2 and Figure 2).

[0131] 3 , DNA sequencing results

[0132] Complete genomes of the 12 isolated bacteriophages were determined. The final annotation is in progress, but based on preliminary data, the presence of functional integrase -encoding genes can be excluded, which is also supported by the plaque morphology results. The presence of integrase genes is a reliable sign of an ability for a temperate life cycle because functional integrase-encoding genes have not yet been described in the genomes of lytic bacteriophages, even in cases where integrons (genetic elements able to capture, express and rearrange mobile gene cassettes in diverse bacteria) were detected [Qi et al. , 2023] . The length of the genomes of the isolated Xaj phages was 45-50 kbp, and the G+C (%) content was around 47.4-47.7% (data not shown). Genomic sequences of Xaj3, Xaj4, Xaj5, Xaj7, Xaj8 and XajPHU2 were nearly identical (99.0-99.9% identity) to the previously sequenced Xaj 2 phage genome. The genome of Xaj 2 phage is available in the GenBank database under accession number KU 197014 (version number: KU 197014.1). Interestingly, some biological features, including host range (see Tables 3 and 4) and efficacy against the host in laboratory trials (Figure 1A), are distinct among these six bacteriophages (compared to each other and to Xaj2), respectively. The genome of bacteriophage XajPHUl is 45290 bp. The G+C ratio of the genome is 35.3%. The genomic sequence is shown in SEQ ID NO: 1. The genome does not contain an integrase gene. The genome of bacteriophage XajPHU 1 does not show any significant similarity to the genome of any other bacteriophage according to NCBI Blast against the nucleotide collection (nr / nt) database, i.e., the genome is completely unique.

[0133] The XajPHUl phage belongs to the Myoviridae family.

[0134] EXAMPLE 2: In vitro efficacy trials of the isolated phages (application experiment)

[0135] A) Materials and methods

[0136] Laboratory efficacy trials of the phages other than phage XajPHUl was carried out as follows. The growth of the bacterial cultures was monitored by measuring the OD600 nm value of the suspensions. The bacterial cultures were incubated in vitro at 27 to 28 °C and shaken at 420 rpm. The optical density (OD) was measured every 15 minutes for 24 hours with a BioTeK Synergy HT Multimode Reader using the Gene 5.0 program in 96-well plates. NB was supplemented with 2 mM MgCb and was used for the efficacy trial experiments. Bacteriophages were applied in MOI 1, 10 and 100, respectively, before starting shaking. The positive control consisted of 185 pL ofNB supplemented with 2 mM MgCb. and 15 pL of overnight suspension ofXaj strain 1155. The negative control consisted of 185 pL of NB supplemented with 2 mM MgCb and 15 pL of phage suspension.

[0137] The phage strain XajPHUl to be used was grown overnight in shaking culture at 28 °C in WALNUT medium. The sterile WALNUT medium preheated to 28 °C was inoculated with the given bacterial strain (see Tables 1 and 4) at time 0 to the appropriate viable cell count. The bacterial cultures were incubated in vitro at 28 °C and shaken at 420 rpm in a BioTeK Synergy HT Multimode Reader, which measured the optical density every 15 minutes for 24 hours using the Gene 5.0 program. The experiments were performed in microtiter plates. For the measurements, 200 pL of MOI 1, MOI 10 and MOI 100 suspensions were measured together with positive and negative controls.

[0138] The positive control consisted of 185 pL ofWALNUT medium and 15 pL of an O / N suspension of Xaj l049 bacteria. The negative control consisted of 185 pL of WALNUT medium and 15 pL of phage suspension. The negative controls were prepared with a concentrated suspension of XajPHUl bacteriophage to ensure that none of the suspensions were infected.

[0139] Three independent experiments were performed, with three technical replicates for each experiment.

[0140] Determination of MOI (multiplicity of virus per bacterial cell): The virus titer measured in a given sample (virus / mL) was divided by the number of viable cells measured in said sample (cells / mL).

[0141] B) Results (Results of the application experiment)

[0142] Laboratory efficacy trials were performed in a multimode reader, as described in the Materials and Methods part. Bacteriophages were co-cultured with the XAJ 1155 bacterium strain. This strain was chosen for the trials because every investigated Xaj bacteriophage produced a high titer. Interestingly, Xaj8 did not form plaque on a solid medium on strain XAJ1155 but could be produced in the liquid culture on this strain. Albeit genomes of Xaj2, Xaj3, Xaj4, Xaj5, Xaj7, Xaj8, and XajPHU2 were nearly identical (see Example 1), these bacteriophages hindered the growth of the host strain with different kinetics (Figure 1A). For example, Xaj4 allowed the host to grow but caused a stationary phase at lower optical density than the control. Xaj2 hindered the growth of the bacteria for 14 hours, but after this point (probably due to a resistance event), the host started to grow. Phage Xaj7 hindered the growth of the Xaj strain for 24 hours effectively.

[0143] Figure IB represents the growth kinetics of the Xaj phages that were components of the cocktail used for the field trials (see Examples 4 and 5 below). (Phage XajPHUl, the 6th phage of the field cocktail, is not shown in this figure; its efficacy trial result is presented separately in Figure 2.) These bacteriophages hindered the growth of strain XAJ 1155 with different kinetics. Xaj bacteriophages were also tested on other Xaj bacterium strains, causing distinct kinetics when investigating their effect on the growth of strain Xaj 1049 (data not shown).

[0144] Figure 2 shows the results of the laboratory efficacy trials of phage XajPHUl. The trials were performed in a multimode reader as described in the Materials and Methods part. The experiment showed that bacteriophage Xaj PHU 1 effectively inhibited the growth of the host bacterium. Maximum efficacy was observed at 12 hours, when the optical density of the sample comprising bacteriophage XajPHU 1 was 43% of that of the control.

[0145] EXAMPLE 3: Composition comprising isolated XajPHUl bacteriophage

[0146] A suspension and a lyophilized composition comprising the XajPHUl phage having a unique genome sequence (see SEQ ID NO: 1) were prepared.

[0147] A) Suspension

[0148] A suspension composition comprising the XajPHUl bacteriophage was produced as follows.

[0149] 1. Propagating Xai phages

[0150] The medium used for culturing the Xaj bacterial strains was Nutrient Broth (NB) solid medium or Nutrient Broth (NB) liquid medium. The NB solid medium contained 0.5% (w / v) peptone, 0.3% (w / v) yeast extract, 0.5% (w / v) sodium chloride, and 1.5% (w / v) agar, and had a pH of 6.8 to 7.2. The NB liquid medium contained 0.5% (w / v) peptone, 0.3% (w / v) yeast extract and 0.5% (w / v) sodium chloride, and had a pH of 6.8 to 7.2. The bacteria were incubated in NB liquid medium at 28 °C, with 160 rpm, on an orbital shaker for 36 to 48 hours.

[0151] On day 1, an ON culture of the host bacteria was inoculated. If the bacteria were on a plate: one inoculum of the bacterial colony was inoculated into 10 mb of medium (measured together in a test tube). If the bacteria were in liquid: 400 pL (OD 0.5-0.6) of bacterial culture was measured into 10 mb of NB liquid medium (measured together in a test tube). The bacteria were incubated for 36 to 48 hours on an orbital shaker with shaking at 160 rpm at 28°C. On day 2, the optical density (OD) was measured with a spectrophotometer. The wavelength of the measurement was 600 nm. The OD value of the optimal bacterial culture was 0.5 to 0.6.

[0152] For the propagation of the XajPHU 1 phage, 600 mL of NB liquid medium and 8.5 mL of the host bacterium ON culture were mixed in a 1000 mL Erlenmeyer flask. The obtained suspension was incubated for 3 hours on an orbital shaker with shaking at 160 rpm at 28°C. After incubation, 8.5 mL of filtered bacteriophage (titer: 1010PFU / mL) and 250 pL of 1 M MgCL filtered solution were added. The phages were incubated at 28°C for 24 hours, placed on an orbital shaker with shaking at 160 rpm, at 28°C.

[0153] Then, an ON culture of the host bacterium was inoculated again, as described above.

[0154] On day 3, Petri dishes were first prepared: NB agar medium was heated in a microwave oven at medium temperature for approx. 15 minutes. The completely, homogeneously thawed NB medium was poured into Petri dishes, then we waited approx. 20 minutes until it solidified.

[0155] After that, a dilution series was prepared as follows. We prepared 11 Eppendorf tubes for the XajPHUl phage (10 for dilution, 1 for the stock solution). We measured 85 pL of lx dilute PBS and 5 pL of 1 M MgCL into each tube. The lx dilute PBS buffer contained 0.2 g potassium chloride, 8 g sodium chloride, 0.24 g KH2PO4 and 1.44 g Na2HPO4 X 12 H2O for a final volume of 1000 mL; its pH was 7.4.

[0156] For the stock solution, the XajPHUl bacteriophage stored in a room at 4°C was filtered through a syringe filter with a pore diameter of 0.2 pm. Then a decimal dilution followed. We pipetted 10 pL of the stock solution into the first Eppendorf tube. We vortexed, suspended it thoroughly, then pipetted 10 pL from the first Eppendorf tube into the second Eppendorf tube, and so on. The final volume in the Eppendorf tubes was 90 pL, except for the 10th Eppendorf tube, in which it was 100 pL.

[0157] Controls were used: the positive control was the original filtered bacteriophage, the titer of which was known; for the negative control, we measured 10 pL of the phage dilution medium (without phage) onto NB solid medium.

[0158] To prepare a bacterial lawn, we pipetted 500 pL of the ON culture inoculated on day 2 (which had an OD value of 0.5 to 0.6 at 600 nm) onto the middle of the Petri dish, and then spread the liquid evenly over the layer with trained movements. We waited until it dried, about 20 minutes.

[0159] The prepared dilution series was dropped onto the Petri dishes with the bacterial lawn. From each Eppendorf tube, 10 pL was dropped to the appropriate label. Moving towards the 10^1, stock solution, the pipette tip was changed between each dilution. Then we had to wait about 20 minutes for drying. After that, the plates were incubated in a thermostat at 28°C for 36 to 48 hours.

[0160] The titer results were then read. The appropriate titer is 1012PFU / mL.

[0161] 2, Centrifugation and filtration of the propagated bacteriophages

[0162] To centrifuge the phages, 40 mL of the phage suspension was measured into sterile centrifuge tubes and then centrifuged at 6000 g for 45 minutes. After that, the phage suspensions were filtered through a filter with a pore diameter of 0.22 pm (can be autoclavable or disposable). This resulted in a filtered suspension comprising the XajPHUl phage.

[0163] B) Lyophilized composition

[0164] For the lyophilized composition comprising the XajPHUl phage, the phages were propagated as described for the suspension composition (see point 1).

[0165] If the titer test result showed that the phage suspension had the appropriate concentration (1012PFU / mL), we could start lyophilizing the phage composition. Lyophilization is essential for longterm storage and easy transportation of bacteriophages.

[0166] Lyophilization was carried out as follows.

[0167] On day 1, we froze the phages and set up the lyophilization machine. The lyophilization was performed with IO10PFU / mL filtered bacteriophage and a 0.5 M sucrose solution in a 1:3 ratio, then placed in a -70°C freezer overnight. Setting up the lyophilization machine: we waited until the machine cooled down to about -110°C, inserted the samples, and then started the process.

[0168] Lyophilization can take 2 to 3 days. According to experience, it is ready when the pressure is 0.004 hPa / mbar. When lyophilization is complete, the finished lyophilized composition is produced.

[0169] EXAMPLE 4: Composition comprising XajPHUl and additional bacteriophages

[0170] The XajPHUl phage can also be used together with other bacteriophages. To demonstrate this, a suspension and lyophilized composition (phage cocktail) comprising a plurality of bacteriophages were prepared.

[0171] This composition contained 6 bacteriophages (Xaj2, Xaj5, Xaj6, Xaj7, Xaj8 and XajPHUl), each with a titer of 1010PFU / mL. This composition is also referred to as the XajPhage product or XajPhage composition in the description.

[0172] A) Suspension composition

[0173] 1. Propagation of Xai phages

[0174] For the phage cocktail, the Xaj phages were propagated as described in Example 3, Part A, point 1, each bacteriophage separately.

[0175] 2, Centrifugation and filtration of the propagated bacteriophages, and mixing of the phage cocktail (phage mix)

[0176] To centrifuge the phages, 40 mL of the phage suspension was measured into sterile centrifuge tubes and then centrifuged at 6000 g for 45 minutes.

[0177] After that, the phage suspensions were filtered through a filter with a pore diameter of 0.22 pm (can be autoclavable or disposable). A separate filter was used for each phage to avoid mixing.

[0178] For the phage cocktail, the 6 different phages were mixed in a ratio of 1 : 1 : 1 : 1 : 1 : 1. This resulted in the suspension composition. B) Lyophilized composition

[0179] For the lyophilized composition, we first prepared the suspension phage cocktail according to part A), i.e., ifwe obtained a phage suspension ofthe appropriate concentration (1012PFU / mL) when reading the titer test result, we prepared the phage cocktail from them in the ratio described in point 2 of part A) of Example 4. After that, we started lyophilizing the phage mix. Lyophilization is essential for longterm storage and easy transportation of the phage mix.

[0180] Lyophilization was carried out as follows.

[0181] On day 1, we froze the phage cocktail and set up the lyophilization machine. The lyophilization was performed with IO10PFU / mL filtered phage cocktail and a 0.5 M sucrose solution in a 1:3 ratio, then placed in a -70°C freezer overnight. Setting up the lyophilization machine: we waited until the machine cooled down to about -110°C, inserted the samples, and then started the process.

[0182] Lyophilization can take 2 to 3 days. According to experience, it is ready when the pressure is 0.004 hPa / mbar. When lyophilization is complete, the finished lyophilized phage cocktail is produced.

[0183] EXAMPLE 5: Field trials

[0184] The XajPHU 1 phage can also be used in combination with other bacteriophages. To demonstrate this, field trials were conducted using a composition comprising a plurality of bacteriophages, prepared according to Example 4.

[0185] The main objective of the trial was to investigate the field efficacy of the bacteriophage cocktail comprising six bacteriophages, described in Example 4, during which the cocktail was applied to two different walnut varieties, in two consecutive years.

[0186] A) Materials and methods

[0187] Field trials were conducted at the Research Institute for Fruit Growing in Pitesti, Romania, in 2021 and 2022. Walnut plants cv. Jupanesti and Roxana were planted in 1988, with a planting distance of 5.0 m x 8.0 m, and a density of 250 trees / hectare. 45 trees were involved in the trial. The XajPhage product (i.e. the composition according to Example 4) contained a mixture of 6 bacteriophages (Xaj2, Xaj5, Xaj6, Xaj7, Xaj8 and XajPHUl), each with a titer of 1010PFU / mL. This composition was applied in the trial by spraying once, twice or three times, sprayed on the leaves of the trees, at a dose of 8 mL / 10 L per spray. The spray volume was as follows: 10 liters / 3 trees / application. The efficacy of the XajPhage mixture was compared with the Cuproxat Flowable product (4 g / L, 10 L / 3 trees / application), sprayed three times. Negative control trees remained untreated. Each experimental group contained three trees, with 3 replicates, i.e., in all five experimental groups (untreated control, XajPhage once, XajPhage twice, XajPhage 3 times, Cuproxat Flowable 3 times) there were 9 walnut trees. The damages degree DD% on different organs was calculated taking into account both disease incidence DISINC% and disease severity DISSEV%, according to the following formula: DD[%] = (DISINC%* DISSEV%) / 100 wherein the disease occurrence, i.e. the disease incidence (DISINC%) was calculated according to the following formula:

[0188] DISINC[%] = (n* 100) / N wherein n is the number of diseased organs and N is the number of assessed organs; and wherein the disease severity (DISSEV%) was calculated as weighted average of the number of diseased organs with different attack severities (percentages), according to the following formula:

[0189] DISSEV[%] = (sum(i*f)) / n wherein i is the percentage of the attack on the organ, f is the number of cases with various attack percentages and n is the number of diseased organs.

[0190] The product efficacy (E%) was calculated for the different organs assessed according to the Abbott formula:

[0191] EAbbott [%] = (1-(T / U))* 100 wherein T is the calculated DD% in the treated variants and U is the calculated DD% in the untreated control variant.

[0192] In addition, in the trial conducted in 2022, the varietal sensitivity index (VSI%) of the compositions was calculated according to the following formula:

[0193] VSI% = DD%Shoots + DD%ieaves + DD* / !) fruits

[0194] B) Results

[0195] Preliminary (non-GLP) six-pack toxicology and ecotoxicology studies were also performed. Based on these preliminary data, we could conclude that XAJPHAGE (i.e., the composition of Example 4) was non-toxic in acute inhalation, eye and dermal irritation, oral and acute dermal toxicity, and skin sensitization studies (category 5, data not shown). Preliminary ecotoxicity tests confirmed that the bacteriophage cocktail was non-toxic against honeybees, fishes (carp and trout), Daphnia magna, algae, and did not hinder the germination of plants (data not shown). These toxicological results are also supported by earlier findings summarized in a review [Stefani et al., 2021],

[0196] Two Juglans regia cultivars (Jupanesti and Roxana) were treated once, twice, or three times with the composition of Example 4 (XAJPHAGE) during two consecutive seasons, respectively. Cuproxat Flowable was used as an efficacy reference. Table 5 and Figure 3 summarize the results of the field trials.

[0197] Table 5. DD (%) reduction results for the Roxana and Jupanesti cultivars in 2021 and 2022

[0198] In 2021 , two treatments of the Roxana cultivar resulted in significant DD (%) reduction compared to the untreated control and achieved a higher reduction than Cuproxat Flowable did (Table 5 and Figure 3A). Next year (2022), two treatments produced similar results to Cuproxat Flowable (significant DD (%) reduction compared to the untreated control), and three treatments protected the plants nearly completely (Table 5 and Figure 3C). It should be noted that the total disease damages were significantly less in 2022 than in 2021 in both cultivars (Table 5 and Figure 3A-D).

[0199] XAJPHAGE was also effective in the Jupanesti cultivar, although to a lesser extent than in the Roxana. In Jupanesti, three treatments were needed for better disease damage reduction than Cuproxate Flowable in both years (Figures 3B and 3D).

[0200] XAJPHAGE caused a higher DD (%) reduction in both cultivars in 2021 and 2022 in leaves, shoots, and fruits (Figure 3A-D).

[0201] Abbott’s efficacy results are shown in Table 6 and Figure 4. The results of the Roxana cultivar are shown in Figure 4A (2021) and Figure 4C (2022), while the results of the Jupanesti cultivar are shown in Figure 4B (2021) and Figure 4D (2022).

[0202] Table 6. Abbott’s efficacy results for the Roxana and Jupanesti cultivars in 2021 and 2022

[0203] The results of the Varietal Sensitivity Index are shown in Table 7. These results indicate the total extent of damage to the organs assessed (shoot, leaf, fruit) due to bacterial infection in a given cultivar. As can be seen from Table 7, in the case of the Jupanesti cultivar, the XAJPHAGE composition applied twice or three times showed better results than the standard Cuproxat Flowable composition applied three times. In the case of the Roxana cultivar, the XAJPHAGE composition applied three times showed the best results, but the two-time application also showed similar results to the standard Cuproxat Flowable composition applied three times.

[0204] Table 7. Varietal Sensitivity Index (VSI%) in 2022.

[0205] Recently, Juglans regia var. Chandler trees were treated with a cocktail containing three bacteriophages [Retamales et al., 2022], In this study, the treatment was implemented six times during the cultivation period. The authors applied the phage cocktails in three different doses. It was concluded that bacteriophages conferred comparable disease reduction as a copper-based product did when the cocktail was applied in the highest dose (albeit trees treated with the copper-based product produced 21% more fruits than those treated with the highest dose of the bacteriophage cocktail).

[0206] Comparing our results, we can observe that the XAJPHAGE cocktail provided better protection to the walnut plants, in both cultivars and in both years, in spite of the smaller number of treatments (3 vs. 6). We do not have enough data to explain this phenomenon, but the effect of cultivar-dependency and different composition of cocktails (including more ingredients in the XAJPHAGE, i.e. 3 phages vs. 6 phages) could not be excluded. The plant - pathogenic bacterium - bacteriophage triad’s interactions should be investigated to explain the cultivar-dependency of the efficacy of bacteriophage-based treatments of phytopathogenic bacteria, as detailed by Stefani et al. [Stefani et al. , 2021], Plants respond to pathogen-associated molecular patterns (PAMPs) of pathogenic bacteria by activating PAMP- triggered immunity (PTI) or effector-triggered immunity (ETI). This is mediated by pathogen-specific receptors [Navarova et al., 2012], Plant responses against phytopathogenic bacteria are often cultivardependent, i.e., the sensitivity of the cultivars can vary against the same plant pathogenic bacterial species or strain, as has been described also in the case of Xanthomonads [Wang et al., 2017; Stefani et al., 2021], On the other hand, different bacteriophages can interact differently with the same bacterial strain (even if it is their host), and the same bacteriophage can have different efficacy against a divergent set of strains of a phytopathogenic bacterial species, as we have also demonstrated by the host-range trials (see Tables 3 to 4) and efficacy trials (see Figure 1). The variability of the interactions between the members of the plant - phytopathogenic bacterium - bacteriophage triad can contribute to the different outcomes of bacteriophage applications and the cultivar-dependency of the results.

[0207] EXAMPLE 6: Additional field trials

[0208] In addition to the field trials described in Example 5, additional field trials were conducted in 2023 and 2024, also on walnut trees (Juglans regia), with the composition comprising the XajPHUl phage and five other bacteriophages (XajPhage bacteriophage cocktail), prepared according to Example 4.

[0209] The trials were conducted on walnut plantations in different countries and locations.

[0210] The main objective of the trials was to demonstrate that the prepared composition is suitable for protection against Xaj bacteria in other geographical areas and in other walnut cultivars, as well. The following is a summary of these trials.

[0211] Table 8. Field trials in 2023 and 2024. In the field trials, in addition to the untreated group, there were 4 treated groups: 3 groups were treated with different doses of the XajPhage composition, while the fourth group was treated with a commercially available product (reference product) as a positive control. BB RSR Disperss NC is the abbreviation of Bouillie Bordelaise (Bordeaux mixture) RSR Disperss NC.

[0212] The Lara cultivar is one of the most sensitive walnut varieties to bacterial infection by Xanthomonas arboricolci pv . juglandis .

[0213] Each treatment (dose) was applied several times (e.g. 6 to 10 times), with an interval of 8 to 13 days between each treatment. The treatments were applied to the foliage (crown).

[0214] Results

[0215] In none of the field trials did the Xaj PHAGE product cause phytotoxicity (at any dose), the product was selective for walnut trees.

[0216] Overall, the results of the trials show that XajPhage significantly reduced infection of leaves compared to the untreated control, generally at all three doses used.

[0217] Regarding the doses, in most cases there was a dose-response relationship, wherein the highest dose caused the greatest effect. However, there were also trials where there was no significant relationship between the applied dose and the response (e.g. 2023, France; 2024, Bulgaria, Stamboliyski). Overall, the Xaj PHAGE product performed statistically similarly well in terms of efficacy and yield at the highest dose as the reference products. However, in some cases this dose produced even better results than the commercially available product (e.g. 2023, Spain - compared to OXIMUR); and there were also cases where even the middle dose (80 mL / ha) proved to be more effective than the reference product (2024, Bulgaria, Stamboliyski - compared to COPRANTOL DUO WG; 2024 Hungary - compared to CHAMPION WG).

[0218] Furthermore, we would like to note that in 2024 in France (Saint-Romans) the reference product, a copper-containing preparation (BB RSR Disperss NC; 12.5 kg / ha, which corresponds to a dose of 2500 g copper / ha per treatment), did not prove to be effective against the Xaj bacterium. This also supports the need for novel compositions in addition to (or instead of) traditional preparations (antibiotics, copper-containing compositions).

[0219] Summarizing the results of the additional field trials: the Xaj Phage product is suitable for controlling bacterial infection by Xanthomonas arboricolci ipv. juglandis, especially at high doses.

[0220] INDUSTRIAL APPLICABILITY

[0221] The isolated Xaj PHU 1 bacteriophage according to the invention, i.e. the bacteriophage deposited under the accession number DSM 34666 at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ); InhoffenstraBe 7B, 38124 Braunschweig, Science Campus Braunschweig-Siid, Germany) on May 25, 2023, is suitable for destroying Xanthomonas arhoricola pv . juglandis bacterial strains. Furthermore, it is also suitable for destroying Xanthomonas arhoricola pv. juglandis bacterial strains that are resistant to an antibiotic and / or to a copper-containing compound.

[0222] The bacteriophage can be used to prevent or treat bacterial infection caused by Xanthomonas arhoricola pv. juglandis, which is optionally resistant to an antibiotic and / or a copper-containing compound, in plants. Preferably, the plant is walnut (Juglans regia) or European hazelnut (Cory his avellana).

[0223] The bacteriophage Xaj PHU 1 isolated by us may be suitable for use as an active ingredient in a bacteriophage -based plant protection product due to its broad host range and the fact that its genome does not encode an integrase gene.

[0224] Since the genome of XajPHUl is unique, it can be infectious even in cases where a lysogenic bacteriophage infects the host bacterium. Furthermore, due to the unique genome, the chance that the host bacterium would be immune to the XajPHUl bacteriophage is small, thus the infection is more likely to be successful.

[0225] The XajPHUl bacteriophage can be used not only alone, but also in combination with other bacteriophages that infect Xanthomonas arhoricola pv . juglandis (Xaj).

[0226] The XajPHU 1 phage and bacteriophage-based biopesticides comprising it can be an effective and green tool for the protection against Xaj, including organic production.

[0227] REFERENCES

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Claims

CLAIMS1. A bacteriophage deposited under the accession number DSM 34666 at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ); InhoffenstraBe 7B, 38124 Braunschweig, Science Campus Braunschweig-Siid, Germany) on May 25, 2023; or a derivative, variant or mutant thereof, the genomic sequence of which is at least 95% identical to the sequence of SEQ ID NO: 1.

2. A bacteriophage deposited under the accession number DSM 34666 at the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ); InhoffenstraBe 7B, 38124 Braunschweig, Science Campus Braunschweig-Siid, Germany) on May 25, 2023, the genomic sequence of which is optionally the sequence of SEQ ID NO: 1.

3. The bacteriophage according to claim 1 or 2, wherein the genomic sequence of the bacteriophage or derivative, variant or mutant thereof does not contain an integrase gene.

4. The bacteriophage according to any one of claims 1 to 3, wherein the bacteriophage or derivative, variant or mutant thereof is capable of killing the bacterium Xanthomonas arboricolci i v. juglandis .

5. The bacteriophage according to any one of claims 1 to 4, which is capable of killing Xanthomonas arhoricola \Y\ .juglandis bacterium resistant to an antibiotic and / or to a copper-containing compound.

6. The bacteriophage according to claim 4 or 5, wherein the bacteriophage capable of killing Xanthomonas arhoricola pv. juglandis bacterium forms plaque in a plaque assay, wherein optionally the plaque assay comprises the following steps: i) preparing a multi-step, for example five-step, decimal dilution from a stock solution comprising the bacteriophage, ii) adding a drop, for example about 0. 1 mL, of the dilutions to a Xanthomonas arhoricola pv. juglandis bacterial lawn, wherein optionally the bacterium is resistant to an antibiotic and / or a copper-containing compound, iii) covering the bacterial lawn with a gel-forming substance, thereby forming a gel, iv) examining the gel after a given time to determine whether plaques have formed, v) if plaques have formed, the bacteriophage is capable of killing Xanthomonas arhoricola pv. juglandis, if plaques have not formed, the bacteriophage is not capable of killing Xanthomonas arhoricola pv. juglandis.

7. A composition comprising a bacteriophage according to any one of claims 1 to 6.

8. A composition comprising a bacteriophage according to claim 2.

9. The composition according to claim 7 or 8, which comprises additional bacteriophage(s), preferably additional bacteriophage(s) against Xanthomonas arboricolci i v.juglandis .

10. The composition according to claim 8, which comprises additional bacteriophage(s) against Xanthomonas arhoricola i v.juglandis .

11. Use of a bacteriophage according to any one of claims 1 to 6 or a composition according to any one of claims 7 to 10 on a plant for the prevention or treatment of a bacterial infection caused by Xanthomonas arhoricola i v.juglandis.

12. The use according to claim 11 , wherein the plant is a walnut tree (Juglans regia) or a European hazelnut (Corylus ave liana), preferably a walnut tree.

13. The use according to claim 11 or 12, wherein the Xanthomonas arhoricola pv . juglandis is a bacterial strain resistant to an antibiotic and / or to a copper-containing compound.

14. The use according to any one of claims 11 to 13, wherein the composition according to claim 9 or 10 is used.

15. A method for preventing or treating a bacterial infection of a plant caused by Xanthomonas arhoricola pv. juglandis, wherein the method comprises using a bacteriophage according to any one of claims 1 to 6 or a composition according to any one of claims 7 to 10 on the plant or on a part of the plant or on a location of the plant.

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