Bioproduct based on lytic bacteriophage for the control of xanthomonas campestris pv. campestris in brassicas

Abstract

The invention relates to the use of a lytic bacteriophage, stabilised in a carrier, of the family Myoviridae, Foxunavirus genus (deposit IDAC 261122-01), which acts as a biocontroller of Xanthomonas campestris pv. Campestris (Xcc), a phytopathogen responsible for the main disease affecting brassicas globally, angular leaf spot or black rot. In addition, the invention relates to the preparation of a bioproduct comprising the phage and an agronomically acceptable carrier, such as alginate and / or chitosan.

Description

[0001] BIOPRODUCT BASED ON LYTIC BACTERIOPHAGE FOR THE CONTROL OF XANTHOMAS CAMPESTRIS PV. CAMPESTRIS IN BRASSICAS

[0002] DESCRIPTIVE MEMORY

[0003] SCOPE

[0004] The present invention is directed to the field of agriculture, more particularly, it relates to the development of an environmentally friendly management strategy against angular spot or black rot of brassicas (Xanthomonas campestris pv. campestris), through the use of lytic bacteriophages that act as biocontrol agents of this bacteria and its disease.

[0005] BACKGROUND

[0006] Angular leaf spot or black rot of brassicas is a disease caused by the bacterium Xanthomonas campestris pv. campestris (Xcc). It is important for brassica cultivation worldwide, as it causes losses in crop yield and quality. In Chile, for example, Brassica oleracea species represent around 15% of the annual vegetable cultivation area. This is the most damaging bacterial disease affecting the Brassicaceae family, as it causes economic losses and affects most species belonging to this family. For example, in cabbage crops, losses reaching 100% of the harvest have been recorded; and in the case of cauliflower, losses in quality and yield of between 10-50% are known. Like brassicas, angular leaf spot or black rot is cosmopolitan and occurs in different climatic zones.

[0007] The pathogen responsible for this disease, Xcc, can survive in soil, plant debris and weeds, be transmitted by seeds, irrigation, rain and work tools; and can colonize the seed through flowers, during harvest or in storage. The range of species it can colonize is wide, and it has been reported as pathogenic to most members of the brassicas such as turnips, cabbage, kale, broccoli, cauliflower, radish, kohlrabi, among others. It usually causes leaf spots, chlorosis, necrosis and plant death. Angular leaf spot or black rot is a vascular disease and its initial symptoms are chlorotic spots in a "V" shape, which begin at the edges and culminate in necrosis of the entire leaf, including the vascular system.

[0008] Currently, there is limited capacity to control this disease, and it is mainly based on copper-based products, which produce resistance in the pathogen and are ecotoxic to crop soils. On the other hand, there are antibiotics, which are producing resistance wherever they are applied; for example, the use of streptomycin has led to the emergence of resistant strains of Xcc. In this scenario, the study of alternative management strategies based on biological control has been promoted, including the use of bacteriophages or antagonistic bacteria, for example, of the genera Bacillus, Pseudomonas and non-pathogenic strains of Xcc for disease control.

[0009] Specifically, bacteriophages, or phages, are bacteria-specific viruses that utilize their host's metabolism to replicate internally. Their development has emerged as a viable biological control alternative for different bacterial genera, exhibiting advantages over chemical control, such as specificity in control and zero generation of bacterial resistance. Biocontrol is carried out using phage therapy and aims to reduce or prevent losses caused by the disease, a strategy that is in line with the global trend toward using clean and environmentally friendly plant protection tools. Phages are natural, biodegradable products that could even be adopted for organic brassica crops, both for fresh consumption and seed production.

[0010] Unlike chemical control measures for this disease, phage therapy does not cause harm or damage to humans and animals that may be constantly exposed to them. These biocontrol agents, due to their high specificity for the hosts they infect, also pose no threat to other beneficial bacteria in agriculture. Furthermore, they only persist in large numbers in the environment if their host is present, so they do not accumulate, unlike copper-based bactericides and fungicides. Another fundamental difference with copper-based bactericides is that there is evidence of phage mobility within plant cells (translocation from roots to upper leaves). Therefore, unlike copper, they can exert their action systemically in the plant, providing opportunities for curative or eradication mechanisms of action, in addition to preventive ones.

[0011] Considering the above, the present invention proposes the use of a lytic bacteriophage as a biocontroller of the main disease that affects brassicas worldwide, called angular spot or black rot, whose causal agent is the bacterium Xanthomonas campestris pv. campestris.

[0012] Prior art

[0013] Despite the significant economic impact of Xcc disease, there are few studies currently addressing the issue, demonstrating that it is a difficult pathogen to manage. Additionally, within the Xanthomonas genus, each species and even each pathovar exhibits distinct behavior and very specific hosts. Therefore, biological tools for controlling one pathovar are not necessarily applicable to another.

[0014] In the publication by Nagai Hirofumi, (2017), entitled "Improved control of black rot of broccoli caused by Xanthomonas campestris pv. Campestris using bacteriophage and a nonpathogenic Xanthomonas sp. strain", the use of a mixture of a phage for Xcc, which is not taxonomically identified, and a non-pathogenic Xanthomona is described. The phage disclosed in the publication has limited efficacy, where the incidence of the disease decreases only to 69%, so its combined use with the non-pathogenic Xanthomona is proposed, where with this combination the incidence of the disease decreases to 59%.

[0015] Regarding patent documents, Chinese patent application CN113201504 discloses a phage to combat the Xanthomonas campestris mango pathotype, which infects mangoes. In this case, the pathogen to be biocontrolled is different from that of the present invention.

[0016] On the other hand, patent application US2019116799, whose applicant is the University of

[0017] Chile discloses the use of bacteriophages for the biocontrol of Xanthomonas a. pv juglandis in walnut trees, that is, the aim is to control another species of Xanthomonas. The phages used are species of the Podoviridae and Siphoviridae families, different from the phage of the present invention, which is of the Myoviridae family, genus Foxunavirus.

[0018] Finally, patent document US2020093139A1 refers to a method of delivering phages to a plant susceptible to colonization by pathogenic bacteria such as Xylella or Xanthomonas. The emphasis of this document is not on the phage used but on the composition that allows the phage to be successfully administered, which comprises a mixture of two water-soluble polymers to which the lyophilized phage and nutrients are added, and capsules are formed, which can be administered in a preparation into the plant tissue.

[0019] From the documents presented it is clear that there is no phage in the art, nor a bioproduct, that allows for successful biocontrol of Xcc in brassicas.

[0020] BRIEF DESCRIPTION OF THE FIGURES

[0021] Figure 1: Thermal stability of phage Xcc_phage 27. Phage stability is shown after 1 h of incubation at different temperatures, presented as Log PFU / mL. The phage was suspended in SM buffer at the indicated temperatures, and the viral titer was then calculated using the double-layer technique. Error bars indicate the standard deviation calculated on three replicates.

[0022] Figure 2: pH stability of phage Xcc_phage 27. Stability is shown after 1 and 24 hours at different pH, presented as Log PFU / mL. Phage were suspended in pH-adjusted SM buffer and incubated at 4°C for 16 hours, and then viral titer was calculated using the double-layer technique. Error bars indicate standard deviation calculated on three replicates.

[0023] Figure 3: UV light stability of phage Xcc_phage 27. Phage stability to UV light exposure (UVA and UVB) over time was evaluated and presented as Log PFU / mL. Phage were suspended in SM buffer. Error bars indicate standard deviation calculated over three replicates. Figure 4: Biocontrol curves (Killing curves) for phage Xcc_phage 27 demonstrating the decrease in OD63nm over time at different MOLs. The average OD630nm of three independent replicates is represented by error bars showing the standard deviation. Experiments were performed in nutrient medium at 28 °C with Xcc 12-3 as host.

[0024] Figure 5: Transmission Electron Microscopy. Photograph of the bacteriophage of the invention, showing the icosahedral morphology with a characteristic 4>G tail.

[0025] Figure 6: Genome annotation of bacteriophage 4>G. Gene names encoding metabolism-related proteins and structural proteins are highlighted, while the larger arrow below highlights genes related to the lytic cycle. No genes associated with a lysogenic life cycle were identified; in particular, integrase was absent, indicating a strictly lytic infection cycle.

[0026] Figure 7. Phage spot test (DG) present in leaf tissue of cabbage plants. 1-5: filtrates from 5 cabbage plants taken at random. A: Spot test day 1 post inoculation. B: Spot test day 2 post inoculation. C: Spot test day 7 post inoculation. CP: Positive control phage (DG). CN: Negative control.

[0027] Figure 8. Spot test of the phage (DG present in cabbage seedlings. 1-6: filtrates from 6 randomly taken seedlings. A: Spot test week 1 post inoculation. B: Spot test week 2 post inoculation. CP: Positive control phage G. CN: Negative control.

[0028] Figure 9. Viral phage titer (DG) present in cabbage seedlings 1 week post inoculation. P1-P6: filtrates from 6 randomly selected seedlings. CN: Negative control. Columns that share the same letter do not show significant differences according to the Tukey test (p<0.05).

[0029] Figure 10. In vitro stability of 4>G in contact with fungicides. The variation in viral titer was evaluated at three phage exposure times (24 and 48 hours, and 7 days) to three fungicides: Polyben, Benomyl, and Switch. Tests were performed with phage Xcc_phage 27 at three different viral titers: le9, le8, le7 (UFP / mL). In each assay, the viral titer was quantified in three replicates. Statistical analysis was performed using the Shapiro-Wilk normality test and then an analysis of variance (ANOVA). Different letters indicate significant differences between treatments. It can be seen that exposure of the phage to the different agrochemicals resulted in a 1 log decrease in viral titer at 24 hours, regardless of the initial titer at which the assay was performed. No further change in viral titer was recorded at the following viral particle counting points (48 hours and 7 days).

[0030] Figure 11. In vitro stability of 4>G in contact with insecticides. The variation in viral titer was evaluated at three phage exposure times (24 and 48 hours, and 7 days) to three insecticides: Karate, Bull, and Coragen. Tests were performed with phage Xcc_phage 27 at three different viral titers: le9, le8, le7 (UFP / mL). In each assay, the viral titer was quantified in three replicates. Statistical analysis was performed using the Shapiro-Wilk normality test and then an analysis of variance (ANOVA). Different letters indicate significant differences between treatments. It can be seen that exposure of the phage to the different agrochemicals resulted in a 1 log decrease in viral titer at 24 hours, regardless of the initial titer at which the assay was performed. No further change in viral titer was recorded at the following viral particle counting points (48 hours and 7 days).

[0031] DETAILED DESCRIPTION OF THE INVENTION

[0032] The invention relates to a bioproduct for biocontrol of Xanthomonas campestris pv. campestris (Xcc), which consists of a lytic phage, Myoviridae family, in an agronomically acceptable carrier, where the phage is internally called Xcc_phage 27 G, or 4>G and has been deposited with the International Depositary Authority of Canada, National Microbiology Laboratory, Public Health Agency of Canada located at 1015 Arlington Street Winnipeg, Manitoba Canada R3E 3R2 and has the deposit number IDAC 261122-01 of November 28, 2022. In studies carried out after the deposit, which are shown in the examples, the inventors were able to determine that the genus of the phage of the invention is foxunavirus.

[0033] As discussed in the background, Xcc affects brassicas, a family of vegetables that includes cabbage, broccoli, cauliflower, turnips, kale, collards, radishes, and swede. Therefore, the bioproduct, or biocontrol composition, of the invention is primarily used to prevent, treat, and control the angular leaf spot or black rot disease of brassicas, caused by the pathogen Xcc.

[0034] Although there is knowledge about phage therapy and the use of different types of phages for the biocontrol of the Xanthomas species, the type of phage described and evaluated by the present invention for the lysis of Xanthomonas campestris pv. campestris (Xcc) in particular, with the high efficacy demonstrated herein, has not yet been disclosed. Likewise, a bioproduct comprising said phages, in addition to agriculturally acceptable carriers or adjuvants, which acts as a natural bactericide, has not been disclosed.

[0035] In a specific embodiment, the main characteristics of the use of phage therapy through the application of a bioproduct according to the characteristics of the present invention, and which can help control the angular spot disease or black rot of brassicas are:

[0036] It is biodegradable, non-carcinogenic and non-mutagenic.

[0037] It leaves no residue and has no safety limits. It is a selective therapy, meaning it only affects the target species.

[0038] It provides a diverse and complex mode of action through the synthesis of metabolites, toxins, antagonism, competition, etc., which makes the development of resistance difficult.

[0039] In another embodiment, the invention relates to the preparation of a bioproduct comprising the lytic bacteriophage of the invention, for use as a naturally occurring bactericide. The bioproduct also comprises agriculturally acceptable carriers or adjuvants.

[0040] For the development of the invention, the genetic diversity and pathogenicity of Xcc isolates present in crops of horticultural species of Brassica oleracea and related weeds were evaluated. To this end, the inventors took plant samples from 36 plots destined for the production of seeds and fresh consumption of brassicas distributed between the regions of Valparaíso and Ñuble, Chile, and were collected to isolate Xcc and bacteriophages. Thirty Xcc isolates were identified using selective media and specific amplification of fragments of the hrpF and estA genes. Furthermore, phage isolation was carried out using the double agar layer technique, obtaining lytic phages with distinct plaque morphologies, isolated from different geographical areas and with differential specificity with respect to Xcc isolates.

[0041] In this way, a collection of bacteriophages associated with Xcc isolates obtained in Chile was established, coming from various environments (soil, water, plant material), and various affected horticultural species.

[0042] Subsequently, the sensitivity to bacteriophages was analyzed in a collection of Xcc bacterial strains or pathovars collected in Chile. Parameters were evaluated that allowed for adequate selection of the bacteriophage of the invention and evaluation of the biocontrol capacity of each bacteriophage under in vitro and greenhouse conditions.

[0043] In this way, a bioproduct was prepared comprising the bacteriophage according to the present invention, as well as carrier and / or stabilizing agents that allow for obtaining a stable final product. The stabilizers will allow for a uniform dispersion of the agents contained in the bioproduct, maintaining its integrity. In a preferred embodiment, the bioproduct can be presented in solid, liquid, gel, or emulsion form.

[0044] Agriculturally acceptable stabilizing agents to be incorporated may be selected from the group consisting of alginates and chitosans.

[0045] Alginates are polysaccharides extracted from brown algae. The main alginate-producing algae species are Macrocystis pyryfera, Laminaria hyperborea, Laminaria digitata and Ascophyllum nodosum.

[0046] Chitosans are biocompatible and biodegradable natural polymers.

[0047] In an additional embodiment, the bioproduct may also comprise other carriers or adjuvants such as fluidifying agents and emulsifying agents, among others.

[0048] Thus, the invention relates to a biocontrol composition, or bioproduct, for agricultural use to control diseases caused by Xanthomonas campestris pv. campestris, comprising the bacteriophage, Xcc._Phage 27, deposit number IDAC 261122-01 and an agronomically acceptable carrier. Wherein the composition is formulated in the form of a liquid, oil dispersion, powder, dry wettable powder, gel, dispersible granule or dry wettable granule. And where the phage 27 G is present in a concentration of between 10 4 to 10 8 UFP / mg or per 10 4 to 10 8 UFP / mL, in compositions for direct application. Additionally, more concentrated compositions for storage and distribution, such as 10 9 to 10 15UFP / mg or per 10 9 to 10 15 PFU / mL

[0049] The agronomically acceptable carriers of the composition are chosen from: buffer solution at pH 6 to 7.5; mineral oil, alginate, chitosan, maltodextrin, trehalose, mannitol, zeolite, adjuvants, and mixtures thereof. Preferably, the agronomically acceptable carriers are a mixture of alginate and chitosan.

[0050] In a second aspect, the invention relates to the use of said biocontrol composition to prevent or control diseases caused by Xanthomonas campestris pv. campestris (Xcc). That is, to prevent or control angular leaf spot or black rot of brassicas caused by Xcc; in plants or crops of cabbage, broccoli, cauliflower, turnip, kale, cabbage, radish and / or swede. Wherein the composition of the invention is applied by spraying onto plants or seedlings that are diseased or susceptible to being infected with Xcc. Alternatively, the composition of the invention is applied in the irrigation water onto plants or seedlings that are diseased or susceptible to being infected with Xcc; or it is used as a coating on seeds.

[0051] The strain of the invention has demonstrated significant advantages, including its high lytic capacity against different variants of the Xcc pathogen, and its stability against UV light, pH changes, and temperature changes, making it suitable for use in agricultural conditions. It is also resistant to the action of chemical pesticides frequently used in brassicas, specifically fungicides commonly used in these crops.

[0052] EXAMPLES

[0053] Example 1. Obtaining the phage of the invention.

[0054] Plant and soil samples were collected from horticultural and seedbed plots in the Valparaíso and Ñuble regions of Chile for isolation of the pathogen and its bacteriophages. All Xcc strains were verified by RT-PCR of the hrpF gene. A total of 196 bacterial colonies were isolated from the sampled plots, which, based on their morphology, color, and growth, were considered Xcc candidates. Of these, 178 were isolated from plant material of the following plant varieties: cabbage, kale, weeds, broccoli, and cauliflower.

[0055] Subsequently, macerated plant samples with symptoms were enriched with different Xcc hosts for subsequent filtration and evaluation on cell lysis plates using the double agar technique. Candidate bacteriophages were reisolated and purified in an Xcc host three times to ensure their consistent lytic activity. Once their lytic nature was confirmed, they were observed by transmission electron microscopy (TEM) for taxonomic classification.

[0056] In a next step, the host range was evaluated using the double agar layer drop technique (spot test), 30 strains of Xcc and 9 strains of native bacteria (non-Xcc) were analyzed with the previously isolated lytic bacteriophages.

[0057] The drop or spot test technique was used as described by Khan Mirzaei & Nilsson 2015, the test consists of preparing a plate with NBY (Nutrient medium supplemented with yeast extract) with a double layer of soft agar (50% w / v) plus the bacteria to be evaluated, in this case Xcc. Once the double layer is dry, 5 pL drops of the phage are deposited on it, the plate is incubated at 28 ° C for 24 hours, after this period if the phages have lytic activity on the bacteria, a lysate or halo should be observed in the place where the drop was deposited.

[0058] Finally, 9 bacteriophages were isolated from the analyzed samples, and their lytic capacity was verified for 30 different strains of the Xcc bacteria.

[0059] Table 1: Genomic identification of isolated phages

[0060] Of the phages identified in Table 1, phage Xcc_phage 27 (CDG) was selected, which in addition to having excellent lytic properties, had the best behavior against changes in T° (see Figure 1), pH (see Figure 2) and prolonged exposure to UV light (see Figure 3) compared to the other isolated phages.

[0061] Table 2 shows the lytic results of the phage of the invention against different strains of the basic pathogen Xcc. The table indicates the origin of the pathogen evaluated. Table 2: Host range of lytic bacteriophages for Xcc <DG. Identificación de las bacterias sobre lascuales tienen actividad lítica el fago (DG; * indica lisis.

[0062] As can be seen, the phage of the invention was effective against 25 of 30 Xcc strains of different origins, demonstrating its significant capacity as a biocontrol agent.

[0063] The biological characterization of the selected phage is detailed below. Example 2. Properties of the phage of the invention.

[0064] Biological characterization of phage Xcc_phage 27

[0065] As we already mentioned, the thermal stability of the phage of the invention was studied, for which phage suspensions were incubated in SM buffer at a concentration of 1.2xl0 5 PFU / mL at different temperatures for 1 hour, and then their viability was assessed by calculating the viral titer using the double-layer technique. They were subjected to temperatures of 20, 30, 40, and 50°C. The results are shown in Figure 1, where it can be seen that increasing temperature does not have a significant effect on the average viability of the phage of the invention.

[0066] The stability of the phage at different pH levels was then evaluated. Phage suspensions were incubated for 16 hours at 4°C at a concentration of 2x10 5PFU / mL in SM buffer adjusted to different pHs, ranging from 1 to 13. After incubation, phage viability was assessed by measuring viral titer using the double-layer technique. The results are shown in Figure 2, which shows that the phage exhibited high stability between pHs 2 and 12.

[0067] Finally, the stability of the phage against UV light (UVA and UVB) was studied over time, presented as Log PFU / mL. The phage was suspended in SM buffer at a concentration of 1x10 5 PFU / mL, and their viability was determined at 2, 4, 8, and 12 hours of UV exposure. The results are shown in Figure 3, where it can be seen that UV exposure does not significantly decrease the average viability of the phage of the invention.

[0068] Lytic activity of the phage of the invention.

[0069] A suspension of Xcc bacteria was prepared at a concentration of lxlO 8CFU / mL in NBY medium (Nutrient medium supplemented with yeast extract) and a quantity of phage suspension of the invention in different concentrations between lxlO was added to this. 5 and lxlO 3 CFU / mL, which gives a ratio of 0.001, 0.0001 or 0.00001 phages / bacteria

[0070] These mixtures of Xcc bacteria and phages were incubated and the concentration of bacteria was evaluated by measuring the absorbance at 630 nm of the suspension. Controls of bacteria without phage, phage without bacteria, culture medium and SM buffer were included in the assay, in order to demonstrate that the effect of decreasing bacterial growth is due to the presence of phage at different concentrations. The results are shown in Figure 4. Where it can be seen that at all the phage concentrations evaluated it is capable of inhibiting the growth of the bacteria.

[0071] Phage Xcc_phage 27 presents optimal multiplex of infection (MOI), equivalent to the ratio between the number of bacteria and the number of phages, of 0.001 according to the graph in Figure 4.

[0072] Figure 5 is a photograph of the phage of the invention, using Transmission Electron Microscopy (TEM). The icosahedral morphology with a characteristic 4>G tail can be seen.

[0073] In order to taxonomically classify the phage of the invention, the inventors sequenced the genome of 4>G, and subsequently made an annotation of said genome, which is shown in Figure 6. From the identification of genes it was possible to determine that the strain of the invention has a strictly lytic infection cycle, this is evident from the fact that the integrase enzyme is not present.

[0074] Additionally, from the sequencing of the Xcc_phage 27 phage it was established that it has 98% homology with phages of the Foxunavirus genus, so it can be established that this is the genus of the phage of the invention.

[0075] Example 3: Systemic movement of phage <DG ¡n planta

[0076] In an additional test, cabbage plant substrates were inoculated with the bacteriophage of the invention to evaluate the movement of viruses from the roots to the aerial part of the plant.

[0077] For this purpose, cabbage plants of the Copenhagen Market cultivar were grown six weeks post-emergence in 190 mL pots with sterile substrate (3:1, peat:perlite) to demonstrate the ability of the phage (PG) to move systemically. The plants were watered by automatic irrigation four times a day for 6 min, and two foliar applications of Engeo® 247 ZC (Syngenta), a broad-spectrum insecticide, were made before phage application. Phage uptake by the plants (5 plants and three replicates) was evaluated by adding 5 mL of the lysate to the substrate at a concentration of 10 8 PFU / mL (TI) and a negative control group (T2) consisting of adding SM buffer without phages. Subsequently, the presence of phage in leaf tissue was determined for 1, 2, and 7 days post-inoculation. Leaf tissue samples measuring 2 cm were taken. 2from 3 random plants and surface sterilized with 10% sodium hypochlorite for 20 min, followed by three rinses with sterile distilled water. Leaf cuts were made under sterile conditions, considering individual sterile bags for each experimental group. Leaf samples were macerated using a mortar and pestle and then adding 2 mL of SM buffer pH 7.5. This macerate was centrifuged and filtered (0.22 pm) to obtain the filtrates for each group (TI and T2). Finally, 5 pL of these filtrates were taken to inoculate NBY plates by performing a spot test. The plates were previously inoculated with a bacterial lawn of 200 pL of Xcc and 5 mL of soft agar, and then incubated for 18 ± 2 h at 28 °C.

[0078] The results are shown in Figure 7, where lysis plaques are observed from leaf tissue samples of cabbage plants. In this sense, it was possible to isolate the phage (DG) during 1 and 2 days post inoculation in 5 plants taken at random, on the other hand, it was not possible to isolate the phage (DG) at 7 days post inoculation (C). However, these results indicate that the phage (DG) is systemic and is present in leaf tissue at least in the first 48 h after its application to the plant substrate and in the absence of its host.

[0079] Additionally, as a complementary test, cabbage seedlings of the Copenhagen Market cultivar were grown for two weeks post-emergence in 50-cell seedbeds with sterile substrate (3:1, peat:perlite) to evaluate the ability of phages to move systemically and determine their concentration in plant tissue. To do this, the viral titer was determined by adding 5 mL of the lysate to the substrate at 10 °C.8 PFU / mL per seedling (TI, 20 seedlings and three replicates) and a negative control group (T2) consisting of the addition of SM buffer without phages. Subsequently, 6 seedlings were randomly selected from each treatment and surface sterilized with 10% sodium hypochlorite for 20 min, followed by three rinses with sterile distilled water. The samples were macerated using a mortar and pestle and 1 mL of SM buffer pH 7.5 was added. This macerate was centrifuged and filtered (0.22 pm) to obtain the filtrates for each treatment. Finally, 5 pl of these filtrates were used to inoculate NBY plates by performing a spot test. If lysis plaques were observed, the phage concentration (DG) was determined by a double agar layer test of serial dilutions of the filtrates with 200 pL of Xcc and 5 mL of soft agar. The final phage concentration in the seedlings was determined up to 1 week post inoculation.

[0080] The results are shown in Figure 8, where lysis plaques are observed in the filtrates from the cabbage seedlings. The spot test performed in the first week post-inoculation shows that the phage is present in all six randomly selected seedlings (A), while in the second week it was only possible to obtain it from a single seedling (B).

[0081] In each case where the spot test was positive, the viral titer of the phage (DG) was calculated as shown in Figure 9. In all 6 seedbeds, the viral titer exceeded 1x10. 4 PFU / mL, with significant differences observed with respect to the negative control (without phages) in the Tukey test with 95% confidence.

[0082] Example 4. Composition of the invention.

[0083] A formulation was prepared that included the selected phage, that is, phage Xcc_phage 27, together with the stabilizing agent alginate to provide stabilization to the phage within the formulation.

[0084] For which a 2% w / v sodium alginate solution was prepared, which was mixed with the phage suspension lxlO 9 UFP / mL at 20% v / v, then alginate beads were formed using a 5% w / v calcium chloride solution.

[0085] Example 5. Evaluation of lytic activity in the presence of fungicides.

[0086] To evaluate whether the lytic effect of the phage on the pathogenic bacterium Xcc is maintained in the presence of fungicides commonly applied to brassica crops, an in vitro experiment was designed to evaluate the variation in the concentration of the phage Xcc_phage 27 over time, in the presence of different fungicides.

[0087] Specifically, the variation in viral titer was evaluated at three times of phage exposure (24 and 48 hours, and 7 days) to three different fungicides: Polyben®, Benomyl® and Switch®, all of them commonly used in the management of fungal diseases in cabbage crops. The active ingredients of these fungicides are: Polyben®: Benomyl 50% w / w, as well as Benomyl®: Benomyl 50% w / w and Switch®: cyprodinil 37.5% w / w and fludioxonil 25% w / w. The tests were performed with the phage at three different viral titers: 10 9 , 10 8 , 10 7 (UFP / mL). In each assay, the viral titer was quantified in 3 replicates.

[0088] In brief, the methodology of the experiment consisted of mixing 500 pL of the phage and 500 pL of fungicide at field concentration: Polyben (lg / L), Benomyl (2.6 g / L) and Switch (lg / L). The test was carried out in cell culture plates and kept at room temperature throughout the evaluation time (7 days). All the aforementioned combinations were tested. After 24 hours, a lysis plate count (viral titer) was performed using the double agar layer technique. This test was repeated after 48 hours and after 7 days.

[0089] The results are shown in Figure 10. Under all conditions, exposure of the phage to the different agrochemicals (fungicides) resulted in a 1-log decrease in viral titer at 24 hours, regardless of the initial titer at which the assay was performed. No further change in viral titer was recorded at subsequent viral particle count points (48 hours and 7 days).

[0090] This result demonstrates that the phage of the invention is sufficiently stable against the agrochemicals to which they might be exposed under field conditions.

[0091] Example 6. Evaluation of lytic activity in the presence of insecticides.

[0092] To evaluate whether the lytic effect of the phage on the pathogenic bacterium Xcc is maintained in the presence of fungicides commonly applied to brassica crops, an in vitro experiment was designed to evaluate the variation in the concentration of the phage Xcc_phage 27 over time, in the presence of different insecticides.

[0093] Specifically, the variation in viral titer was evaluated at three exposure times of the phage (24 and 48 hours, and 7 days) to three different insecticides: Karate®, Bull® and Coragen®, all of them commonly used in cabbage crop management. The active ingredients of these insecticides are Karate®: Lambda Cyhalothrin, Bull®: Gama Cyhalothrin and Coragen®: Chlorantranoliprol. The tests were performed with the phage at three different viral titers: 10 9 , 10 8 , 10 7 (UFP / mL). In each assay, the viral titer was quantified in 3 replicates.

[0094] In brief, the methodology of the experiment consisted of mixing 500 pL of the phage and 500 pL of fungicide at field concentration: Karate (0.2 mL / L), Bull (2 mL / L) and Coragen (lmL / L). The test was carried out in cell culture plates and kept at room temperature throughout the evaluation time (7 days). All the aforementioned combinations were tested. After 24 hours, a lysis plate count (viral titer) was performed using the double agar layer technique. This test was repeated after 48 hours and after 7 days.

[0095] The results are shown in Figure 11. Under all conditions, exposure of the phage to the different agrochemicals (insecticides) resulted in a 1-log decrease in viral titer at 24 hours, regardless of the initial titer at which the assay was performed. No further change in viral titer was recorded at subsequent viral particle count points (48 hours and 7 days).

[0096] This result demonstrates that the phage of the invention is sufficiently stable against the agrochemicals to which they might be exposed under field conditions.

Claims

CLAIMS 1) A biocontrol composition for agricultural use to control diseases caused by Xanthomonas campestris pv. campestris, CHARACTERIZED in that it comprises bacteriophage phage 27, deposit number IDAC 261122-Oly and an agronomically acceptable carrier. 2) A composition according to claim 1, CHARACTERIZED in that it is formulated in the form of a liquid, oil dispersion, powder, dry wettable powder, gel, dispersible granule or dry wettable granule. 3) A composition according to claim 2, CHARACTERIZED in that phage 27 is present in a concentration of between 10 4 to 10 15 CFU / mg or per 100ml 4 to 10 15 CFU / mL. 4) A composition according to claim 2, CHARACTERIZED in that the agronomically acceptable carriers are chosen from: buffer solution at pH 6 to 7.5; mineral oil, alginate, chitosan, maltodextrin, trehalose, mannitol, zeolite, adjuvants and mixtures thereof. 5) A composition according to claim 4, CHARACTERIZED in that the agronomically acceptable carriers are a mixture of alginate and chitosan. 6) Use of the biocontrol composition according to any of claims 1 to 5, CHARACTERIZED in that it serves to prevent or control diseases caused by Xanthomonas campestris pv. campestris (Xcc). 7) Use according to claim 6 CHARACTERIZED in that it serves to prevent or control angular spot or black rot of brassicas produced by Xcc. 8) Use according to claim 7 CHARACTERIZED in that it serves to prevent or control angular spot or black rot in plants or crops of cabbage, broccoli, cauliflower, turnip, kale, cabbage, radish and / or kohlrabi. 9) Use according to claim 8 CHARACTERIZED in that it is applied by spraying on plants or seedlings that are sick or susceptible to being infected with Xcc. 10) Use according to claim 8 CHARACTERIZED in that it is applied in irrigation water on plants or seedlings that are sick or susceptible to being infected with Xcc. 11) Use according to claim 8 CHARACTERIZED in that it is applied as a coating on seeds.

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