WO2023233182A1

WO2023233182A1 - Biological inoculant with biocontrol action against xanthomonas arboricola

Info

Publication number: WO2023233182A1
Application number: PCT/IB2022/055094
Authority: WO
Inventors: Pablo Dagoberto NÚÑEZ CERDA; María Camila MORALES ITURRA; Sofía Shantal BUSTOS MOLINA; María Fernanda FLORES ECHEVERRÍA
Original assignee: Agroadvance Spa
Priority date: 2022-05-31
Filing date: 2022-05-31
Publication date: 2023-12-07

Abstract

Provided is a formulation corresponding to a biological inoculant focused on controlling diseases caused by phytopathogens in fruit crops of the type Prunus, hazelnut and walnut, in particular phytopathogens of the species Xanthomonas arboricola. The composition is formed by a complex of microorganisms (fungi, bacteria and viruses), which are characterised in that they have powerful biocontrol capacity, which is strongly amplified by the synergic action of the microbial components thereof. The composition according to the invention comprises strains of the bacteriophage Prado virus (Autographiviridae family), of the fungus Trichoderma atroviride and of the bacterium Bacillus velezensis, and is characterised in that it allows diseases caused by X. arboricola to be prevented and/or controlled. The composition of the invention is harmless to humans and the environment.

Description

BIOLOGICAL INOCULANT WITH BIOCONTROLLER ACTION AGAINST XANTHOMONA ARBORICOLA

TECHNICAL FIELD

The present invention is framed in the agricultural industry and the biological control of phytopathogens. In particular, the present invention is aimed at a biocontrol formulation against diseases caused by the phytopathogen Xanthomonas arboricola.

BACKGROUND OF THE INVENTION

The species Xanthomonas arboricola corresponds to gram-negative, phytopathogenic bacteria that cause diseases in a wide variety of plants. They are classified into the following pathovars (pv.): corylina, juglandis, fragariae, populi, pruni and celebensis. The species Xanthomonas arboricola pv. Juglandis (Xaj) is the causal agent of the disease known as black plague and brown apical necrosis (BAN), mainly affecting walnut trees. Pathovar corylina (Xac) mainly affects hazel trees, causing the disease known as Bacterial blight and Gray necrosis (GN). On the other hand, the species Xanthomonas arboricola pv. pruni affects all trees of the genus Prunus, which includes cherry, plum, peach, nectarine, apricot and almond trees producing the disease known as bacterial spot.

Black Death

As indicated above, Xanthomonas arboricola pv. juglandis (Xaj) causes the disease known as black plague in walnut, attacking leaves, nuts, buds, catkins and young branches. The lesions begin as small dark brown spots with a yellowish halo. The infection then spreads and large areas of dark, dead tissue appear.

The symptoms caused by Xaj can be easily observed on fruits as single or confluent necrotic spots; on leaves, as single spots or sometimes very large wilted areas; on twigs and branches, like cankers that develop quickly and crack. Production losses are particularly serious, and can affect more than 50% of the fruits, because they fall very easily during the initial stage of their development. The disease is considered a limiting factor for walnut production in most areas of the world.

Control of the Black Death is particularly cumbersome and relies on copper treatments (when permitted) and antibiotic sprays. The use of copper-based compounds (copper sulfate, copper hydroxide or cuprous oxide) is frequently useless, due to the presence of highly resistant strains, which has inspired the search for other solutions.

It has been shown that Xaj can multiply epiphytically and invade walnut trees mainly through stomata, leaf scars, or wounds. It has also been described that dormant buds They are hibernation sites for Xaj. Furthermore, the existence of alternative host reservoirs, such as asymptomatic plant species, could also be relevant for the dissemination and transmission of Xaj to walnut plants.

Brown Apical Necrosis

In addition, Xaj is also a primary causal agent of another complex disease in walnut called brown apical necrosis or BAN. This disease causes spots and damage to the apices and shell of fruits, which promotes the colonization of opportunistic fungi, among which Fusarium and a group of three different taxa of Alternaria stand out, among other fungi. The infection of these fungi causes brown spots and produces premature fruit drop, generating losses close to 20% in plantations.

External symptoms begin to appear after fruit set at the apical end and become more evident as the fruit grows. The lesions are usually small and dark brown or black in color, not moist, and progress to a size of approximately 2mm to 15mm, with relatively regular and circular margins. Regarding internal symptoms, the infection progresses through the tissues and can reach the seed, causing brown or black rot of the seeds.

Thus, BAN disease consists of a sequential problem, initiated by a primary infection of the Xaj bacteria in young nuts, and continuing with the subsequent secondary infection of the opportunistic fungal pathogens, Fusarium spp. and Alternaria spp, which grow as saprophytes on tissues infected by the bacteria, thus intensifying the symptoms and severity of the disease.

Many aspects of apical necrosis are still unknown, so traditional control strategies may not be efficient enough.

In this context, the present invention initially arises as an alternative for biocontrol of Xanthomonas arboricola pv. juglandis, however, once the product capable of controlling Xaj was obtained, the inventors tested it against other harmful species. Wherein the invention is a biological product made up of a set of beneficial microorganisms within which there are specific strains of bacteriophages, fungi and bacteria, with a broad and powerful capacity to act against these phytopathogens of the Xanthomonas arboricola species.

Bacteriophages are viruses that exclusively infect prokaryotic organisms (bacteria and archaea), so their use is presented as an effective alternative to treat diseases caused by bacteria. Bacteriophages act by infecting bacterial cells and subsequently using them through a sequential and highly regulated enzymatic mechanism. Fungi of the genus Trichoderma belong to the division Ascomycetes and the order Hipocreales, they are widely distributed in soils, have green spores and can be found throughout the world. Trichoderma spp. They are colonizers of cellulosic materials, they can often be found where decomposing plant material is available, as well as in the rhizosphere of plants, where they can induce systemic resistance against pathogens and compete against harmful microorganisms by establishing mutualistic interactions with plants. Among the mechanisms by which Trichoderma species establish interactions with other organisms, competition and mycoparasitism stand out, which makes them excellent biocontrol agents both for their antagonistic properties against other fungi and for their ability to colonize diverse habitats. and successfully establish themselves in those recipient communities where they have been introduced.

On the other hand, species of the Bacillus genus are gram-positive bacteria widely distributed in the environment, they have a unique ability to replicate rapidly, resist adverse environmental conditions and have a broad spectrum of biocontrol capacity.

State of the art

Given the importance of these pathogens, in the state of the art there are other products to try to control them, below are the documents considered closest, where none of them anticipates the invention.

Document US2019116799 A1 presents a bactericidal composition based solely on bacteriophages for the control of Methods for the isolation, propagation and application of bacteriophages for the prevention, treatment or reduction of symptoms due to Xaj are described. Similarly, in the document by S. Romero-Suarez et al. (2012) studied bacteriophages present in walnut trees, which can infect Xanthomonas arboricola pv. Juglandis, among which are bacteriophages of the Podoviridae and Siphoviridae species that could be used in the biocontrol of this bacterial phytopathogen. Furthermore, in the work carried out by D. Dómótór et al. (2016) studied bacteriophages present in walnut trees, which can infect Xanthomonas arboricola pv. Juglandis, among which are bacteriophages of the Podoviridae and Siphoviridae species, with a lytic effect. The phages were sequenced, and it was found that one of them, Xaj24, has a genome similar to the Prado-phage.

The inventors of the present invention consider that only the application of phages is not sufficient for the control of diseases caused by X. arboriola pv. juglandis, which explains that, despite these documents, the disease is still very present in walnut trees around the world. Thus, although the state of the art shows that there is currently a tendency to resort to pest biocontrol solutions instead of traditional and harmful chemical pesticides, none of the documents analyzed anticipates the solution developed by the inventors to the phytopathogen Xa, which is especially difficult to control.

DESCRIPTION OF THE INVENTION

The present invention corresponds to a biocontrol formulation that is composed of a mixture of 3 different microorganisms: bacteriophage Pradovirus (family Autographiridae) (Phage/phage 13) deposited under IDAC number 181021-01 on October 21, 2021 fungus Trichodermas atroviride ( strain 205) deposited under IDAC number 181021-02 on October 21, 2021 Bacillus velezensis bacteria (strain PC), deposited under IDAC number 181021-04 on October 21, 2021

Where the phage was deposited together with the phytopathogenic bacteria

All deposits were made in Canada, at the International Depositary Authority of Canada.

As we have indicated, pests caused by Xa are very difficult to manage. The bacteria must be controlled, and at the same time the associated opportunistic fungi. Faced with this complex panorama, the inventors developed a biocontrol mixture that includes 3 different types of microorganisms, which surprisingly act in synergism and allow comprehensive control of diseases caused or initiated by this bacteria. The invention comprises the combined use of the specified strains, which in turn were selected for their individual properties, as will be shown in the examples.

During the development of the invention, each microorganism was analyzed separately against the pathogen of interest and the study of microcosms of different combinations of the selected microorganisms was also carried out. In this way the invention refers to:

A biocontrol formulation for agricultural use to prevent or treat diseases associated with bacteria Bacillus velezensis strain PC deposit number 181021-04. Where the formulation is formulated in the form of a liquid, oil dispersion, powder, dry wettable powder, dispersible granule or dry wettable granule. It is preferably formulated in powder form, in a maltodextrin matrix. Where the fungus Trichodermas atroviride strain 205 deposit number 181021-02 can be found in the form of spores. In a second aspect, the invention points to a process for preparing a biocontrol formulation, which includes the stages of mixing: a. a composition comprising the bacteriophage Pradovirus Phage/tago 13 deposit number 181021-01 in a concentration of between 1.0x10 ⁶ and 1.0x 10 ¹² PFU/mL, b. a composition comprising the fungus Trichodermas atroviride strain 205 deposit number 181021-02 in a concentration between 1.0xl0 ⁵ and 1.0x 10 ⁹ CFU/g, and c. a composition comprising the bacteria Bacillus velezensis strain PC deposit number 181021-04 in a concentration of between 1.0x10 ⁵ and 1.0x10 ⁹ CFU/mL or between 1.0x10 ⁵ and 1.0x10 ⁹ CFU/g, and stirring to obtain a homogeneous dispersion of at least 1 liter of formulation.

Preferably, there is a process for preparing the biocontrol formulation, which includes the stages of mixing: a. a composition comprising the bacteria Bacillus velezensis strain PC deposit number 181021-04 in a concentration of between 1.0xl0 ⁵ and 1.0x10® CFU/mL, b. 1 g of a composition comprising the fungus Trichodermas atroviride strain 205 deposit number 181021-02 in a concentration between 1.0x10 ⁵ and 1.0x10 ⁹ CFU/g, c. 1 mL of a composition comprising the bacteriophage Pradovirus Phage/tago 13 deposit number 181021-01 in a concentration of between 1.0x10 ⁶ and 1.0x10 ¹² PFU/mL, and shake to obtain a homogeneous dispersion of at least 1 liter of formulation.

In addition, there is a second preferred process for preparing the biocontrol formulation, which includes the mixing steps: d. a composition comprising the bacteriophage Pradovirus Phage/phage 13 deposit number 181021-01 in a concentration of between 1.0x10 ⁶ and 1.0X 10 ¹² PFU/mL, e. 1 g of a composition comprising the fungus Trichodermas atroviride strain 205 deposit number 181021-02 in a concentration between 1.0x10 ⁵ and 1.0x10 ⁹ CFU/g, and f. 1 g of a composition comprising the bacterium Bacillus velezensis strain PC deposit number 181021-04 in a concentration of between 1.0xl0 ⁵ and 1.0x10® CFU/g, and stir to obtain a homogeneous dispersion of at least 1 liter of formulation.

Finally, the last alternative is a process for preparing the biocontrol formulation, which includes the stages of mixing: a. a composition comprising the bacteria Bacillus velezensis strain PC deposit number 181021-04 in a concentration of between 1.0xl0 ⁵ and 1.0x10® CFU/mL, b. 1 g of a composition comprising the fungus Trichodermas atroviride strain 205 deposit number 181021-02 in a concentration between 1.0x10 ⁵ and 1.0x10 ⁹ CFU/g, c. a composition comprising the bacteriophage Pradovirus Phage/tago 13 deposit number 181021-01 in a concentration of between 1.0xl0 ⁶ and 1.0x 10 ¹² PFU/mL, and stir to obtain a homogeneous dispersion of at least 1 liter of formulation .

The aforementioned process, and its preferred alternatives, optionally comprises a stage where the final dispersion is subjected to drying or lyophilization to obtain a dry powder, optionally with formulation adjuvants.

Where the dry powder can be mixed with formulation auxiliaries such as surfactants, dispersants and theological agents to obtain a dispersion in oil, dispersible granule or dry wettable granule.

In a third aspect, the invention aims at a method to prevent or control diseases caused by phytopathogenic fungi. In one embodiment the formulation is applied by spraying on the plants or seedlings, or fruits. The application can be foliar or root application on plants or seedlings that are sick or susceptible to being infected with Xanthomonas arboricola and/or phytopathogenic fungi. Where plants or seedlings are diseased or susceptible to infection, with

Where these diseases are, for example: the black plague or brown apical necrosis produced by Xanthomonas arboricola pv. juglandis, mainly in walnut, or gray necrosis caused by Xanthomonas arboricola pv. corylina (Xac) on hazelnuts, or bacterial spot caused by Xanthomonas arboricola pv. pruni in fruit trees of the genus Prunus. For which the formulation of the invention is applied by spraying on plants or seedlings that are sick or susceptible to being infected, with Xa. In another embodiment, the formulation of the invention is applied in irrigation water to plants or seedlings that are sick or susceptible to being infected with Xa.

Where advantageously the formulation of the invention controls both the presence of Xa and opportunistic phytopathogenic fungi. Where the opportunistic fungi are Neofusicoccum spp., Diaporthe spp., Rhizoctonia spp., Aspergillus spp., Phytophthora spp., Fusarium spp., Rhizopus spp., and/or Alternaria spp. In particular, opportunistic fungi are Fusarium destruens and/or Alternaria alte mata.

Although each phytopathogenic pathovar attacks a particular host, for example, Xaj, mainly infects walnut trees, as indicated in the background, it has been seen that the pathogen can be present in other plants or trees, which do not develop the disease. , but they act as reservoirs or vectors. Therefore, the formulation of the invention can be applied to species susceptible to being infected by some Xa, diseased or healthy plants, to prevent or control the disease, and also on nearby trees, in order to prevent its spread.

Formulation

The formulation comprising the described microorganisms may be in the form of a liquid, oil dispersion, powder, dry wettable powder, dispersible granule or dry wettable granule. Preferably, the formulation is in the form of a wettable liquid or powder.

The concentration of the bacteriophage Pradovirus Phage/tago 13 deposit number 181021-01 in the formulation is between 1.0x10 ⁶ and 1.0x10 ¹² PFU/mL.

The concentration of the fungus Trichodermas atroviride strain 205 deposit number 181021-02 in a concentration between 1.0x10 ⁵ and 1.0x10 ⁹ CFU/g, and

The concentration of the bacteria Bacillus velezensis strain PC deposit number 181021-04 in a concentration of between 1.0x 10 ⁵ and 1.0X 10 ⁹ CFU/mL or between 1.0x10 ⁵ and 1.0x10 ⁹ CFU/g,

Among the vehicles or additives that can be incorporated into the formulation are the wetting agent, the dispersing agent, the disintegrating agent, the surfactant agent and the filler. In a preferred embodiment, the humectant and thickener corresponds to: maltodextrin, trehalose, anhydrous betaine, mannitol and/or zeolite.

The scope of the invention will become clearer in light of the following illustrative examples thereof.

DESCRIPTION OF THE FIGURES

Figure 1 . Monitoring of the BAN microorganism population: (i) Xaj (ii) Alternaria alternata and (iii) Fusarium destruens, in experimental microcosms through qPCR. The control microcosms with only pathogen are observed in the first column. The microcosms with application of the formulation of the invention are observed in the second column for each sampling time. Significant differences between treatments at each time were obtained using the Bonferroni post-test (a=0.05; P<0.05) and are represented with asterisks on each bar of the graphs. The data correspond to the average number of copies of each gene, and their respective standard deviations.

Figure 2: Inhibition in the growth of Xanthomonas arboricela treated with Bacillus PC and liquid formulation of the invention. The data are graphed as a function of the average diameter of the inhibition zone (mm) for isolates T3H and N7 of Xanthomona arborícela pv juglandis and for A7 and H1.2 corresponding to Xanthomona arborícela pv corylina (n=5). The tests were carried out on plates in YPD agar medium. Figure 3: Effect on the growth of Xanthomonas arboricola treated with Phage 13 and liquid formulation of the invention under evaluation at 24 hours post inoculation of the bacteriophage. The bacterial count is expressed as Log 10 (CFU/mL) for isolates T3H and N7 of Xanthomona arboricola pv juglandis and for A7 and H1.2 corresponding to Xanthomona arboricola pv corylina (n=5). The tests were carried out in YPD liquid medium.

Figure 4: Effect on the growth of pathogenic fungi of BAN (brown apical necrosis) and GN (gray necrosis) treated with Bacillus PC and liquid formulation of the invention. The data are graphed based on the average growth of the pathogenic fungus evaluated, corresponding to Fusarium, Alternaria and Diaporhe (n=5). The tests were carried out on plates in PDA medium.

Figure 5: Effect on the growth of pathogenic fungi of BAN (brown apical necrosis) and GN (gray necrosis) treated with Trichoderma 205 and liquid formulation of the invention. The data are graphed based on the average growth of the pathogenic fungus evaluated, corresponding to Fusarium, Alternaria and Diaporhe (n=5). The tests were carried out on plates in PDA medium.

EXAMPLES

Example 1: Obtaining the components of the biocontrol formulation of the invention.

Next, the selection methodology for each microorganism and the growth conditions used are described. Notwithstanding the above, a person skilled in the art may cultivate the strains under other conditions or with slight modifications to those described, remaining within the scope of the invention, which is defined by the specific strains that are used.

A.1 Selection of Trichoderma strains.

The isolates identified as positive for the Trichoderma genus were evaluated in plate biocontrol assays with PDA medium. For this test, Trichoderma was inoculated 2 cm from the edge of the plate, and additionally a pathogenic fungus (Fusarium sp. or Alternaria sp.) was added 2 cm from the opposite edge of the plate. The plates were incubated at 27°C for 6 to 10 days and subsequently the percentage of inhibition of the growth of the biocontrol fungus on the pathogenic fungus was quantified. Of the total number of fungi analyzed, 3 isolates were selected for their remarkable biocontrol capacity. Where finally the one with the best result was chosen, which was identified as Trichodermas atroviride (strain 205) and was deposited under number 181021-02.

A.2 Growth conditions of Trichoderma strain

For growth on solid medium, rice was used as the main substrate. In this stage, around 400g of previously sterile rice were inoculated with 20mL of liquid culture of Trichoderma fungi prepared from a suspension of mycelium in sterile water. It was allowed to grow at 27°C in culture chambers for 7 days, then the rice was allowed to dry at room temperature for 7 days. B.1 Selection of Bacillus strains.

With the strains initially identified as Bacillus sp. A plate biocontrol test was carried out against Fusarium and Alternaria fungi and Xa bacteria (Xaj and Xac), to determine which are the best candidates for the formulation of a product for biocontrol. For this, each of the isolates was cultured in liquid YPD medium at 27°C, 180rpm for 24h and then its concentration was adjusted to 10 ⁶ CFU/mL, and a 25uL aliquot was added on a lawn with the corresponding pathogen ( Xaj, Xac, Fusarium and Alternaria). The plates were incubated at 27°C for 24-48h in the case of Xaj and Xac and for 6 days in the case of Fusarium and Alternaria.

The selection of the isolates was carried out by quantifying the zone of inhibition observed between the biocontroller and the pathogen. In the case of biocontrol against phytopathogenic fungi, the selection was carried out by analyzing the inhibition of the biocontroller towards the fungus.

Of the total Bacillus analyzed, 3 isolates were selected for their remarkable biocontrol capacity. From this selection, a molecular identification was carried out through massive sequencing using the ¡Ilumina technique, where it was determined that all the bacteria corresponded to strains of Bacillus velezensis. Finally, the strain with the greatest inhibition against the 3 pathogens was chosen, which was identified as Bacillus velezensis (strain PC), deposited under number 181021-04.

B.2 Growth conditions and viability of Bacillus strains.

Growth of vegetative cells and spores was evaluated in CFU/mL, OD600 and pH in liquid culture in DSM and YPD medium. For this, flasks were inoculated with YPD medium for each of the selected isolates and left shaking at 180rpm, 27°C for 18h. From these flasks, 1 mL was taken and a 250 mL flask was inoculated with 100 mL of each medium, in triplicate for each isolate. These flasks were left shaking at 160rpm, 32°C for 7 days. The indicated parameters were evaluated at the initial time, 24h, 48h, 5 days and 7 days. Both media show good results and can be used interchangeably.

Additionally, the viability of the strains was evaluated in three storage conditions: 4°C, 25° and 40°C for periods of up to 2 years. It was determined that there is no loss of viability in any of the conditions evaluated.

C.1 Selection of phage strains.

With the phages obtained, an evaluation of the host range was carried out, in this way it was determined which of all the isolated phages is capable of lysing more Xa strains. 16 Xa strains and 21 selected phages were used. The achieved titer of each phage on an Xa lawn was evaluated using the double agar layer technique. From the quantification data of the viral titer of each phage evaluated on each of the Xa isolates, 4 bacteriophages were selected. for the formulation prototype. These selected phages were evaluated in different combinations on grass for each of the 16 Xa bacterial isolates.

Of the total bacteriophages analyzed, 3 isolates were selected for their outstanding biocontrol capacity, corresponding to strains F13, strains F29 and F11. From this selection, a molecular identification was carried out through massive sequencing using the ¡Ilumina technique. The results indicated that these belong to the Autographiviridae family

C.2 Phage growth and viability conditions.

A decay curve of a strain of Xaj bacteria was carried out in the presence of phage 13, 29 or 11. For this, 250 mL flasks were inoculated with 100 mL of liquid YPD medium and 1 mL of culture of the Xaj bacteria was added and left stirring at 27°C and 160rpm until reaching an OD600 of 0.2. Once this OD value was reached, 1mL of phage was added and left stirring at 140 rpm for 18h.

The viral titer achieved (PFU/mL), the bacterial concentration (CFU/mL), the OD600 and the pH were evaluated for each amplification round. Measurements were made every 4 hours until the 24-hour process was completed. From this evaluation, a phage was selected as a potential biocontroller of Xaj.

For this selected phage, viability tests over time were carried out. In this way, phage F13 was finally selected, (Phage/phage 13) which was identified as Pradovirus (Autographiviridae family) deposited under number 181021-01.

As we indicated, the strains with the best biocontrol properties were deposited in an appropriate institution, in this case the “International Depositary Authority of Canada”, obtaining the deposits already mentioned at the beginning of the description:

A: Bacillus velezensis strain of bacteria (PC strain), deposited under No. 181021-04 on October 21, 2021

B: Strain of the fungus Trichodermas atroviride (strain 205) deposited under No. 181021-02 on October 21, 2021

C: Strain of the Pradovirus bacteriophage (family Autographiviridae) (Phage/phage 13) deposited under No. 181021-01 on October 21, 2021

Example 2: Formulation in liquid format

To obtain the biocontrol formulation of the invention, the following formulations of each of the components were mixed, which have the following concentrations:

1 .0x10 ⁵ -1 .0x10 ⁹ CFU/mL of PC strain (B. velezensis), in DSM medium, pH 6.7; 1 g of a composition comprising spores of the fungus Trichodermas atroviríde strain 205 deposit number 181021-02 in a concentration between 1.0*10 ⁵ and 1.0*10® CFU/g in rice substrate; and

10 ⁷ PFU/mL of Phage 13, in SM buffer medium (50 mM Tris-HCI; 8 mM MgSC (7H2O); 100 mM NaCl), pH 7.5.

The mixture was then stirred to obtain a homogeneous dispersion up to a total volume of 1 liter.

Example 3: Powder formulation

The formulation obtained in the previous example was subjected to lyophilization, in a 15% maltodextrin matrix.

This powder formulation is reconstituted in water before being applied to crops.

Example 4: Evaluation of the formulation against brown apical necrosis produced by Xanthomonas arboricola pv. juglandis (Xaj) and the fungi Fusarium spp. and Alternaria spp in microcosm assays.

Experimental microcosms composed of microorganisms belonging to the BAN disease were constructed; i) Xanthomonas arboricola pv. juglandis, i) Fusarium destruens and iii) Alternaria alternata.

The assay setup was carried out in sterile 20 mL penicillin bottles, which contained 0.3 g of sterile rock wool as a fixation matrix for the growth of microorganisms. Vogel medium (supplemented with 0.5% yeast extract, 0.5% dextrose and 30 pg/mL chloramphenicol) was used as culture medium, which was added to the rock wool until reaching 60%. its water retention capacity. The microcosms were inoculated with 1x10 ⁴ CFU/mL of Xaj and 1x10 ⁴ spores/mL of each fungus (Fusarium destruens and Alternaria alternata) and sealed with cotton plugs. After 7 days of inoculating the isolates belonging to the BAN, and therefore establishing the microorganisms in the experimental system, the complex of beneficial microorganisms was inoculated in the microcosms, composed of; i) strains of the genus Tríchoderma, i) strains of the genus Bacillus and iii) infective bacteriophages of Xaj bacteria. The product was applied at a concentration of 1.5 g/L. Additionally, control microcosms composed only of BAN pathogens were set up. All microcosms were performed in triplicate. The microcosms were maintained for a period of 30 days post-product application (dpa), obtaining samples through destructive sampling at 0, 3, 7, 10, 20 and 30 dpa.

The content of the microcosms was obtained through destructive sampling, in order to minimize the disturbance of the microcosms and avoid the risk of contamination. The samples were collected by moistening the contents of each penicillin bottle with 3 mL of 1X Vogel medium. Placing it in a sterile Petri dish and mechanically homogenizing it using tweezers and a spatula under sterile conditions. Subsequently, the samples were placed in 2 mL tubes for subsequent DNA extraction.

gDNA extraction and qPCR were performed for each BAN microorganism during the 6 sampling times.

The results (see Figure 1) showed that the BAN pathogenic population composed of Xaj, A. alternata and F.destruens, begins to decrease 3 days after applying the formulation of the invention in the microcosms, with significant differences in F.destruens. Additionally, between 7 and 30 days after application, significant decreases in the populations of Xaj, A. alternata and F.destruens were observed.

Example 4: Evaluation of the formulation of the invention against Xanthomonas arborícela pv. juglandis (Xaj) and Xanthomonas arborícela pv. corylina (Xac).

In order to test the effect of the invention with respect to different pathovars, plaque and inhibition zone tests were carried out. Plates were obtained with YPD agar medium, covered with a lawn of Xaj or Bacillus PC, which is also present in the formulation of the invention, at a concentration of 1.0x10 ⁷ CFU/mL.

In parallel, independent experiments were carried out, where the effect of the phage used in the invention was evaluated, in comparison with the formulation of the invention and with a control without inhibitor, in this case YPD liquid medium was used to grow the Xa bacteria.

Plate experiment.

Different strains of Xaj (T3H and N7) ^and different strains of . The plates were incubated for 24 hours at 37°C, and the zone of inhibition was measured. The results are shown in Figure 2. Firstly, it can be seen that both the bacterial strain PC by itself, and the combination of the invention, have an inhibitory effect on the pathogens Xaj and Xac, with the greatest inhibition achieved by the formulation of the invention, Figure 2.

Experiment in liquid medium.

5 mL of YPD liquid medium was inoculated with 10 ⁷ of each pathogenic bacteria Xa, and incubated for 24 hours at a temperature of 27°C. Subsequently, 0.1 mL of the liquid formulation obtained in Example 2 or 0.1 mL of a composition of 10 ⁷ PFU/mL of Fago 13 was added. The tubes were incubated for 18 hours at a temperature of 27°C and the bacterial count in each tube. The results are shown in Figure 3. It was observed that, although the phage itself has an inhibitory effect on the four strains of Xanthomonas arboricola evaluated, from 2 different pathovars, this is significantly less than that achieved by the formulation of the invention. .

These results demonstrate that the formulation of the invention can control Xa of different pathovars. It is especially significant to note that, in both conditions evaluated, the effect of the formulation of the invention is of the same order of magnitude in the 4 different strains of Xanthomonas arboricola tested.

Example 5: Evaluation of the formulation of the invention against pathogenic fungi of BAN (brown apical necrosis) and GN (gray necrosis).

In order to test the effect of the invention on the opportunistic fungi that cause BAN and GN, plaque tests were carried out, in this case evaluating the growth of the pathogen. Plates were obtained with PDA agar medium, supplemented either with liquid formulation obtained in example 2, and for comparison, supplemented with a solution of Bacillus PC, which is also present in the formulation of the invention, at a concentration of 1, 0x10 ⁷ CFU/mL. Finally, a control without inhibitory agent was included.

In a set of independent experiments, plates were obtained with PDA agar medium, supplemented either with the liquid formulation obtained in example 2, and for comparison, supplemented with 0.1g of Trichoderma 205 spores, which is also present in the formulation of the invention. Finally, a control without inhibitory agent was included.

Three phytopathogenic fungi that cause diseases associated with primary infection by Xanthomona arboricola were evaluated: Fusarium, Alternaria and Diaporthe, each in 5 independent experiments. The plates were incubated for 24 hours at 37°C, and the growth zone was measured.

The results of the comparison with Bacillus PC are graphed in Figure 4. The results show that the growth of each of these phytopathogenic fungi decreased significantly in the presence of the bacterial strain PC by itself, as compared to the combination of the invention, The best result being the formulation of the invention. Likewise, the results of the comparison with Trichoderma 205 are graphed in Figure 5. It can be seen that both the fungus and the formulation of the invention significantly decreased the growth of all the phytopathogenic fungi evaluated, with the best result being that achieved by the formulation of the invention

These results demonstrate that the formulation can control the fungi associated with BAN and GN.

Example 6: Formulation in alpha liquid format

To obtain the biocontrol formulation of the invention, the following formulations of each of the components were mixed, which have the following concentrations: 1 .0x10 ⁵ -1 .0x10 ⁹ CFU/mL of Bacillus velezensis strain PC, in DSM medium, pH 6.7

1 g of spores of the fungus Trichodermas atroviride strain 205 deposit number 181021-02 in a concentration between 1.0*10 ⁵ and 1.0*10® CFU/g in rice substrate,

1 mL of a composition of 10 ⁷ PFU/mL of bacteriophage Pradovirus Phage/Fago 13, in SM buffer medium (50 mM Tris-HCI; 8 mM MgSO ₄ (7H ₂ O); 100 mM NaCl), pH 7.5 , was stirred to obtain a homogeneous dispersion up to a total volume of 1 liter.

Example 7: Formulation in beta liquid format

To obtain the biocontrol formulation of the invention, the following formulations of each of the components were mixed, which have the following concentrations: 10 ⁷ PFU/mL of bacteriophage Pradovirus Phage/Fago 13, in SM buffer medium (50 mM Tris-HCI ; 8 mM MgSO ₄ (7H ₂ O); 100 mM NaCl), pH 7.5,

1 g of a composition comprising between 1.0x10 ⁵ - 1.0x10® CFU/g of Bacillus velezensis strain PC, in DSM medium, pH 6.7 was stirred to obtain a homogeneous dispersion up to a total volume of 1 liter.

Claims

1. A biocontrol formulation for agricultural use to prevent or treat diseases associated with 02 and the bacteria Bacillus velezensis strain PC deposit number 181021-04.

2. A biocontrol formulation according to claim 1, CHARACTERIZED because it is formulated in the form of a liquid, oil dispersion, powder, dry wettable powder, dispersible granule or dry wettable granule.

3. A biocontrol formulation according to claim 2, CHARACTERIZED because it is formulated in powder form.

4. A biocontrol formulation according to claim 3, CHARACTERIZED because it is formulated in a maltodextrin matrix.

5. Method for preventing or controlling diseases caused by

6. Method according to claim 5 CHARACTERIZED because it is applied by spraying on plants or seedlings or fruits.

7. Method according to claim 5 CHARACTERIZED because it is applied foliar or root to plants or seedlings that are sick or susceptible to being infected, with Xanthomonas arboricola and/or phytopathogenic fungi.

8. Method according to claim 5 CHARACTERIZED because it comprises applying the formulation of claim 1 to plants or seedlings that are sick or susceptible to being infected, with Xanthomonas arboricola of the pathovars (pv.): corylina, juglandis, fragariae, populi, pruni and/or celebensis.

9. Method according to claim 8 CHARACTERIZED because it comprises applying the formulation of claim 1 to plants or seedlings that are sick or susceptible to being infected with the black plague or brown apical necrosis produced by Xanthomonas arboricola pv. juglandis., where the plants are walnut trees.

10. Method according to claim 11, CHARACTERIZED because it comprises applying the formulation of claim 1 to plants or seedlings that are sick or susceptible to being infected with gray necrosis produced by Xanthomonas arboricola pv. corylina, where the plants are hazel.

eleven . Method according to claim 5 CHARACTERIZED because it comprises applying the formulation of claim 1 to plants or seedlings that are sick or susceptible to being infected with the phytopathogenic fungi Neofusicoccum spp., Diaporthe spp., Rhizoctonia spp., Aspergillus spp., Phytophthora spp. , Fusarium spp., Rhizopus spp., and/or Alternaria spp.

12. Method according to claim 11 CHARACTERIZED because the opportunistic fungi are Fusarium destruens and/or Alternaria alternata.

13. Process for preparing the biocontrol formulation according to claim 1 CHARACTERIZED because it comprises the stages of mixing: a. a composition comprising the bacteriophage Pradovirus Phage/tago 13 deposit number 181021-01 in a concentration of between 1.0x10 ⁶ and 1.0x 10 ¹² PFU/mL, b. a composition comprising the fungus Trichodermas atroviride strain 205 deposit number 181021-02 in a concentration between 1.0xl0 ⁵ and 1.0x 10 ⁹ CFU/g, and c. a composition comprising the bacteria Bacillus velezensis strain PC deposit number 181021-04 in a concentration of between 1.0x10 ⁵ and 1.0x10 ⁹ CFU/mL or between 1.0x10 ⁵ and 1.0x10 ⁹ CFU/g, and stirring to obtain a homogeneous dispersion of at least 1 liter of formulation.

14. Process according to claim 13, CHARACTERIZED because the final dispersion is subjected to drying or lyophilization to obtain a dry powder, optionally with formulation adjuvants.

15. Process according to claim 14, CHARACTERIZED in that the dry powder is mixed with formulation auxiliaries to obtain an oil dispersion, dispersible granule or dry wettable granule.