New bacteriophages against erwinia amylovora infection and use of same

Abstract

The present invention relates to new bacteriophages with SEQ ID NO.: 1-2 against Erwinia amylovora infections and use of same for treating and / or preventing an Erwinia amylovora infection in a crop.

Description

[0001]NOVEL BACTERIOPHAGES AGAINST ERWINIA AMYLOVORA INFECTIONS AND THEIR USE DESCRIPTION The present invention relates to novel bacteriophages against Erwinia amylovora infections and their use for the treatment and / or prevention of Erwinia amylovora infections in crops. More specifically, in a first aspect, the invention provides novel bacteriophages against Erwinia amylovora infections, a phytopathogen that causes fire blight in pear and apple trees, as well as in ornamentals. Bacterial diseases caused by Erwinia amylovora affect all pome fruit crops and some woody ornamentals under high humidity conditions. Furthermore, this phytopathogen infects plants through their aerial parts, making it possible to treat them with bacteriophages.Current conditions of increased seasonal climate instability and intense global trade favor the emergence of crop diseases. The solution to this problem lies in optimizing crop production in a sustainable and environmentally friendly manner. To this end, in addition to providing balanced and beneficial fertilization for both soil and plants, one of the major challenges facing 21st-century agriculture is eliminating or minimizing the impact of diseases caused by plant pathogens on crops. These pathogens can cause diseases in plants before, during, or after harvest. Diseases associated with plant pathogens, besides significantly reducing crop yields, also diminish the quality of agricultural products and derived foods.The harmful effects of plant pathogens on crops also cause enormous economic losses. The most significant direct economic impact of a pest or disease is the loss of production or reduced yield, which considerably diminishes the profits from agricultural activity. These economic losses caused by pests can be complex and extend beyond the immediate impact on the directly affected agricultural products. Bacterial diseases develop rapidly when conditions are favorable. Furthermore, they are easily transmitted from one plant to another or from one plot to another via water (irrigation tape, rain, etc.), wind, through the use of contaminated tools used for crop maintenance (pruners, harvesters, etc.), or via any living organism that comes into contact with infected plants and can carry the pathogen to other healthy plants.In particular, the phytopathogenic bacterium Erwinia amylovora is present worldwide, but its frequency / intensity is more pronounced in Europe, North Africa, the Middle East, and the Americas. This bacterium is considered a quarantine pathogen by the EPPO (European and Mediterranean Plant Protection Organization). Bacteriophages, on the other hand, are considered non-living biological entities consisting of nucleic acid enclosed by a protein capsid. As obligate parasites of bacteria, they are unable to reproduce without their host. The first proposed phage therapy for treating infections dates back to 1919, almost a decade before the discovery of penicillin (Chanishvili N., “Phage therapy--history from Twort and d'Herelle through Soviet experience to current approaches”, Adv Virus Res 2012; 83: 3-40).Later, with the development of antibiotics, this type of strategy was abandoned due to the enormous technical difficulties at the time. Today, with technological advancements and the problems associated with antibiotics, it is re-emerging as a tool with great potential. Bacteriophages are simple and incredibly diverse. They are widely distributed in all ecosystems; specifically, it is estimated that, on average, there are 1.5 x 10²³ bacteria in the soil. 8 phages / g of soil (Kevin E. et al; 2003, “Elevated Abundance of Bacteriophage Infecting Bacteria in Soil”, Applied and Environmental Microbiology, p. 285-289 Vol.69) and can be counted up to 10 10Phages / g in certain soils. Furthermore, bacteriophages are highly specific; that is, each virus has a limited capacity to infect, ranging from a few species to only a few strains of the same species. This characteristic is very important, since it greatly reduces potential side effects by not significantly affecting the target microbiota. Phage therapy is also self-dosed. The application of chemical inhibitors requires minimum concentrations of inhibitory substances and their stability to ensure they continue to function. Phages are self-replicating entities within their target bacteria. The presence of few bacteria will result in few infections and a limited number of new phages being generated. The more bacteria present and the more they are multiplying, the more pronounced the phage parasitism will be, and the number of viruses will grow exponentially, multiplying the desired effect.Furthermore, the use of bacteriophages leaves no polluting chemical residues, as they are essentially organic matter. These entities have a limited lifespan in the soil in the absence of their host. This lifespan is subject to various factors such as temperature, humidity, soil composition, and so on. Extensive documentation exists on the use of this type of technology, especially in pest control, where it has seen the greatest development. There is a well-recognized need to develop new, environmentally friendly control strategies to combat bacterial crop diseases. Current control measures involving the use of traditional chemicals or antibiotics are losing their effectiveness due to the natural development of bacterial resistance to these agents. Moreover, there is a growing awareness that their use is not environmentally sound.Bacteriophages have garnered increasing research interest in recent years as a realistic and environmentally friendly means of controlling bacterial diseases in crops, with some bacteriophage-based products already commercially available. This biological control with bacteriophages offers advantages over chemical controls, as customized bacteriophage cocktails can be tailored to target specific disease-causing bacteria, readily adapting to bacterial resistance that may develop over time. The use of phages as a tool may initially appear to be a forced approach, particularly in soil-based settings. However, studies of interactions between viruses and their target bacteria in various habitats reveal that these types of ecological interactions are naturally abundant in different ecosystems.Phages are considered the most abundant biological entity on Earth and play a fundamental role in regulating bacterial populations. For example, phages are responsible for the death of approximately 20% to 40% of all bacteria on the ocean surface every 24 hours (Wittebole X. et al; 2014, “A historical overview of bacteriophage therapy as an alternative to antibiotics for the treatment of bacterial pathogens”, Virulence; 5: 226-235). The effect of these interactions between bacteria and bacteriophages at the soil level is also widely described. Soil provides an incredible range of niches occupied by many species of microorganisms, leading to a similar diversity in the variety of viruses that parasitize these microorganisms. In agricultural soils, viral abundance is significantly correlated with bacterial abundance (Williamson et al.).(Kleczkowska J; 2007, “Incidence of lysogeny within temperate and extreme soil environments”, Environ Microbiol 9:2563–2574). This indicates that the presence and abundance of susceptible hosts is a key factor controlling viral abundance. When host organisms are present, particularly in large numbers, new viruses can be continuously produced and released into the soil matrix through lytic infections. In the absence of susceptible hosts, the abundance of extracellular viruses is controlled by the physical and chemical properties of the soil environment. The effect of this virus-bacteria balance has been demonstrated, for example, with phages that parasitize bacteria of the genus Rhizobium (Kleczkowska J; 1971, “Genetic changes in rhizobium bacteria and in their bacteriophages during coexistence”, Plant Soil 35(1):47–56).Further evidence of the high frequency of these types of soil infections and their ecological importance was demonstrated in experiments conducted by Allen et al. in 2010. In habitats with high amounts of soluble organic carbon, the microbial mass did not increase even with the addition of more consumable substrates; however, the application of anti-phage substances generated a substantial increase in the number of microorganisms. This demonstrates that these soil populations were in a controlled equilibrium thanks to the constant presence of phages. Generally speaking, the fight against bacterial diseases in crops consists mainly of preventing their occurrence through cultural practices (soil and tool disinfection, crop rotation, etc.) and the use of copper-based products (copper oxychloride, copper sulfate, copper hydroxide, etc.) or carbamates (mancozeb).These tools are purely preventative and have very limited effectiveness once symptoms of bacterial infection appear. Today, a wide range of biological control strategies against pathogenic microorganisms are available on the market, using bacteria capable of inhibiting the growth of these microorganisms. However, these are primarily preventative strategies; the vast majority have no curative effect once an infection is established. Until recently, the only curative tools available to farmers for combating bacterial diseases were antibiotics (streptomycin, oxytetracycline, gentamicin, etc.). Now, most of these antibiotics, which were used routinely until the early 2000s, face various restrictions on their use and even outright bans in some territories, such as the European Union.Furthermore, in areas where antibiotics continue to be used extensively, the emergence of bacteria resistant to these obsolete phytopharmaceuticals is becoming increasingly noticeable. US Patent 11089785, for example, provides a biopesticide having antimicrobial activity consisting of Bacillus amyloliquefaciens subsp. plantarum 71 (NRRLB-67021) and its metabolites, and one or more agriculturally acceptable excipients, wherein said metabolites comprise bacillaene, difficidin, and macrolactin polyketide metabolites, and which can be applied against, among many other microorganisms, Erwinia amylovora. Similarly, US Patent 20180020676 provides a composition comprising a biologically pure culture of Bacillus velezensis RTI301 deposited as ATCC No.PTA-121165, or a mutant thereof that has all the identifying characteristics thereof, for application to a plant to one or both benefit plant growth or confer protection against a pathogen, including Erwinia amylovora. Currently, when a tree is infected by Erwinia amylovora, the most effective strategy is to cut the affected branch first, and if this is not enough to prevent the spread of the disease, the tree is uprooted and burned to prevent the disease from spreading to other healthy trees.The invention aims to avoid the use of chemical compounds and eliminate the disadvantages of known prior art products by providing novel bacteriophages against Erwinia amylovora infections according to SEQ ID NO: 1 and SEQ ID NO: 2, herein referred to as bacteriophages Phi-EF1 and Phi-EF2 (or ɸEF1 and ɸEF2), respectively (deposited in the German DSMZ collection under deposit numbers DSM 34933 and 35078, respectively). The invention also relates to the use of the bacteriophages according to SEQ ID NO: 1-2, herein referred to as bacteriophages Phi-EF1 and Phi-EF2, respectively (deposited in the German DSMZ collection under deposit numbers DSM 34933 and 35078), for the treatment or prevention of Erwinia amylovora infection in culture, using the described phages in solution form or individually. or in combination.Thus, in a preferred embodiment of the use of bacteriophages Phi-EF1 and / or Phi-EF2 according to SEQ ID NO: 1-2, respectively, these are applied in aqueous solution at a dose of between 10. 6 and 10 7Plaque-forming units (PFUs) of Phi-EF1 and / or Phi-EF2 per milliliter of water, applied foliarly. For use, the bacteriophages of the invention according to SEQ ID NO: 1-2, as described herein, may be formulated together with a pH stabilizer, a UV protectant, or a suitable adjuvant. A preferred example of a stabilizer is citric acid, for example, added to the solution of the bacteriophage(s) in a proportion of 1 to 5% w / v. Where applicable, the UV protectant is present in the solution of the bacteriophage(s) at a concentration of 0.1 to 20% w / v. These UV protectants may be selected, for example, from carrot extract, casein, soy peptone, or aromatic amino acids. In this case, the adjuvants, for example selected from amino acids, lecithin or glycerol, are present in the solution of the bacteriophage(s) at a concentration of 0.1 to 8% by weight / volume.The amino acids would enhance the illicithor effect of the product; that is, in addition to the direct effect of the phages on the pathogens, the amino acids would help strengthen the plant by activating its immune system. Furthermore, the lecithin would act as an emulsifier, facilitating foliar application and the spreading of the droplets across the leaves due to its ability to reduce the surface tension of the product. The invention is described below based on the following tests and examples of its implementation, and with reference to the following figures, in which: Fig. 1: Transmission electron microscopy of suspensions of the bacteriophages of the invention obtained according to example 1. Fig. 2: Photographs showing the result of the in vivo application of the bacteriophages of the invention.Examples: Isolation and characterization of bacteriophages ɸEF1 and ɸEF2. First, due to the importance of the disease it causes in plantations worldwide, the bacterium Erwinia amylovora (CECT 222; ATCC 29850) was chosen as the host bacterium to search for environmental phages capable of efficiently infecting and lysing it. Sixty-five agricultural environmental samples of different origins (soil, irrigation water, crop and pruning debris) were selected from different days and locations in the provinces of Barcelona and Teruel as the sources for searching for potential phages. These environmental samples were centrifuged and filtered using a 0.22 µm pore size filter with low protein adsorption to remove the bacterial fraction (in the case of solid samples, a prior 1:4 homogenization step in sterile PBS liquid medium was necessary). Next, they contacted Erwiniaamylovora using the double layer agar method.During this process, different concentrations of environmental phage suspensions and bacterial cultures of the phytopathogen were tested at different growth stages. At the time of contact between the phage suspension and the tested phytopathogen, the optimal condition (resulting in a greater number of transparent lysis plaques sufficiently separated to facilitate isolation) was established as exponential growth of the phytopathogen and a low concentration of the phage suspension. After contacting the different bacterial strains with the various environmental phage suspensions using the "double-layer" technique, approximately 120 lysis plaques were selected for the different strains. The selected lysis plaques were those that showed the most transparent lysis (generated by presumably virulent phages following the lytic cycle), which are optimal for the application of phages as a biocontrol tool.Furthermore, an attempt was made to select phages that produced lysis plaques of varying sizes and morphologies in order to obtain phages from different species. To isolate the selected phages, the area of ​​the double agar layer containing the lysis plaque was cut out and resuspended in sterile saline buffer. The resuspended plaque was treated with 10% chloroform to eliminate bacterial debris and the presence of phages with lipids in their capsid. The suspension was centrifuged, and the supernatant containing the phages was recovered. The collected phages were verified using the so-called "drop test," which consists of adding a 15 µl drop of each suspension and its decimal dilutions onto a monolayer containing soft agar and the respective host bacteria.After incubation, this step is performed to verify that the phages are indeed infectious, capable of producing infection visualized by a zone of lysis across the entire area. The resulting phage suspensions, presumably lytic, were analyzed by transmission electron microscopy (Figure 1). Phage ɸEF1 is of the Myoviridae type (long contractile tail). Myovirus phages (formerly family Myoviridae according to the latest classification by the International Committee on Taxonomy of Viruses 2023) typically have high lytic capabilities, which is suitable for our purposes. However, the ɸEF2 phage is of the Podovirus type (formerly family Podoviridae) with a short tail. The ɸEF1 and ɸEF2 phages were also genetically characterized by massive sequencing, ensuring that both phages were virulent, completely lacking genes associated with lysogenic phages (see Sequence List).Functionality of phages ɸEF1 and ɸEF2. Both phages showed in vitro a high capacity of virulence against Erwinia amylovora (CECT 222; ATCC 29850). When confronting the phages (10⁷ CFU / ml) with respect to a bacterial culture of 10⁸ CFU / ml, results of total lysis were observed at a phage concentration of 10. 7 and 10 6 UFP / ml; at 10 5 UFP / ml shows semi-confluent lysis or fewer than 100 bacterial colonies. Below that concentration (10 4UFP / ml) total lysis is not observed, but only isolated bald patches and a formed mat. In vivo efficacy assay on pear surface. An assay was performed with the phages ɸEF1 and ɸEF2 and the phytopathogen Erwinia amylovora on the surface of fresh pears. In the assay, pears were used in which a cut was made on their surface with a sterile knife. The following conditions were applied and inoculated to this cut: A. Negative control: the cut wound of a pear was inoculated with 100 µl of sterile water; B. Phage control: the cut wound of a second pear was inoculated with a suspension of the phages ɸEF1 (10 7 UFP / ml) and ɸEF2 (10 7 UFP / ml), using 50 µl of each phage. C. Positive control: the wounds were inoculated with a suspension of 100 µl of the phytopathogen Erwinia amylovora (10 8CFU / ml). D. Phage + Bacteria Test: The cut was inoculated with a suspension of phages ɸEF1 and ɸEF2 (107 PFU / ml, 50 µl of each phage) and, after waiting one hour for the phage application to dry, the cut area was inoculated with 100 µl of the phytopathogen. The pears were kept at room temperature for 9 days. As shown in the photographs in Figure 2, inoculation with phages alone (B) has no effect on the pear. In the pear infected with Erwinia amylovora (C), the development of the phytopathogen can be seen, significantly affecting the pear. Finally, when the mixture of both phages is used preventively and then the phytopathogen is applied (D), it is demonstrated that the phages are able to almost completely stop the bacterial infection, preventing it from spreading throughout the fruit.

Claims

CLAIMS 1. Bacteriophages according to SEQ ID NO: 1 and SEQ ID NO: 2, designated bacteriophages Phi-EF1 and Phi-EF2.

2. Use of the bacteriophages according to SEQ ID NO: 1-2, according to claim 1, for the treatment or prevention of an Erwinia amylovora infection in a culture, the described phages being used in solution form, individually or in combination.

3. Use of the bacteriophages according to claim 2, wherein they are applied in aqueous solution at a dose of between 10 6 and 10 7PFU (plaque-forming units).

4. Use of the bacteriophages according to claim 2, wherein the aqueous solution further includes a pH stabilizer selected from citric acid, added to the solution of the bacteriophage(s) in a proportion of 1 to 5% w / v.

5. Use of the bacteriophages according to claim 2, wherein the aqueous solution further includes a protectant against ultraviolet radiation in a concentration of 0.1 to 20% w / v.

6. Use of the bacteriophages according to claim 2, wherein the aqueous solution further includes a suitable adjuvant selected from amino acids, lecithin, or glycerol, or combinations thereof, present in the solution of the bacteriophage(s) in a concentration of 0.1 to 8% w / v.

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