New bacteriophage against xanthomonas campestris and / or xanthomonas citri infections and use of same

Abstract

The present invention provides a new bacteriophage with SEQ ID No.: 1 against Xanthomonas campestris and / or Xanthomonas citri infections and use of same for treating and / or preventing a Xanthomonas campestris and / or Xanthomonas citri infection in a crop.

Description

[0001] NEW BACTERIOPHAGE AGAINST INFECTIONS CAUSED BY XANTHOMONAS CAMPESTRIS AND / OR XANTHOMONAS CITRI AND THEIR USE

[0002] DESCRIPTION

[0003] The present invention relates to a novel bacteriophage against infections caused by Xanthomonas campestris and / or Xanthomonas citri and its use for the treatment and / or prevention of infections caused by Xanthomonas campestris and / or Xanthomonas citri in a crop.

[0004] More specifically, in a first aspect, the invention provides a new bacteriophage against infections by Xanthomonas campestris and / or Xanthomonas citri, phytopathogens that cause the diseases known as "black rot" and "citrus canker" respectively.

[0005] 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.

[0006] 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.

[0007] In particular, bacteria of the genus Xanthomonas are phytopathogens that can have a significant negative impact, causing necrosis in the leaves of a wide variety of plants worldwide. They are distributed globally, and several strains are considered quarantine pathogens by the EPPO (European and Mediterranean Plant Protection Organization).

[0008] Within this genus is the species Xantomonas campestris, which causes a disease called black rot, considered by numerous authors to be the most destructive in cruciferous plants such as cauliflower or broccoli worldwide.

[0009] Black rot in cruciferous plants, caused by Xanthomonas campestris, is one of the most economically important diseases worldwide, affecting most species of the Brassica genus, as well as other vegetables, ornamental plants, and weeds, in which it can survive during cold periods. The responsible pathogen thrives in warm, humid climates, with an optimum temperature range of 27°C–35°C, and its impact is particularly severe in tropical and subtropical regions with high humidity.

[0010] In addition, the X. campestris attack can be quickly followed by other soft rots caused by bacteria such as Erwinia carotovora or Pseudomonas sp., as secondary pathogens.

[0011] Another species of Xanthomonas, formerly classified as Xanthomonas campestris pv citri, is now called Xanthomonas citri, which is also notable for its pathogenicity. This bacterium is the causal agent of citrus canker, one of the most important citrus diseases worldwide. This disease causes severe defoliation, dieback of branches, and premature fruit drop, resulting in a loss of commercial value.

[0012] However, it can also pose a problem in cold climates, such as coastal areas of North America and northern Europe. Therefore, if temperature is not an obstacle to its growth, it becomes a serious threat to cruciferous vegetable crops worldwide.

[0013] 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). Subsequently, with the development of antibiotics, this type of strategy was abandoned due to the enormous technical difficulties of the time. Today, with technological advancements and the problems associated with antibiotics, it is re-emerging as a tool with great potential.

[0014] 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 10¹⁰ 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 1 ° phages / g in certain soils.

[0015] 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 microbiota to which they are not directed.

[0016] Similarly, phage therapy is self-administered. 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. 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.

[0017] 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 when their host is absent. This lifespan is subject to various factors such as temperature, humidity, soil composition, etc.

[0018] There is extensive documentation 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. Furthermore, there is a growing awareness that their use is not environmentally sound.

[0019] Bacteriophages have garnered increasing research interest in recent years as a realistic and environmentally friendly means of controlling bacterial diseases in crops, and some bacteriophage-based products are 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, easily adapting to bacterial resistance that may develop over time.

[0020] The use of phages as a tool may initially appear to be a forced situation promoted in the soil environment. However, the study of interactions between viruses and their target bacteria in different habitats reveals that these types of ecological interactions are very abundant naturally in various 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., 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 infections in the soil and their ecological importance was shown in experiments carried out by Alien 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 the phages.

[0021] In general, the fight against bacterial crop diseases consists primarily 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 only preventative and have very limited effectiveness once symptoms of bacterial infection appear.

[0022] Today there is a wide range of biological control strategies on the market against pathogenic microorganisms using bacteria with the ability to inhibit the growth of pathogenic microorganisms, but these are basically preventive strategies; the vast majority do not have a curative effect once the infection is established.

[0023] Until recently, the only curative tools available to farmers to fight bacterial diseases were antibiotics (streptomycin, oxytetracycline, gentamicin...).

[0024] Now, most of these antibiotics, which were widely used until the early 2000s, face various restrictions on their use and even outright bans in some regions, such as the European Union. Furthermore, in places where antibiotics continue to be used extensively, the emergence of bacteria resistant to these now-obsolete phytopharmaceuticals is becoming increasingly noticeable.

[0025] EP2603083, for example, provides a bismuth thiol-based composition as an antiseptic with antimicrobial activity, and can be applied against, among many other microorganisms, those of the Xanthomonas species.

[0026] The invention aims to avoid the use of chemical compounds and eliminate the disadvantages of known prior art products, providing a new bacteriophage against Xanthomonas campestris and / or Xanthomonas citri infections according to SEQ ID NO: 1, herein called bacteriophage Phi-XF1 (or (>XF1), (deposited in the German collection DSMZ with deposit number DSM 34882).

[0027] The invention also relates to the use of the bacteriophage according to SEQ ID NO: 1, herein called bacteriophage Phi-XF1 (deposited in the German collection DSMZ with deposit number DSM 34882) for the treatment or prevention of an infection by Xanthomonas campestris and / or Xanthomonas citri in a culture, using the described phage in the form of an aqueous solution.

[0028] Thus, in a preferred embodiment of the use of the bacteriophage Phi-XF1 according to SEQ ID NO: 1, it is applied in aqueous solution at a dose of between 106 and 10 7 PFU (plate-forming units) of Phi-XF1 per milliliter of water, in foliar form.

[0029] For use, the bacteriophage of the invention according to SEQ ID NO: 1 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 bacteriophage solution in a proportion of 1 to 5% w / v.

[0030] In this case, the UV protectant is present in the bacteriophage solution at a concentration of 0.1 to 20% by weight / volume. This UV protectant can be selected, for example, from carrot extract, casein, soy peptone, or aromatic amino acids.

[0031] In this case, the adjuvants, for example, selected from amino acids, lecithin, or glycerol, are present in the bacteriophage solution at a concentration of 0.1 to 8% by weight / volume. The amino acids would enhance the illicithal 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. On the other hand, the lecithin would act as an emulsifier, facilitating foliar application and the spreading of the droplets across the leaves, as it reduces the surface tension of the product.

[0032] The invention is described below based on the following tests and examples of its implementation and with reference to the following figures, in which:

[0033] Fig. 1: Transmission electron microscopy of a suspension of the bacteriophage of the invention obtained according to example 1.

[0034] Fig. 2: Photographs showing the result of the in vivo application of the bacteriophage of the invention. Examples

[0035] Isolation and characterization of bacteriophage XF1

[0036] Firstly, because of the importance of the disease it develops in plantations around the world, the bacteria Xanthomonas campestris pv campestris (CECT 97) and Xanthomonas citri (CECT 4428) were chosen as host bacteria to search for environmental phages capable of infecting and destroying them efficiently.

[0037] As samples of environmental origin where to look for possible phages, 65 agricultural environmental samples of different nature (soil, irrigation water, crop and pruning remains) were chosen from different days and places in the provinces of Barcelona and Teruel.

[0038] These environmental samples were centrifuged and filtered through a 0.22 pm pore size filter with low protein adsorption to remove the bacterial fraction before putting them in contact with the different phytopathogens Xanthomonas campestris and Xanthomonas citri using the double layer agar method (in the case of solid samples with a prior step of homogenization (1 :4) in sterile PBS saline buffer).

[0039] During this process, different concentrations of environmental phage suspensions and the bacterial culture of the phytopathogen were tested at different growth stages. At the time of contact between the phage suspension and the tested phytopathogen, the most optimal condition (resulting in a greater number of transparent lysis patches, sufficiently separated to facilitate their isolation) was established as exponential growth of the phytopathogen and a low concentration of the phage suspension.

[0040] After exposing different bacterial strains to various environmental phage suspensions using the "double-layer" technique, approximately 150 lysis plaques were selected for each strain. The selected plaques were those exhibiting 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. Additionally, an attempt was made to select phages that produced lysis plaques of varying sizes and morphologies in order to obtain phages from different species.

[0041] To isolate the selected phages, the area of ​​the agar double layer containing the lysis plaque was cut out and resuspended in saline buffer. The resuspended plaque was treated with 10% chloroform to eliminate bacterial debris and the presence of phages with lipid capsids. The suspension was centrifuged, and the supernatant containing the phages was recovered. The collected phages were verified using the "drop test," which involves adding a 15 pL 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 they are indeed phages and that they are infectious, capable of causing infection visualized by a zone of lysis across the entire area.

[0042] The obtained phage suspensions, presumably lytic, were analyzed by transmission electron microscopy (Figure 1). The phage <|)XF1 is of the Myoviridae type (long contractile tail). Myovirus-type phages (formerly family Myoviridae according to the latest classification of the International Committee on Taxonomy of Viruses 2023) usually have high lytic capabilities, which is suitable for our purposes.

[0043] The phage <|)XF1 was also genetically characterized by massive sequencing (see Sequence List).

[0044] Phage functionality <¡)XF1

[0045] The phage exhibits in vitro a high capacity for virulence against Xanthomonas campestris (CECT 97) and Xanthomonas citri (CECT 4428).

[0046] Facing the Phage (10 7 PFU / ml) with respect to a bacterial culture of 10 8 CFU / ml, results of total lysis were observed at a phage concentration of 10 7 and 10 6UFP / ml; at 10 5 UFP / ml shows semi-confluent lysis or fewer than 100 bacterial colonies. Below that concentration (10 4 UFP / ml) total lysis is not observed, but only isolated bald patches and a formed mat. This protocol was performed using the bacteria Xanthomonas campestris (CECT 97) and Xanthomonas citri (CECT 4428), obtaining the same results.

[0047] In vivo efficacy trial on leaves of the Brassica napobrassica plant

[0048] An assay was performed with the phage <|)XF1 and the phytopathogen Xanthomonas campestris on the surface of four leaves of a plant from the Brassicaceae family.

[0049] In the experiment, punctures were made on the leaf surfaces of the plant and these wounds were inoculated under the following conditions: a) Negative control: the wounds of one leaf were inoculated with sterile water; b) Phage control: the wounds of a second leaf were inoculated with a suspension of the phage <|)XF1 (10 7 UFP / ml). c) Positive control: the wounds were inoculated with a suspension of the phytopathogen Xanthomonas campestris (10 8 CFU / ml). d) Phage + bacteria test: wounds were inoculated with a suspension of phage (|)XF1 (10 7 UFP / ml) and, after waiting two hours for the phage application to dry, the same area was inoculated with the phytopathogen Xanthomonas campestris (10 8 CFU / ml).

[0050] As shown in the photographs in Figure 2, neither the “negative control” nor the “phage control” produced any negative effects on the leaf, confirming the phage's harmlessness to plant tissue. In the case of inoculation of wounds with the phytopathogen X. campestris, the necrosis caused by the bacterium on the leaf tissue is clearly visible. Finally, when using the phage XF1 preventively and then inoculating the phytopathogen, the phage is able to contain the X. campestris infection, producing minimal lesions compared to the positive control of bacterial infection.

Claims

CLAIMS 1. Bacteriophage according to SEQ ID NO: 1, called Phi-XF1 bacteriophages.

2. Use of the bacteriophage according to SEQ ID NO: 1 according to claim 1 for the treatment or prevention of an infection by Xanthomonas campestris and / or Xanthomonas citri in a culture, using the described phage in solution form.

3. Use of the bacteriophage according to claim 2, wherein it is applied in aqueous solution at a dose of between 10 6 and 10 7 PFU (plate forming units).

4. Use of the bacteriophage according to claim 2, wherein the aqueous solution further includes a pH stabilizer selected from citric acid, added to the bacteriophage solution in a proportion of 1 to 5% by weight / volume.

5. Use of the bacteriophage according to claim 2, wherein the aqueous solution further includes a protectant against ultraviolet radiation in a concentration of 0.1 to 20% by weight / volume.

6. Use of the bacteriophage 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 bacteriophage solution at a concentration of 0.1 to 8% w / v.

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