BLIGHT IN RICE

The invention relates to a concentrate and a preparation containing the concentrate mentioned as active ingredient for the prevention and treatment of rice bacterial leaf blight, and method for the prevention and fighting down of rice bacterial leaf blight by using the concentrates and the preparations containing it as active ingredient.

It is known that the bacteriophages (in short: phages) are viruses able to infect bacteria. It was first described in 1915 by W. Twort, then in 1917 by Felix d' Herelle, that there exist "micro organisms" which infect bacteria too. At present more than 5100 bacteriophages are recorded in the literature (Ackermann, Arch. Virol. 2001 (146): 843-857), and phages were isolated against almost all major pathogens, bacteria species important from environmental and biotechnological aspects.

Even twenty years before the discovery of the first antibiotic - from the moment of the discovery of phages - the thought emerged that these viruses could be used in the fight against the bacterial pathogens (see the literary work "Arrowsmith" of Sinclair Lewis). Since the penicillin was discovered by Sir Alexander Fleming in 1928, and especially after that in the second half of the 1940s the widespread application of the antibiotics began, the researches directed for the therapeutic application of the phages fell in the background. The reason for that - in addition to the discovery of the antibiotics - was that at that time very little knowledge was known about the phages since the molecular biological techniques suitable for characterizing them didn't exist at that time. In their absence the use of the phages would have been risky, and the separation of the different phage strains from each other was technically unsolvable in the practice. Because of these reasons, considering the antibacterial therapy, the developed Western world voted for the antibiotics discovered at that time, which still could be made cheaply. At the same time, however, the bacterial evolutionary process accelerated, because of which many multi-resistant pathogenic strains have now emerged, the appearance of which makes treatments difficult. For example, in less than 5-year period from the start of the use of penicillin in medicine about 50% of the Staphylococcus aureus isolates became penicillin resistant. That's why methicillin was introduced in 1959, but methicillin resistant Staphylococcus aureus (MRS A) strains were found already in 1961. Nowadays we are there, that even against the two effective agents used for the treatment of MRSA strains, the vancomycin and teicoplanin, decreased sensitivity or even resistance can be detected in some MRSA strains.

During the widespread use of antibiotics, in addition to the occurrence of the multi- resistance it has also became obvious that during the application of several antibacterial chemotherapeutic agents side effects occur in the patient. The followings are included in the symptoms: gastrointestinal disorders, nausea, sickness, redness, joint and muscular pain, hy- persensitivity reactions, while in more severe cases, nervous system, hepatic and hematopoietic disorders, pseudomembranous colitis and anaphylactic shock may develop. Because of the toxic effects mentioned above the use of some antibiotics, which previously proved to be effective, was banned or their uses were limited.

The fight against the pathogenic bacteria never ends: while the pharmaceutical in- dustry is spending bigger and bigger amount of money for the development of newer and more effective antibacterial agents, the bacteria become resistant against these groups of chemotherapeutic agents in shorter and shorter period of time.

However, if instead of and in addition to using antibiotics, phages are (also) used as alternative solution, then we get such a cost-effective system, which is easy to maintain and further develop in the future, the basis of which is the co-evolution relation between the bacterium and its phage, where the development of the phage resistance of the microorganism acts as a selection pressure for the appearance of newer phages. The pledge of the future efficiency of this phage mediated antibacterial therapy is ensured by the phages themselves, since the phages "are taking care" that new phage strains are created which can attack also the resistant bacteria targeted by us and want to eliminate. The new phages generated this way can be isolated, compared to the time required for the development of new antibiotics, in a shorter period and with lower costs, and after the appropriate characterization can be prepared and marketed in the form of a new and efficient preparation.

The above phenomena are valid not only for the field of human medicine, but these are valid even for the field of plant protection.

It is known that in a large part of our world the rice belongs to the basic foods, in some continents it is the important daily nutrition for a large part of the population, but in other places it is an essential part of nutrition. Just that is why rice cultivation, preserving the health of rice crops is of paramount importance in agriculture.

The rice plant has diseases, for which appropriate solutions are not yet available for the prevention and treatment.

That is why our interest turned to the diseases frequently occurring in the rice cultivation.

The Xanthomonas oryzae pv. oryzae is a Gram-negative bacterium belonging to the Xanthomonadaceae family, causing the bacterial leaf blight disease of rice, mainly in Asia and West Africa. The fight against the pathogen is currently performed with streptomycin, and by creating and planting resistant varieties. But in many cases the treatments are unsuccessful, mainly due to the development of resistance described above, but in many cases the use of the antibiotic, and the creation of the genetically modified resistant rice plant is solicitous (the regulations do not allow the genetic modification of plants everywhere).

Bacteriophages were first isolated in 1960 from Xanthomonas oryzae pv. oryzae (Kuo, Huang, Wu and Chen, Can. J. Microbiol. 1968 (14): 1139-1142). Although others have already proposed the use of bacteriophages for plant protection purposes (see for example Erskine, Can J. Microbiol. 1973 (19): 837-845; Goodridge, Trends Biotechnol 2004 (22): 384-385), this has not been realized in practice until today (for reasons mentioned in the introduction). An agent that is against the Xanthomonas oryzae pv. oryzae and uses bac- teriophages as we know it does not yet exist.

When studying the patent literature, it has been found that up to now no patent description can be found in the USPTO patent data base, which for overcoming the above problem would aim the creation of a technical solution using bacteriophages against Xanthomonas oryzae pv. oryzae.

But in relation to the disease of the rice plants mentioned above the defence has no established practice. According to the teaching obtainable from the following literature the solution can be the destruction of the plant residues serving as the source of infection, and the cultivation of varieties less susceptible to the disease: Leung, Hei, et al. "Using genetic diversity to achieve sustainable rice disease management." Plant disease 87.10 (2003): 1156-1169.

The aim of the invention is from one aspect to create concentrates suitable for killing the Xanthomonas oryzae pv. oryzae and preparations containing it, which contain exclusively one or more bacteriophage strain as active ingredient. Additionally, from the other aspect the aim of the invention is a method for the prevention and fighting down of the rice bacterial leaf blight.

The invention is based on the discovery that the disadvantages originating from the prior art, detailed above, can be eliminated with the phage strains suitable for killing the Xanthomonas oryzae pv. oryzae. This concept is not obvious to a person skilled in the art, and the person skilled in the art couldn't get teaching or guidance for this, since such attempts for the prevention of the rice bacterial leaf blight have not become known so far, and what is more, according to the literature quoted above, the person skilled in the art was specifically motivated in a different direction to develop a solution to the problem.

According to the invention the concentrates suitable for killing the Xanthomonas oryzae pv. oryzae are characterized by the fact that they contain one or more selected and characterized anti-Xanthomonas oryzae pv. oryzae bacteriophages, or essentially consist of these.

The preparation according to invention can be a liquid (solution, emulsion, suspen- sion) or solid (powder, granulate). The essence of it is that it contains the concentrate of the invention as active ingredient, in 10 ⁶ - 10 ¹⁵ PFU/ml, or in case of a solid preparation in 10 ³ - 10 ¹² PFU/mg quantity, calculated for the total amount of the preparation, optionally with one or more usual auxiliaries and/or additives used in plant protection. Such materials can easily be selected by a person skilled in the art by his(her) obligatory knowledge, optionally in a combination suitable for the plantation, for the local conditions. In addition to the solid or liquid carriers such materials are additives promoting the effect, for example UV protection additives, but other known additives may also be considered, such as - but not limited to - surfactants, surface tension reducing agents, adhesion enhancers, absorption enhancers, and the like.

The concentrate of the present invention is generally applied in a solid or liquid form together with a carrier, preferably formulated to the preparation according to the invention, the application can be carried out from a pressurized container (for example spraying device) or without pressure (for example below the plants in the soil, or in the flooding water of the rice field).

The method of the invention for preventing and controlling rice bacterial leaf blight is characterized by that the preparation or the concentrate of the invention is applied to the area endangered or to be treated by using 10 ¹¹ -10 ¹³ PFU/ha active ingredient content. The concentrates and preparations of the invention preferably contain several phage strains, since this way the problems resulting from the phage resistance can be reduced.

The concentrate of the invention, and therefore the preparation contains genetically and morphologically characterized phages, tested for host-specificity. All the phages to be used according to the invention were isolated by the Applicant. It is important to mention that theoretically very large number of phage strains may be suitable for use in the preparation, considering the genetic variability of the phages, therefore we do not consider it essential to define more accurately the phage strains in the description. Since the phages are genetically extremely variable, this is the basis for their ability to adapt very quickly to the changes of the bacterial strains. Accordingly, therefore many and many kinds of phage strains can be isolated, or created with another procedure, which are useful for killing the Xanthomonas oryzae pv. oryzae. Since the result of the genetic change is a new phage strain, therefore it is not expedient to define the phage strains used more closely, since it is not excluded, that at the end of the phage treatment the phage strain included originally in the preparation cannot be re- isolated from the environment, but instead a new phage strain can be recovered, developed from the original one.

In the following, the present invention is further illustrated with reference to embodiments illustrated in the examples and figures in which:

Figure 1 is the flow chart of the preparation of the concentrate, while

Figure 2 is an application experiment, in multimode reader.

It should be emphasized that the exemplary embodiments and the drawings serve only to give a better understanding of the invention without limiting its scope.

Example

PSA medium. As it is described in this publication: Tsuchiya, Phytopathology, 1982 (72): 43-46.

Phage isolation. The origin of the phage samples was the paddy water of flooded rice fields from Vietnam and the Philippines. The samples were enriched in liquid cultures. 5 flasks containing 60-60 ml of PSA medium were inoculated with 200-200 μΐ overnight cultures of six different Xanthomonas oryzae pv. oryzae strains. 50 ml sample was filtered sterile on a 0.22 μιη pore-size filter, 40 ml of the filtrate was added to the PSA in the flasks, and 1.2 ml sterile glycerol and 60 μΐ 1M MgCl ₂ x 6H ₂ 0 were added, and were incubated for 24 hours in a thermoshaker at 28 °C. The samples were filtered through sterile gauze, and centrifuged at 2600xg for 40 minutes at 4 °C. After the removal of the supernatant there was a new filtration, on a microfilter of 0.22 μηι pore-size. The phages were purified by a three- pass passage with plaque isolation technique.

Transmission electron microscopy. High titer liquid phage cultures were centrifuged with 8000xg, separating this way the host cells from the phages. Following the aspiration of the supernatant containing the phages there was another centrifugation: 16,000xg, 5 °C for 1 hour. The precipitate was suspended in a drop of sterile distilled water. One drop of suspension was put on a 200x200 nickel grid and allowed to sediment for 2 hours. Contrast staining of the phages on the grid was carried out with 3% phosphotungstic acid for 40 seconds. Following the removal of the staining solution the grid was dried on air. The micro- scope was calibrated with catalase crystals, on the magnification used for taking pictures of phages. Each phage isolate was subcultured three times, each generation was microscopically tested. The accelerating voltage of the transmission electron microscope was 64 kV, magnification was 20,000x.

DNA extraction. 1.5 ml culture was centrifuged at 10,500xg for 10 minutes at 4 °C. 1.2 ml was aspirated from the supernatant and was treated with DNasel and RNase for 30 minutes at room temperature. 100 μΐ 0.5 M EDTA (pH=8.0) was added. Incubation at 75 °C for 10 minutes and following the addition of 10 μΐ ProteinaseK (20 mg/ml) incubation at 65 °C for 1 hour. The samples were divided into two parts (600-600 μΐ), and since then on the experiment was carried out in parallel with both samples. 60 μΐ 7.5 M ammonium acetate and 600 μΐ phenol chloroform were added to the samples. The samples were shaken, then centrifuged at 17,800xg for 10 minutes at 4 °C, and 500 μΐ was aspirated from the supernatant. The DNA was precipitated with 1 ml 96% ethanol, and the samples were incubated at - 20 °C for 1 hour, then centrifuged at 20,800xg for 15 minutes at 4 °C temperature. After the aspiration there were two washes with 70% alcohol. Following drying the samples were dis- solved in 100 μΐ sterile distilled water.

Application experiment. The phage strain intended for use was cultivated in overnight culture at 28 °C, in PSA medium, in a shaken culture. The PSA culture medium pre- warmed to 28 °C was inoculated in the 0 time with the given bacterial strain for the proper viable cell count (described at the application example), and with the phage strain / phage strains described at the application example for the MOI (described at the application example) on microtiter plate, in 200 μΐ final volume. The plates were shaken at 28 °C in Biotek Synergy HT Multimode reader with medium speed, and photometered for 48 hours in every 15 minutes on the 600 mm wavelength.

The viable cell count of the Xanthomonas oryzae pv. oryzae cells was determined from the samples, by diluting 200 μΐ of the samples 0-10 ⁸ fold (in lOx dilution steps) with sterile PSA culture medium or with sterile physiological saline, and 100-100 μΐ from each dilution or just from one or two dilutions were plated to sterile PSA culture medium. The plates were incubated at 28 °C, and after 12-60 hours (when they became countable) the bacterial colonies were counted (in those dilutions, in which they were countable). The number of the colonies was calculated back for 1 ml of the concentrated sample.

The virus titer was determined as follows. 100 μΐ of the sample was diluted 0-10 ¹² fold (in lOx dilution steps) with sterile PSA culture medium or with sterile physiological saline, and 10-10 μΐ from each dilution or just from one or some dilutions were dropped to a plate containing a Xanthomonas oryzae pv. oryzae strain, sensitive for the given virus strain, which bacterial strain was plated 12-48 hours earlier to PSA medium, and pre-incu- bated at 28 °C. The plates were incubated at 28 °C, and from the 12 ^th hour were frequently checked, to be able to count the number of the separately growing phage plaques (virus plaques) in the given drop. The number of the separately growing phage plaques (virus plaques) was calculated back for 1 ml of the concentrated sample.

Determination of the MOI (number of viruses for one bacterial cell): The virus titer (virus/ml) measured in the given sample was divided with the viable cell count (cell/ml) measured in the given sample.

Genome sequencing. The DNA library was prepared with Nextera XT kit, according to the manufacturer's instructions. DNA sequencing was performed with an Illumina MiSeq equipment, with V3, 600 cycle kit, paired end, with at least 100-fold coverage, according to the manufacturer's instructions. The genome of the phages was compiled with Geneious 8.0 software, or with MyPro pipeline.

The process of producing the product is shown in Figure 1.

It can be seen from flow chart 1, that during the preparation of the product first the virus strains are isolated. Following this even the virus strains are separated from each other, during which we first examine them with electron microscopy, or their plaque morphologic characteristics are observed. The separated virus strains are tested for host specificity. We continue working only with those virus strains, which have a sufficiently wide spectrum against Xanthomonas oryzae pv. oryzae strains unable to attack the bacteria which are genetically close to this bacterium (for example Xanthomonas arboricola pv. juglandis or Xanthomonas campestris pv. campestris).

The genome of the viruses tested for host specificity are sequenced, mainly for determining whether they contain some unwanted sequence (for example known antibiotic resistance or pafhogenicity-island gene), or a sequence, which refers to the ability of the integration into the genome of the host bacterium. If this test is positive, the given virus strain is not used anymore.

Subsequently, strains considered to be suitable are subjected to application experiments, in which the effect of the given virus strain on the viable count number of the Xanthomonas oryzae pv. oryzae strains is determined, and taking long-term kinetics is also suitable for providing information on the extent of resistance developing later.

The phage strains presenting the most preferred properties in the application experiments are selected for the product.

It is to be noted that Figure 1 is the most preferred embodiment of the product. At the same time the product can also be prepared if the separation of the strains is carried out with different methods (for example with PCR). Further it is a less preferred embodiment but may result in the preparation of the same products, if the "separation of the strains", "determination of the host specificity", "genome sequencing" and "application experiments" steps are interchanged with one another in any order.

Biological example (efficiency)

Altogether more than 15 anti-Xanthomonas oryzae pv. oryzae phage strains were isolated up to now (isolation is continued in the future). These were tested with electron microscope. The overwhelming majority of phages belong to the Myoviridae, while the other phages belong to the Podoviridae family, but it cannot be excluded that in the future phages belonging to a different phage family also became part of the preparation.

The genome of the phages was sequenced, and sequencing of the genome of 8 phages is currently underway. In 2 cases of sequenced phages, we found that the gene of the inte- grase enzyme can be found in the genome, therefore these phages were destroyed.

The phages were tested for host specificity. All our phages were absolutely specific for Xanthomonas oryzae pv. oryzae. The result of the application experiment of 8 phages can be seen on Figure 2. Based on the data of the figure all the 8 phages tested effectively reduced the cell number of the Xanthomonas oryzae pv. oryzae in the 48 hours interval, therefore they can be used as component of the plant protection agent.

Figure 2 illustrates an application experiment in multimode reader. Rows A, H, and columns 1 and 12: are empty, column 2: negative control (PSA nutrient solution), column 3: positive control, bacteria treated with the following phages: column 4: 5F; column 5: 6F; column 6: 13F; column 7: 15F, column 8: 16F; column 9: 20F; column 10: 21F; column 11: RG18. Rows B, C, D: Xanthomonas oryzae pv. oryzae marked 641, while rows E, F, G: Xanthomonas oryzae pv. oryzae treated strain. As it can be seen, the tests were conducted in 3-3 parallels.

Preparation example

The following plant protection agent is an example for an efficient preparation: In a 100 ml bottle, which should be stored at 2-8 °C, light protected until use:

• 5F bacteriophage, 10 ¹¹ PFU

· 6F bacteriophage, 10 ¹¹ PFU

• 13F bacteriophage, 10 ^u PFU

• 15F bacteriophage, 10 ¹¹ PFU

• 20F bacteriophage, 10 ¹¹ PFU

In a 1000 ml bottle that can be stored at room temperature

· 0.5 g/L sodium alginate

• 1 raM sodium ferulate.

The content of the 1000 ml bottle is diluted in 500 ml water, filled up to 900 L. The content of the 100 ml bottle is added to it with agitation, then filled up to 1000 L, and subsequently applied to 1 ha rice field with spraying.