Ralstonia solanacearum bacteriophage, bacteriophage composition thereof and application of bacteriophage composition in prevention and treatment of bacterial wilt infection diseases

Abstract

The invention discloses a Ralstonia solanacearum bacteriophage, a bacteriophage composition thereof and application of the Ralstonia solanacearum bacteriophage in prevention and treatment of bacterial wilt infection diseases, the Ralstonia solanacearum bacteriophage is named as Ralstonia solanacearum bacteriophage PQ04, the preservation number of the Ralstonia solanacearum bacteriophage is CGMCC No.45898, and the Ralstonia The strain is preserved in the China General Microbiological Culture Collection Center on March 22, 2024. The bacteriophage PQ04 is high in titer, has the characteristics of high splitting capacity and wide splitting spectrum on ralstonia solanacearum, also has relatively high ultraviolet resistance and relatively high temperature resistance, is high in activity stability, can be prevented from being influenced by natural environments such as high temperature and ultraviolet irradiation, and is wide in application range; the compound has the potential of being used as a biopesticide for preventing and treating ralstonia solanacearum, can be applied to prevention and treatment of diseases caused by ralstonia solanacearum infection in field plant planting, is safe, free of toxic and side effects, green and environment-friendly, does not generate drug-resistant bacteria, and has a very good market application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a Ralstonia solanacearum phage, a phage composition thereof and application thereof in preventing and treating diseases caused by bacterial wilt infection. Background Art

[0002] Ralstonia solanacearum is the second most pathogenic bacteria in the world and one of the most serious pathogens on Solanaceae plants. Ralstonia solanacearum is widely found in water bodies, soil and non-plant hosts such as weeds. It has a wide host range. In addition to infecting Solanaceae plants such as tomatoes, peppers, tobacco, and potatoes, it also infects more than 400 species of plants from more than 50 families, including monocots such as bananas and ginger, and dicots such as mulberry and eucalyptus. It has caused devastating disasters, which are particularly serious in tropical, subtropical and temperate regions. Infection with bacterial wilt can cause harvest losses of up to 100% for bananas, 90% for tomatoes and potatoes, and 30% for tobacco. Severe bacterial wilt can lead to the complete failure of the above crops.

[0003] Due to the wide distribution of Ralstonia solanacearum, a wide range of hosts that can be infected, and complex population diversity, it is impossible to fundamentally solve the occurrence of bacterial wilt disease. There are currently three main methods reported for the prevention and control of bacterial wilt disease, namely physical control, chemical control, and biological control. Physical control mainly focuses on the pathogenicity and survival conditions of Ralstonia solanacearum, exposes and disinfects the soil of the land to be planted, and performs reasonable rotation and intercropping to avoid the peak period of disease and reduce artificial damage to plant roots and stems during planting. Chemical control mainly uses chemical agents to quickly kill pathogens, but it also causes damage to beneficial organisms, changes the original microbial community structure and soil physical and chemical properties, and excessive use of pesticides leads to increased resistance of plant pathogens, serious pesticide residues in the soil, and environmental and food pollution. At present, there are many studies on biological control methods, and phage therapy has begun to be valued as a means of preventing and controlling Ralstonia solanacearum infection in plants. Phages have gradually become one of the research hotspots for the development of new drugs.

[0004] Bacteriophages are a type of virus that specifically infects bacteria and are widely distributed in environments where host bacteria exist. Compared with traditional drugs, bacteriophages have the following advantages: First, bacteriophages are highly specific and only work on host bacteria, reducing the harm to other bacteria and maintaining the balance of the soil microbial environment; second, bacteriophages are widely present in the natural environment and have good compatibility with the environment. They do not produce substances that pollute the environment, which meets the requirements of green development; third, bacteriophages reproduce rapidly in an exponential form, avoiding the high concentrations of traditional pesticides that are required to be effective, resulting in high pesticide residues in the soil and polluting the environment; fourth, bacteriophages have a short R&D cycle, low cost, and are easy to mass produce and transport. Therefore, the study of bacteriophages provides new ideas for the prevention and control of bacterial wilt, and is of great significance for the development of new approaches and new preparations for the prevention and control of bacterial wilt.

[0005] Based on the above advantages of bacteriophages, they have been gradually developed as pharmaceutical preparations or environmental disinfection and have been applied in some fields, such as the prevention and treatment of Ralstonia solanacearum. For example, the patent with publication number CN 117187192 A applied by the applicant in 2023 discloses a strain of Ralstonia solanacearum phage PQ43, which can effectively prevent and treat Ralstonia solanacearum infection in tobacco.

[0006] However, the existing Ralstonia solanacearum phages have poor ultraviolet tolerance and their activity is significantly reduced when used in the field. In addition, there are few existing broad-spectrum Ralstonia solanacearum phage resources and few corresponding phage preparations on the market. Therefore, the existing technology needs to be further improved. Summary of the invention

[0007] In view of the above problems, the present invention provides a Ralstonia solanacearum phage PQ04, a phage composition thereof and use thereof in preventing and treating diseases infected by bacterial wilt. The Ralstonia solanacearum phage has a wide lysis spectrum, and is resistant to high temperatures and has excellent ultraviolet light resistance. It can be used to prepare pharmaceutical preparations, plant compound fertilizers, environmental disinfectants and detection kits for treating or preventing diseases infected by Ralstonia solanacearum. While solving the problem of bacterial wilt infection, it avoids the problems of antibiotic residues and pathogen resistance caused by the use of antibiotics.

[0008] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a Ralstonia solanacearum phage isolated from the soil of a tobacco field in Hunan Province, which has a wide lysis spectrum, is resistant to high temperatures, and is resistant to ultraviolet rays. The phage is named PQ04.

[0009] The phage was deposited at the General Microbiology Center of the China Culture Collection Administration on March 22, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.45898.

[0010] Electron microscopy showed that the phage PQ04 has a polyhedral head structure and a non-contractile tail. The head is 60-75 nm in size and the tail is about 2-3 nm long. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), the morphology of the phage PQ04 of the present application conforms to the characteristics of the Brachyura family and belongs to the Brachyura phage.

[0011] In the present application, the above-mentioned bacteriophage PQ04 includes mutants with point mutation, deletion mutation or addition mutation with a homology higher than 98% or 99% and maintaining substantially the same bactericidal activity. Since bacteriophages are very prone to mutation during replication, the mutants of the above-mentioned bacteriophages are also within the scope of protection claimed in the present application. The sequence of bacteriophage PQ04 can be obtained by sequencing the biological material deposited according to the present invention by a known method. For those skilled in the art, it does not require creative labor to screen out mutants with extremely similar properties according to the bacteriophage provided by the present invention.

[0012] The bacteriophage PQ04 has excellent high temperature resistance. It still maintains its original activity after being exposed to 40-60℃ for 1 hour, and its potency is maintained at 1.10×10 8 PFU / mL, and still maintains high activity.

[0013] The phage also has excellent acid-base stability. Its activity is stable in the pH range of 4.0 to 10.0. At pH 3.0, it acts for 1 hour and the titer is 10 8 PFU / mL, and maintained high activity at pH 11 for 3 hours.

[0014] Phage PQ04 is a phage with a wide lysis spectrum and strong lysis performance. The total lysis rate of 172 strains of Ralstonia solanacearum from different sources preserved in the laboratory is 90.12%. Among them, the lysis rate of Ralstonia solanacearum from tobacco and tomatoes is relatively high, both above 90.00%. Compared with the phage PQ43 disclosed in the company's authorized patent (see publication number CN 117187192A), PQ04 has a higher total lysis rate and a wider lysis spectrum.

[0015] More importantly, the bacteriophage PQ04 has a strong tolerance to ultraviolet light. After irradiation with ultraviolet light for 2 hours, the bacteriophage still maintains its original activity, with a titer of 2.80×10 8 PFU / mL; and the phage has high storage stability in high temperature environment, and its titer remains at 10 after being stored at 37℃ for 1 month. 8PFU / mL; therefore, the phage PQ04 has a wide range of applications and high outdoor activity stability. It can avoid the influence of natural environments such as high temperature and ultraviolet radiation. It can be applied to plants or soil by spraying, root irrigation, and stem application, and still maintain a high phage activity, which can effectively block and prevent infection with Ralstonia solanacearum.

[0016] In the second aspect, the present application also provides a phage composition, which includes the aforementioned Ralstonia solanacearum phage and PQ04 other phages. In practical applications, in order to further broaden the lysis spectrum of the phage preparation, give full play to the differences in the lysis spectrum of different phages, and complement each other's advantages, the above-mentioned phage PQ04 and other phages can be used in combination, such as with PQ43 (see patent publication number CN 117187192A), which is also a phage of Ralstonia solanacearum. In this way, the lysis spectrum can be further broadened, the killing of Ralstonia solanacearum in plants or soil environments can be improved, and a variety of plant wilt diseases, such as tobacco, pepper, tomato or ginger, can be better prevented and treated.

[0017] Preferably, the phage composition comprises phage PQ04 and phage PQ43. The deposit number of the Ralstonia solanacearum phage PQ43 is CGMCC No 23813, and the specific information of the phage can be found in the patent publication number CN 117187192A.

[0018] In a third aspect, the present application also provides the use of the above-mentioned Ralstonia solanacearum phage or the above-mentioned phage composition in preventing and controlling plant diseases caused by Ralstonia solanacearum infection.

[0019] Preferably, the diseases caused by Ralstonia solanacearum infection include tobacco bacterial wilt, tomato bacterial wilt, pepper bacterial wilt, ginger bacterial wilt, eggplant bacterial wilt and the like.

[0020] The control includes prevention and treatment. The term "prevention" herein refers to all actions including suppressing or delaying the disease by administering the composition. The term "treatment" herein refers to all actions including improving or ameliorating the disease by administering the composition.

[0021] In a fourth aspect, the present application further provides a phage pharmaceutical preparation, the active ingredient of which includes the aforementioned Ralstonia solanacearum phage PQ04 or the aforementioned phage composition; preferably, the phage pharmaceutical preparation also includes other antibacterial or bactericidal active ingredients, and the content of phage PQ04 in the reagent is ≥10 8 PFU / mL.

[0022] The application method of the bacteriophage drug preparation is: using the bacteriophage or its composition as a therapeutic drug, mainly in a variety of ways such as spraying, root irrigation, stem application, etc. to act on plants or soil.

[0023] Optionally, the phage drug preparation further comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein refers to a carrier or diluent that does not cause significant stimulation to plants and does not eliminate the biological activity and characteristics of the administered active component. If necessary, other conventional additives, such as antioxidants, buffers, and antibacterial agents, etc., may be added. When combined with a diluent, a dispersant, a surfactant, a binder, and / or a lubricant, the composition of the present invention may also be prepared into emulsifiable concentrates, suspensions, wettable powders, powders, granules, aqueous solutions, etc., and other intermediate dosage forms, etc.

[0024] In a fifth aspect, the present application also provides a biological pesticide, the active ingredient of which includes: the aforementioned Ralstonia solanacearum phage or the aforementioned phage composition. The application method can act on plants or soil by soaking, spraying, root irrigation, stem application and other methods. In a sixth aspect, the present application further provides an environmental disinfectant, the active ingredient of which includes the bacteriophage PQ04 or the aforementioned bacteriophage composition as described above; preferably, the concentration of the bacteriophage is 10 8 PFU / mL or above.

[0025] Optionally, the environmental disinfectant is used to disinfect and sterilize the surface or soil of plants such as tobacco and tomatoes, thereby preventing the spread of Ralstonia solanacearum from the source and effectively preventing and treating bacterial wilt. This disinfectant can be used to replace antibiotics or traditional disinfection products, and it does not cause damage to plants or pollution to the environment.

[0026] In a seventh aspect, the present application also provides a detection kit, which includes the aforementioned bacteriophage PQ04. Experiments have found that the bacteriophage PQ04 has lysis specificity for host bacteria, and the bacteriophage PQ04 of the present invention can be applied to the rapid detection of Ralstonia solanacearum, including but not limited to the detection of Ralstonia solanacearum in the form of test strips, test strip boxes, etc., or the screening of target pathogens in clinical samples, effectively ensuring the sensitivity of the detection.

[0027] The present invention has the following beneficial effects: 1. The present invention provides a self-screened Ralstonia solanacearum phage PQ04, which not only has a high titer, but also exhibits strong UV resistance and temperature resistance; at the same time, it exhibits strong lysis ability and a wide lysis spectrum against Ralstonia solanacearum; the Ralstonia solanacearum phage PQ04 has strong activity stability, can avoid the influence of natural environments such as high temperature and ultraviolet radiation, has a wide range of adaptability, and can be used to prevent and control diseases caused by Ralstonia solanacearum infection in field plant cultivation.

[0028] Based on the above characteristics, the bacteriophage PQ04 can be prepared into pharmaceutical preparations, plant biological pesticides, disinfectants and detection kits for the prevention and treatment of Ralstonia solanacearum. It can not only effectively prevent the spread of Ralstonia solanacearum disease in tobacco, tomatoes, peppers and other plants, but also has strong lysis ability, and can more effectively control the incidence of bacterial wilt disease during plant cultivation.

[0029] 2. The bacteriophage PQ04 in the present invention is obtained from natural soil, will not cause soil pollution, has high safety, avoids environmental pollution problems caused by the use of chemical pesticides, and has the characteristics of being green and environmentally friendly; in addition, the bacteriophage has a high explosive volume and strong reproductive ability, is easy to develop and produce on an industrial scale, and effectively reduces costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a plaque image of bacteriophage PQ04; Figure 2 This is an electron microscope image of bacteriophage PQ04; Figure 3 The results of thermal stability test of bacteriophage PQ04; Figure 4 The pH stability test results of bacteriophage PQ04; Figure 5 is the one-step growth curve of bacteriophage PQ04; Figure 6 Analysis of the UV tolerance of bacteriophage PQ04. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0032] Example 1 Screening and Isolation of Ralstonia solanacearum Phage 1. Experimental methods (1) Resuscitation culture of host bacteria and preparation of proliferation medium Ten strains of Ralstonia solanacearum preserved in our laboratory were selected, and the freezing liquid was dipped into a sterilized inoculum and streaked on TTC medium for recovery. The culture was cultured in a constant temperature incubator at 30℃ for 24-48h to obtain a single colony; a single colony was picked and inoculated into 5mL of NB broth, and cultured at 30℃ with shaking at 220rpm / min for 16-24h to obtain a fresh Ralstonia solanacearum liquid.

[0033] (2) Phage isolation and purification Take an appropriate amount of soil sample from a tobacco base in Hunan, add it to an appropriate amount of broth medium, add 10 strains of Ralstonia solanacearum, put the mixed solution into 30°C 220rpm / min shaking culture for 12h, centrifuge at 11000rpm for 5min, and then filter it with a 0.22μm sterile microporous filter membrane to obtain phage proliferation liquid; The phage proliferation liquid was diluted in a 10-fold ratio, and appropriate gradient phage dilution liquid was mixed with 10 strains of Ralstonia solanacearum in a 1:1 ratio. After incubation at 37°C for 5 minutes, 200 μL of the mixture was placed on the upper agar (agar concentration of 0.7%), and after mixing, it was quickly poured onto the lower agar (agar concentration of 1.5%) plate, shaken and placed flat until the culture medium solidified, and placed in a 37°C incubator for inverted culture for 4 to 6 hours to obtain a double-layer plate with plaque formation.

[0034] Pick a single plaque from the double-layer agar medium where plaques are formed, and place it in 1 mL of NB broth and culture it in a constant temperature shaker at 28°C, 220 rpm / min for about 30 minutes to obtain a phage extract. Take the phage extract and mix it evenly with the corresponding plaque-forming Ralstonia solanacearum proliferation solution in a ratio of 1:1, incubate at 37°C for 5 minutes, draw 200 μL and place it on the upper agar, mix it and quickly pour it on the lower agar plate, shake it evenly and place it flat until the culture medium solidifies, place it in a 37°C incubator and culture it upside down for 4-6 hours, and then obtain a double-layer plate with plaques formed again. Use sterilized tweezers to pick a single plaque from the double-layer culture medium where plaques are formed and place it in 1 mL of NB broth, and culture it in a constant temperature shaker at 28°C, 220 rpm / min for about 30 minutes to obtain a phage extract. Repeat the above steps 3 times to obtain the purified phage extract.

[0035] (3) Phage titer determination Take an equal amount of purified phage extract and host bacteria proliferation liquid in 5 mL of liquid NB medium, and culture at 30°C, 220 rpm / min with shaking until the liquid becomes clear. Centrifuge the clear liquid at 11000 rpm for 10 min, take the supernatant, and filter it with a 0.22 μm sterile microporous filter membrane to obtain phage proliferation liquid, and use the double-layer plate method to determine the titer of the newly isolated phage.

[0036] (4) Screening of UV-resistant Ralstonia solanacearum phages The newly isolated Ralstonia solanacearum phages were adjusted to a uniform initial concentration (5.00 × 10 8 PFU / mL), 5mL of Ralstonia solanacearum phage suspension was spread in a sterile culture dish, placed on a clean bench 30cm away from the UV lamp for 2h, and the titer change was detected by double-layer plate method. At the same time, other phages were selected to perform UV resistance test according to the above method, including: Ralstonia solanacearum phage PQ43 (see patent with publication number CN 117187192A), Escherichia coli phage BP7 (see patent with announcement number CN103289963B), Proteus phage PF07 (see patent with publication number CN 116286678A), and Salmonella phage PC127 (see patent with publication number CN 116083374A), and the titer changes of each phage were compared.

[0037] (5) Screening of thermotolerant Ralstonia solanacearum phages The newly isolated Ralstonia solanacearum phages were adjusted to a uniform initial concentration (5.00 × 10 8 PFU / mL), stored in a high temperature environment at 37°C for 1 month, during which the phage titer changes were regularly measured. At the same time, other phages were selected to perform high temperature resistance tests according to the above method. Other phages included: Ralstonia solanacearum phage PQ43 (see patent with publication number CN117187192A), Escherichia coli phage BP7 (see patent with announcement number CN103289963B), Proteus phage vB_PmiM_PF07 (see patent with publication number CN 116286678A), and Salmonella phage PC127 (see patent with publication number CN 116083374A), and the titer changes of each phage were compared.

[0038] 2. Experimental results and analysis (1) According to the above experimental method, 6 Ralstonia solanacearum phages were screened using 10 isolates of the host bacteria of Ralstonia solanacearum, which were numbered PQ01-PQ06. All 6 Ralstonia solanacearum phages formed clear plaques on double-layer agar plates, surrounded by halos with clearly visible edges, with a diameter of about 0.5 mm to 1.5 mm; the titer was between 5.00×10 8 PFU / mL-3.50×10 10 PFU / mL.

[0039] (2) The comparison of the experimental results of the newly isolated 6 strains of Ralstonia solanacearum phage PQ01-PQ06 and 4 existing phages is shown in Table 1. From the results in Table 1, it can be seen that the comprehensive performance of Ralstonia solanacearum phage PQ04 is the best. After being subjected to ultraviolet resistance for 2 hours and stored in a high temperature environment of 37°C for 1 month, its activity and lysis rate are much higher than those of other Ralstonia solanacearum phages screened in the same batch and other four existing phages (PQ43, PF04, BP7 and PC127).

[0040] The above results show that the phage has high activity stability and can adapt to more stringent field application environments, providing guarantees for later field clinical applications, avoiding the influence of high temperatures and strong ultraviolet rays in the natural environment, while also effectively reducing the cost of use and storage.

[0041] Table 1 Stability test of 6 newly isolated Ralstonia solanacearum phages

[0042] Example 2 Morphological Observation and Identification of Ralstonia solanacearum Phage PQ04 1. Experimental methods Take 20 μL of phage sample and drop it on a copper mesh with a carbon-coated film. Wait for it to precipitate naturally for 15 minutes. After blotting it with filter paper, stain it with 2% (W / V) phosphotungstic acid (PTA) for 1-2 minutes. After blotting it with filter paper, observe and photograph it under a transmission electron microscope.

[0043] 2. Experimental results and analysis like Figure 2 As shown, the bacteriophage PQ04 has a polyhedral head structure and a non-contractile tail. The head diameter is 60-75 nm and the tail length is about 2-3 nm. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), the phage morphology of the present application conforms to the characteristics of the Brachyura family and belongs to the Brachyura phage.

[0044] Example 3 Whole genome analysis of Ralstonia solanacearum phage PQ04 The genome of phage PQ04 was extracted, and whole genome sequencing and sequence analysis were performed. The results are as follows: (1) The PQ04 genome is 38797 bp in length, with a G+C content of 62.61% and an A+T content of 37.39%. The base contents of C, G, A, and T are 31.44%, 31.17%, 20.82%, and 16.57%, respectively. The online annotation results of the whole genome RAST showed that the genome contained 50 open reading frames (ORFs). Among these 50 open reading frames (ORFs), 16 structural proteins were found, mainly including the structure and packaging proteins of the phage (capsid protein, capsid and scaffold protein, tail spike, structural protein, phage fiber protein, topoisomerase large subunit, etc.), phage lysis-related proteins (phage lysin), DNA replication and modification-related proteins (DNA polymerase, DNA helicase, DNA binding protein, endonuclease, etc.), and other functional proteins (phage repressor protein, dual infection immunity protein, etc.). At the same time, among the 50 ORFs, 43 start codons are ATG, 4 start codons are GTG, 1 start codon is ATC, and 2 start codons are GAT. The software tRNAscan-SE analysis showed that the genome contained tRNA genes. The online tool CGE server analysis showed that the genome did not contain drug resistance genes and virulence genes. The PHASTER analysis showed that the genome did not contain lysogeny-related genes.

[0045] (2) In the genome of bacteriophage PQ04: the gene sequence of the tail fiber protein related to phage host recognition is shown in sequence 1 in the sequence listing; the sequence of the topoisomerase large subunit protein gene is shown in sequence 2 in the sequence listing; the sequence of the DNA polymerase gene is shown in sequence 3 in the sequence listing; the sequence of the lysin gene related to cleavage ability is shown in sequence 4 in the sequence listing. For specific relevant information, see Table 2 below.

[0046] Table 2 Gene sequence information

[0047] Example 4 Determination of the Optimal Multiplicity of Infection (MOI) of Ralstonia solanacearum Phage PQ04 against Ralstonia solanacearum 1. Experimental methods Pick a single Ralstonia solanacearum colony and inoculate it into 5 ml NB broth medium, culture it at 30°C and 220 rpm for 16-19 hours to obtain bacterial solution, and determine the bacterial concentration by pouring method; detect the phage titer by double-layer plate method. Add the phage titer to the host bacteria concentration in a ratio of 1:1, 0.1:1, 0.01:1, 0.001:1, 0.0001:1, 0.00001:1, 0.000001:1, 0.0000001:1, and 0.0000001:1 to a test tube containing 5 mL NB broth, and culture it at 30°C and 220 rpm for 4-12 hours until the liquid changes from turbid to clear. Take an appropriate amount of clear liquid and centrifuge it at 11000rpm / min for 10min. Take the supernatant and filter it through a 0.22μm sterile microporous filter membrane. Determine the phage titer in the filtrate by the double-layer plate method. The MOI (number of phages / number of bacteria) with the highest phage titer is the optimal multiplicity of infection for the phage.

[0048] 2. Experimental results As shown in Table 3, when MOI was 0.00001, the titer of PQ04 reached the highest, 3.60×10 11 PFU / mL, that is, the optimal MOI is 0.00001; this result not only provides data reference for the large-scale industrial production of later products, but also shows that the cost of industrial batch production of this phage is low.

[0049] Table 3 The titer of Ralstonia solanacearum phage PQ04 at different infection multiplicity

[0050] Example 5 Temperature stability of Ralstonia solanacearum bacteriophage PQ04 1. Experimental methods: 2.00×10 10 PFU / mL Ralstonia solanacearum phage PQ04 proliferation solution was placed at 40℃, 50℃, 60℃, 70℃, and 80℃, and three parallel samples were made at each temperature. After 20min, 40min, and 60min, the samples were immediately cooled in an ice bath after the reaction ended, and then the phage titer at different temperatures was detected by the double-layer plate method. The PQ04 phage thermal stability curve was drawn with temperature as the horizontal axis and the logarithm of the phage titer as the vertical axis.

[0051] 2. Experimental results and analysis like Figure 3 The results showed that the titer of phage PQ04 was relatively stable after 60 minutes of exposure at a temperature between 40℃ and 60℃; it still maintained some activity after 60 minutes of exposure at 70℃; and the titer remained at 1.10×10 8PFU / mL, and still maintains a high activity. It can be seen that Ralstonia solanacearum phage PQ04 has strong heat resistance and can adapt to higher temperature environments.

[0052] Example 6 pH stability of Ralstonia solanacearum bacteriophage PQ04 1. Experimental methods Take three sterile test tubes and add 4.5 mL of NB broth with different pH values ​​(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). Then place the test tubes in a 37°C water bath. After the temperature stabilizes, add 500 µL of 2.00×10 10 PFU / mL phage proliferation solution, mix well and incubate in a 37℃ water bath for 1 h, 2 h, and 3 h. After the reaction, immediately add an appropriate amount of 1 mol / L HCl or NaOH to the mixture to make the pH value of the mixture about 7, dilute it 10 times, take an appropriate dilution gradient to determine the titer, and set 3 parallel samples for each pH value test tube. Draw the phage pH stability curve with pH as the horizontal axis and the logarithm of phage titer as the vertical axis.

[0053] 2. Experimental results and analysis from Figure 4 The results show that the titer of bacteriophage PQ04 remains at 10 in the pH range of 4.0-10.0. 10 PFU / mL, stable activity; after 3 hours at pH 3.0, its titer remained at 10 4 PFU / mL; after 1 hour at pH 11.0, the titer of the phage was 10 8 PFU / mL, and still maintains a high activity; therefore, bacteriophage PQ04 has strong stability in a wide pH range and can adapt to a wide range of strong acid and strong alkaline environments.

[0054] Example 7 One-step growth curve of Ralstonia solanacearum phage PQ04 1. Experimental methods 1 mL of phage PQ04 proliferation solution and host bacteria proliferation solution with a multiplicity of infection of 10 were mixed thoroughly (the timing started at this time), incubated at 30°C for 5 minutes, centrifuged at 13000 rpm for 30 seconds, and the supernatant was removed as much as possible with a micropipette, and then washed once with 5 mL of NB broth (centrifuged at 13000 rpm / min for 30 seconds), and the supernatant was discarded. The precipitate (total volume of 5 mL) was suspended with preheated NB broth and mixed thoroughly, and quickly placed in a 30°C shaker with 220 rpm / min shaking culture. 150 μL was taken out at time 0 and every 5 minutes, centrifuged at 10000 rpm for 1 minute, and the phage titer was measured by the double-layer plate method after 10-fold dilution with NB broth. Three parallels were made, and the results were averaged. The infection time was used as the horizontal axis and the titer of the phage in the infection system was used as the vertical axis to draw a one-step growth curve to obtain the incubation period and outbreak period of phage PQ04, and calculate the outbreak volume.

[0055] Outbreak volume = total number of phages at the end of the outbreak / total number of bacteria at the beginning of the outbreak 2. Experimental results and analysis from Figure 5 The results show that after phage PQ04 infects the host bacteria, the phage lysis cycle is about 70 minutes, the incubation period is about 20 minutes, and the phage outbreak period is about 50 minutes; after 70 minutes, the number of phages remains basically unchanged and enters a stable period, at which time the titer can reach 10 10 PFU / mL, and the burst volume of bacteriophage PQ04 was 93.

[0056] Example 8 Determination of the lysis spectrum of Ralstonia solanacearum phage PQ04 1. Experimental Materials Host bacteria: 172 strains of Ralstonia solanacearum from different plant sources were selected and preserved in the laboratory (90 strains of Ralstonia solanacearum from tobacco, 47 strains of Ralstonia solanacearum from tomato, 13 strains of Ralstonia solanacearum from pepper, and 22 strains of Ralstonia solanacearum from ginger, see Table 4 below for details).

[0057] 2. Experimental methods: (1) 172 strains of Ralstonia solanacearum from different sources were selected and streaked on TTC medium for recovery. Single colonies were selected for proliferation and culture to obtain fresh bacterial suspension. The phage proliferation suspension PQ04 and 172 strains of Ralstonia solanacearum were prepared and the lysis spectrum of phages was detected by double-layer plate method.

[0058] (2) Comparison of lysis performance with existing bacteriophages: According to the above method, the lysis performance of the existing bacteriophage PQ43 (disclosed in the patent with the applicant's publication number CN 117187192A) on the above 172 strains of Ralstonia solanacearum was simultaneously measured, and the difference in the lysis of the above 172 strains of Ralstonia solanacearum by the two Ralstonia solanacearum phages was compared.

[0059] (3) 170 strains of Ralstonia solanacearum host bacteria used in the patent with publication number CN 117187192A (see Table 5 for details) were selected to perform lysis spectrum detection on bacteriophages PQ04 and PQ43, and the lysis effects of the two phages were compared and analyzed.

[0060] 3. Experimental results and analysis 3.1 Comparative analysis of the lysis effects of two phages on 172 strains of Ralstonia solanacearum from different sources (1) From the results in Table 4, it can be seen that the Ralstonia solanacearum phage PQ04 can lyse 155 of the 172 strains of Ralstonia solanacearum from different sources stored in the laboratory, with a total lysis rate of 95.56%, while the existing Ralstonia solanacearum phage PQ43 can lyse 139 of the 172 strains of Ralstonia solanacearum from different sources, with a total lysis rate of only 80.81%. This shows that the phage PQ04 has a wider lysis spectrum and stronger lysis performance.

[0061] (2) Specifically, phage PQ04 could lyse 86 out of 90 strains of Ralstonia solanacearum in tobacco, with a lysis rate of 95.56%, while phage PQ43 had a lysis rate of 90.00%; PQ04 could lyse 43 out of 47 strains of Ralstonia solanacearum in tomato, with a lysis rate of 91.49%, while PQ43 had a lysis rate of 78.72% for 47 strains of Ralstonia solanacearum in tomato; PQ04 could lyse 8 out of 13 strains of Ralstonia solanacearum in pepper, with a lysis rate of 61.54%, while PQ43 had a lysis rate of 53.85% for 13 strains of Ralstonia solanacearum in pepper; PQ04 could lyse 18 out of 22 strains of Ralstonia solanacearum in ginger, with a lysis rate of 81.82%, while PQ43 had a lysis rate of 63.64% for 22 strains of Ralstonia solanacearum in ginger. The detailed results are shown in Table 4.

[0062] 3.2 Comparative analysis of the lysis effects of two phages on 170 strains of Ralstonia solanacearum From the results in Table 5, it can be seen that the Ralstonia solanacearum phage PQ04 can lyse 155 of the 170 strains of Ralstonia solanacearum in patent CN 117187192A, with a total lysis rate of 91.18%, which is significantly higher than the total lysis rate of PQ43 (85.29%); among them, the lysis rates of PQ04 against type I and type II are relatively high, which are 94.47% and 100.00% respectively, both higher than the lysis rates of PQ43 (90.23% and 80.00%); the specific results are shown in Table 5.

[0063] In summary, the lysis rate of Ralstonia solanacearum phage PQ04 for 172 strains of Ralstonia solanacearum from different sources used in this application is significantly higher than that of phage PQ43; and for the 170 strains of Ralstonia solanacearum selected in patent CN 117187192A, the lysis rate of Ralstonia solanacearum phage PQ04 is also higher than that of Ralstonia solanacearum phage PQ43; This shows that Ralstonia solanacearum phage PQ04 has the characteristics of a wide lysis spectrum and better lysis effect, can effectively prevent and control the infection of Ralstonia solanacearum from different sources, and has a broader application prospect.

[0064] Table 4 Lysis spectrum of 2 strains of Ralstonia solanacearum phage against 172 strains of Ralstonia solanacearum

[0065] Table 5 Lysis spectrum of two Ralstonia solanacearum phages against 170 Ralstonia solanacearum strains

[0066] Example 9 Safety Test of Ralstonia solanacearum Phage PQ04 1. Experimental methods Tomato seedlings with three true leaves and the same growth were selected and transplanted into 9-hole pots. The root system of the seedlings was not damaged during the transplanting process. A blank group, phage group 1, and phage group 2 were set up respectively; 10 seedlings were treated in each group. Blank group: no treatment, phage group 1: phage PQ04 (10 10 PFU / g); Phage group 2: stem injection of phage PQ04 (10 10 PFU / plant); all potted plants were cultured under the same greenhouse conditions. At the end of the experiment, the growth and disease status of tomatoes in each experimental group were counted.

[0067] 2. Experimental results and analysis The tomatoes in the two phage groups and the blank group grew well, without abnormal phenomena such as wilting, indicating that the Ralstonia solanacearum phage PQ04 is safe and reliable, and has no adverse effects on plant organisms.

[0068] Example 10 Phage Lysis Test on Non-Host Bacteria 1. Experimental methods Ten strains of Escherichia coli, ten strains of Salmonella, ten strains of Clostridium perfringens, ten strains of Staphylococcus aureus and ten strains of Proteus were selected, and a lysis experiment was performed according to the lysis spectrum determination method in Example 8 above.

[0069] 2. Experimental results and analysis The double-layer plate plaque method was used to detect that no plaques were found when phage PQ04 was cultured with the above-mentioned non-host bacteria, indicating that phage PQ04 could not recognize all of the above-mentioned non-host bacteria. This shows that the tested phage has extremely strong host specificity and has no damaging effect on the microbial community. The high specificity of this phage to Ralstonia solanacearum can be used to prepare a rapid detection kit for Ralstonia solanacearum, which can be used for simple and rapid detection of Ralstonia solanacearum in the test sample or target environment.

[0070] Example 11 Ultraviolet tolerance test of Ralstonia solanacearum bacteriophage PQ04 1. Experimental methods A tobacco field base in Hunan was selected, and the tobacco had no bacterial wilt. Three areas (A, B, and C) were randomly selected, each containing 9 tobacco seedlings. Each tobacco seedling in each area was irrigated with 1.00×10 10 PFU / mL bacteriophage PQ04, combined with the clinical use of bacteriophages, choose to culture normally for 2 days under the environment of sunlight ultraviolet radiation. Sampling was done once every 6 hours, and 25g of root soil was taken from 5 points in each area, added with 225mL of sterile water for leaching, then centrifuged and filtered, and the filtrate was taken to detect the titer of bacteriophage PQ04 using the double-layer plate method.

[0071] 2. Experimental results and analysis like Figure 6 As shown in the figure, after one day of solar ultraviolet irradiation, the Ralstonia solanacearum phage PQ04 in the three areas A, B, and C were relatively stable, maintaining the original effective titer of 10 10 PFU / g; After 2 days of solar ultraviolet irradiation, the titer of Ralstonia solanacearum phage PQ04 in areas A, B, and C decreased, but still maintained 10 9 The results show that the Ralstonia solanacearum phage PQ04 has a strong tolerance to ultraviolet rays, and can maintain a high phage activity when used in the field, which can effectively avoid the influence of ultraviolet rays and other natural environments on its activity, ensure the antibacterial effect of phage in the field, and improve the prevention and control effect of plant diseases.

[0072] Example 12 Potted Plant Experiment on Control of Tomato Bacterial Wilt by Ralstonia solanacearum Phage PQ04 1. Experimental methods Select tomato seedlings with three true leaves and the same growth pattern and transplant them into 9-hole pots, avoiding damaging the seedling roots during the transplanting process.

[0073] A blank group, a bacteria attack group, and a phage group were set up respectively, and the settings of each group were as follows: ①Blank group: no treatment; ② Bacteria attack group: only inoculated with Ralstonia solanacearum, inoculated in the soil for 107 CFU / g Ralstonia solanacearum; ③ Phage group 1: inoculated with Ralstonia solanacearum and irrigated with phage PQ04 (10 8 PFU / g); ④ Phage group 2: inoculation with Ralstonia solanacearum and stem injection with phage PQ04 (10 8 PFU / strain).

[0074] Each group was treated with 27 seedlings, with 3 replicates and 9 seedlings in each replicate. All potted plants were cultured under the same greenhouse conditions. At the end of the experiment, the incidence and prevention rate of bacterial wilt in each experimental group were counted.

[0075] According to the degree of leaf wilting at the time of disease onset, the disease is divided into 5 levels. Level 0: no symptoms; Level 1: 1%-25% of the leaves of the plant show wilting symptoms and can recover; Level 2: 26%-50% of the leaves of the plant show wilting symptoms and only part of them can recover; Level 3: 51%-75% of the leaves of the plant show wilting symptoms; Level 4: more than 75% of the leaves of the plant show wilting symptoms or even die.

[0076] Incidence rate = number of diseased plants / total number of plants surveyed × 100% Disease index = (Σ number of diseased plants at each level × disease level) / (total number of plants surveyed × highest disease level) × 100 Control rate = (control disease index - treatment disease index) / control disease index × 100% 2. Experimental results and analysis As shown in Table 6, compared with the high incidence rate (92.59%) of the challenge group, the incidence rate of phage groups 1 and 2 using the solanacearum phage PQ04 was reduced by an average of 72.22%.

[0077] Comparing the results of phage group 1 and phage group 2, it can be seen that the control rate of phage group 1 using stem injection of phage PQ04 was 88.89%, which was higher than the 82.71% control rate of phage PQ04 treated with roots, but there was no significant difference in the control of tomato bacterial wilt between the two (p>0.05), indicating that the bacterial wilt phage PQ04 can effectively prevent and control the occurrence of tomato bacterial wilt.

[0078] Table 6 Control situation of each experimental group in potted plant test

[0079] Example 13 Field trial of Ralstonia solanacearum phage PQ04 against tomato bacterial wilt 1. Experimental methods A vegetable greenhouse of a farmer in Shandong was selected for field experiment. The vegetable greenhouse had suffered from severe tomato bacterial wilt in the early stage. The selection of tomato seedlings in this experiment was the same as that in Example 10. A total of 9 treatments were designed, with 3 areas in each treatment and 18 tomatoes in each area. All areas were randomly distributed to reduce the impact of environmental factors on the test results. A control group and a phage group were set up respectively; the dosage and method of phage use were consistent with those in Example 10. The incidence rate and disease prevention rate of each experimental group were statistically analyzed according to the tomato bacterial wilt grading standard in Example 10.

[0080] 2. Experimental results and analysis The results of the field experiment are shown in Table 8. The incidence rate of the control group was 55.56%, and the incidence rates of root irrigation treatment with phage PQ04 and stem injection with phage PQ04 were significantly lower than those of the control group, which were 11.11% and 5.56%, respectively. The disease prevention rate of stem injection of phage PQ04 (95.35%) was higher than that of root irrigation treatment with phage PQ04 (86.04%), and there was no significant difference in the prevention and control of bacterial wilt between the two (p>0.05). This shows that bacterial wilt phage PQ04 has a significant effect on the prevention and control of tomato bacterial wilt in the field and can be promoted as a new biological agent.

[0081] Table 7 Control situation of each experimental group in field test

[0082] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A Ralstonia solanacearum phage, characterized in that: It was named PQ04 and its deposit number is CGMCC No.45898.

2. A bacteriophage composition, characterized in that: It includes the Ralstonia solanacearum phage described in claim 1 and other phages.

3. The bacteriophage composition according to claim 1, characterized in that The other bacteriophages also include Ralstonia solanacearum phage PQ43, and the deposit number of the Ralstonia solanacearum phage PQ43 is CGMCC No 23813.

4. Use of the Ralstonia solanacearum phage according to claim 1 or the phage composition according to claim 2 or 3 in preventing and controlling plant diseases caused by Ralstonia solanacearum infection.

5. The use according to claim 4, characterized in that: The plant diseases caused by the infection of Ralstonia solanacearum include tobacco bacterial wilt, tomato bacterial wilt, pepper bacterial wilt, ginger bacterial wilt, and eggplant bacterial wilt.

6. A phage pharmaceutical preparation, the active ingredient of which comprises the Ralstonia solanacearum phage according to claim 1 or the phage composition according to claim 2.

7. A biological pesticide, characterized in that: The active ingredients include: the Ralstonia solanacearum phage according to claim 1 or the phage composition according to claim 2 or 3.

8. An environmental disinfectant, characterized in that The active ingredients include the Ralstonia solanacearum phage as claimed in claim 1 or the phage composition as claimed in claim 2 or 3; preferably, it also contains other active ingredients for inhibiting or eliminating bacteria in the environment.

9. Use of the biological pesticide according to claim 7 or the environmental disinfectant according to claim 8, characterized in that: The application method is: the biological pesticide or environmental disinfectant is applied to plants or soil in the form of spraying, root irrigation or stem application.

10. A detection kit, characterized in that: It comprises the Ralstonia solanacearum phage as described in claim 1.

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