CN115725512A - Ralstonia solanacearum bacteriophage and composition, kit and application thereof - Google Patents
Ralstonia solanacearum bacteriophage and composition, kit and application thereof Download PDFInfo
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- CN115725512A CN115725512A CN202210940732.3A CN202210940732A CN115725512A CN 115725512 A CN115725512 A CN 115725512A CN 202210940732 A CN202210940732 A CN 202210940732A CN 115725512 A CN115725512 A CN 115725512A
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Abstract
The invention discloses a solanaceae Ralstonia phage and a composition, a kit and application thereof, wherein the solanaceae Ralstonia phage is a solanaceae Ralstonia phage RS-P48 (Ralstonia solanacearum phage RS-P48), and the preservation number is CCTCC NO: M2020748; the solanaceae ralstonia phage RS-P48 is a virulent phage separated from nature, has high tolerance to pH, is suitable for different control environments, and can achieve a good biological control effect on eucalyptus bacterial wilt.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a novel solanaceae ralstonia phage, a composition thereof, and a preparation method and application thereof.
Background
Bacterial wilt is a worldwide bacterial soil-borne disease caused by Ralstonia solanacearum and is widely distributed in tropical, subtropical and some temperate regions. The Ralstonia solanacearum has a wide host range and can infect more than 200 plants of more than 50 families, wherein the solanaceous crops such as potatoes, tomatoes, tobaccos and the like are most seriously damaged. Due to global warming caused by the greenhouse effect, the strain shows a tendency of spreading to cold and cool areas with high latitudes, and brings great threat to crop production.
Ralstonia solanacearum (Ralstonia solanacearum) infects not only solanaceous crops but also non-solanaceous cash crops, which may result in, for example: eucalyptus bacterial wilt and sesame bacterial wilt; pumpkin bacterial wilt; bacterial wilt of strawberries; bacterial wilt of chrysanthemum; bacterial wilt of water spinach and the like. Taking the bacterial wilt of eucalyptus as an example, the infection of the eucalyptus by the ralstonia solanacearum can cause the withering of the whole eucalyptus seedling or the red and purple leaves at the base of the seedling. At present, the control method for the bacterial wilt of crops mainly comprises agricultural control, physical control, medicament control and the like, and although the method plays a certain role in controlling the bacterial wilt, the disease cannot be comprehensively and effectively controlled.
Therefore, there is a need to develop a novel method for controlling the bacterial wilt disease of non-solanaceous cash crops by using the bacteriophage.
Disclosure of Invention
Aiming at the technical current situation, the invention provides 1 strain of bacteriophage of the ralstonia solanacearum and a composition thereof, and the bacteriophage has stronger capability of cracking the ralstonia solanacearum from different crop sources.
The invention provides a solanaceae ralstonia phage and a composition, a kit and an application thereof, and adopts the following technical scheme:
in a first aspect, the invention provides a solanaceae ralstonia phage, which adopts the following technical scheme:
a solanaceae Ralstonia phage is a solanaceae Ralstonia phage RS-P48 (Ralstonia solanacearum phase RS-P48), the preservation number is CCTCC NO: M2020748, the preservation unit is China center for type culture collection, and the preservation date is 2020, 11 and 16 days.
By adopting the technical scheme, the application provides the Ralstonia solanacearum phage RS-P48 which has excellent pH resistance and is preserved in China center for type culture collection with the preservation number of CCTCC NO: M2020748.
Preferably, the Ralstonia solanacearum phage RS-P48 is a virulent phage, has a polyhedral and three-dimensionally symmetrical head and a shorter tail, has the head diameter of 70-80 nm, the tail length of 35-40 nm and the tail diameter of 10-16 nm, and belongs to the phage of the family Autographridae.
By adopting the technical scheme, the appearance of the phage is observed in an electron microscope, so that the phage has a polyhedral three-dimensional symmetrical head and a telescopic tail, the injection of nucleic acid of the head into host bacteria is facilitated, and a special receptor on the surface of the host bacteria can be effectively identified.
Preferably, the Ralstonia solanacearum phage RS-P48 (Ralstonia solanacearum phase RS-P48) has a nucleotide sequence shown in SEQ ID NO. 1.
Preferably, the Ralstonia solanacearum phage RS-P48 (Ralstonia solanacearum phase RS-P48) is cultured for 24h under the condition of multiplicity of infection MOI =0.00000001, and the titer is 9.6 × 10 10 PFU/mL。
By adopting the technical scheme, the multiplicity of infection (MOI) is the ratio of the number of the phage to the number of bacteria, and is an important basis for researching the dose-effect relationship between phage-infected bacteria and produced phage progeny. The solanaceae ralstonia phage can infect solanaceae ralstonia to proliferate and obtain a large amount of progeny phage only by adding a small amount of the solanaceae ralstonia phage. The invention provides a high-quality phage strain source for the industrial production of phage bactericides.
Preferably, the Ralstonia solanacearum phage RS-P48 (Ralstonia solanacearum phase RS-P48) is tolerant at pH = 3-12, and the titer does not decrease more than 4 orders of magnitude within 96 h.
By adopting the technical scheme, the solanaceae ralstonia phage RS-P48 has excellent tolerance at pH3 or pH12, namely under acidic or alkaline conditions, and can play an effective control role when the control environment is between pH =3 and 12.
Preferably, the titer of the Ralstonia solanacearum bacteriophage RS-P48 is 10 at the temperature of 55-75 DEG C 6 PFU/mL or more, and still has 10 after being treated for 2h at 75 DEG C 6 A titer of PFU/mL or greater.
In a second aspect, the invention provides a composition of a Ralstonia solanacearum bacteriophage, which adopts the following technical scheme: a composition of a Ralstonia solanacearum phage, comprising a Ralstonia solanacearum phage RS-P48 (Ralstonia solanacearum phase RS-P48).
Preferably, the composition further comprises other solanaceous ralstonia phages. The other solanaceae Ralstonia phages are any one or more of solanaceae Ralstonia phages GP1 (Ralstonia solanacearum phage GP 1), solanaceae Ralstonia phages GP2 (Ralstonia solanacearum phage GP 2) and solanaceae Ralstonia phages GP3 (Ralstonia solanacearum phage GP 3); the mixture ratio of the components of each composition is equivalent-valence equivalent-volume ratio, and the final concentration of each composition is 10 6 PFU/mL。
Wherein, the preservation number of the Ralstonia solanacearum phage GP1 (Ralstonia solanacearum phage GP 1) is CCTCC NO: M2016633; solanaceae Ralstonia solanacearum phage GP2 (Ralstonia solanacearum phage GP 2) with a preservation number of CCTCC NO: M2016634; the solanaceae Ralstonia solanacearum phage GP3 (Ralstonia solanacearum phage GP 3) has a preservation number of CCTCC NO: M2016635.
As one embodiment of the invention, the Ralstonia solanacearum bacteriophage RS-P48 and other Ralstonia solanacearum bacteriophages are used together as a composition to obtain better killing effect on target bacteria. By way of illustration, the proportional relationship between the Ralstonia solanacearum bacteriophage RS-P48 and other bacteriophages can be determined by a person skilled in the art in connection with the present invention and the actual field of application and general knowledge in the field.
Preferably, the composition further comprises a bactericide. The bactericide is 1200 times of the copper hydroxide water dispersible granule.
As an embodiment of the present invention, the above-mentioned Ralstonia solanacearum bacteriophage RS-P48 and a chemical fungicide are used in combination as a composition. As an exemplary illustration, the proportional relationship between the Ralstonia solanacearum bacteriophage RS-P48 and 1200 times of the copper hydroxide water dispersible granule can be determined by the person skilled in the art by combining the application and the practical application field and the common general knowledge in the field.
Wherein, the used copper hydroxide water dispersible granule (effective component and content thereof: copper hydroxide 46%) is prepared by the following steps: dupont, USA.
In a third aspect, the invention provides a reagent or a kit for a solanaceae ralstonia phage or a solanaceae ralstonia phage composition, which adopts the following technical scheme:
a reagent or kit for a Ralstonia solanacearum phage, the reagent or kit comprising Ralstonia solanacearum phage RS-P48.
A reagent or kit of a composition of a Ralstonia solanacearum bacteriophage contains a composition of a Ralstonia solanacearum bacteriophage RS-P48 (Ralstonia solanacearum phage RS-P48).
Through the technical scheme, the solanaceae ralstonia phage is applied to the rapid detection of solanaceae ralstonia, and the detection comprises but is not limited to the detection of the solanaceae ralstonia in the forms of test paper, reagents, test paper boxes and the like, or the screening of target pathogenic bacteria in clinical samples is carried out, so that the detection sensitivity is effectively ensured.
The solanaceae ralstonia phage RS-P48 can be used for preparing a composition, a reagent or a kit, is applied to the rapid detection of solanaceae ralstonia, and comprises but is not limited to the detection of the solanaceae ralstonia in a target sample in the form of the reagent, the kit and the like, or the screening of target pathogenic bacteria in a clinical sample, so that the detection sensitivity is effectively ensured.
In a fourth aspect, the invention provides an application of a Ralstonia solanacearum bacteriophage, which adopts the following technical scheme:
application of a Ralstonia solanacearum phage RS-P48 (Ralstonia solanacearum phase RS-P48) in 10 3 PFU/mL～10 6 Within the titer range of PFU/mL, the killing rate to the Ralstonia solanacearum reaches more than 94 percent.
In a fifth aspect, the invention provides an application of a composition of a Ralstonia solanacearum bacteriophage, which adopts the following technical scheme:
the application of the composition of the solanaceae ralstonia phage RS-P48 is used as an effective component of a biological disinfectant, a biological medicament or a biological pesticide to prevent but not limit bacterial diseases caused by the solanaceae ralstonia.
By adopting the technical scheme, the solanaceae ralstonia phage RS-P48 and the composition thereof can be used for treating and preventing bacterial infection caused by solanaceae ralstonia, can be applied to various products in the aspects of detection, disinfection, plant protection and the like and applied to industry, and can also be used as biological disinfectants, biological medicaments or biological pesticides for preventing and treating diseases caused by solanaceae ralstonia.
The solanaceae ralstonia phage RS-P48 and the composition thereof can be used as an effective component of various products for environmental disinfection, for example, the solanaceae ralstonia phage RS-P48 and the composition thereof can be used for disinfecting and decontaminating water distribution systems, irrigation facilities, aquaculture facilities, public and private facilities or other environmental surfaces in the forms of liquid soaking, spraying, combined use with aqueous carriers and the like, and can effectively control the growth and activity of target bacteria; liquid immersion, spray forms including but not limited to detergents, disinfectants, decontaminants, and the like; aqueous carriers include, but are not limited to, phosphate buffered saline, TSB medium, LB medium, chloride free water, and the like.
In conclusion, the invention has the following beneficial effects:
1. the solanaceae ralstonia phage RS-P48 is a virulent phage separated from nature, has high tolerance to pH, is suitable for different control environments, and can play a good biological control effect on eucalyptus bacterial wilt;
2. the inventive Ralstonia solanacearum bacteriophage RS-P48 reaches 10 in 24h of culture 10 A titer of PFU/mL or greater;
3. the solanaceae ralstonia phage RS-P48 has good stability, has tolerance under the condition that the pH = 3-12, and has titer reduction not more than 4 orders of magnitude within 96 h;
4. the concentration of the solanaceae Ralstonia phage RS-P48 and the composition thereof in the liquid culture medium is 10 3 PFU/mL of the Ralstonia solanacearum has good killing capacity; when the concentration of the Ralstonia solanacearum bacteriophage RS-P48 is more than or equal to 1 x 10 3 When PFU/mL, the killing rate of the bacteria to the Ralstonia solanacearum reaches more than 94 percent.
Drawings
FIG. 1 is a schematic representation of plaques of the Ralstonia solanacearum bacteriophage RS-P48 prepared in example 1;
FIG. 2 is a schematic diagram showing the results of electron microscopy of the Ralstonia solanacearum phage RS-P48 prepared in example 1;
FIG. 3 is an SDS-PAGE analysis chart of the Ralstonia solanacearum phage RS-P48 prepared in example 1.
Detailed Description
The invention is described in further detail below with reference to the accompanying figures 1 to 3 and examples.
In the following examples, the reference numbers of the strains are the nomenclature numbers of the company.
The solanaceae Ralstonia solanacearum phage RS-P48 (Ralstonia solanacearum phase RS-P48) has the preservation number of CCTCC NO: M2020748, the preservation unit is China center for type culture collection, and the preservation date is 2020, 11 and 16 days.
The solanaceae Ralstonia solanacearum phage GP1 (Ralstonia solanacearum phase GP 1) has the preservation number of CCTCC NO: M2016633, the preservation unit is China center for type culture Collection, and the preservation date is 2016, 11 and 10 days.
The solanaceae Ralstonia solanacearum phage GP2 (Ralstonia solanacearum phase GP 2) has the preservation number of CCTCC NO: M2016634, the preservation unit is China center for type culture Collection, and the preservation date is 2016, 11 and 10 days.
The solanaceae Ralstonia solanacearum phage GP3 (Ralstonia solanacearum phase GP 3) has the preservation number of CCTCC NO: M2016635, the preservation unit is China center for type culture Collection, and the preservation date is 2016, 11 and 10 days.
The address of the depository: wuhan university in Wuhan, china
Pseudomonas solanacearum (Smith) Smith, with a deposit number GDMCC 1.79, is available for purchase by contact with a depository.
In the following examples, the following examples are given,
the formula of the TSB liquid culture medium is as follows: 15g of tryptone, 5g of soybean peptone, 5g of sodium chloride and 1000mL of distilled water;
the formula of the TSA solid culture medium is as follows: 15g of tryptone, 5g of soybean peptone, 5g of sodium chloride, 15g of agar and 1000mL of distilled water;
TSA plate: sterilizing a TSA solid culture medium, pouring the sterilized TSA solid culture medium on an aseptic flat plate, and cooling and solidifying to prepare the TSA flat plate;
the formula of the TSB semisolid agar culture medium is as follows: 15g of tryptone, 5g of soybean peptone, 5g of sodium chloride, 7g of agar and 1000mL of distilled water;
SM liquid formula: 5.8g of sodium chloride, 2g of magnesium sulfate, 1mol/L of Tris-HCl 50mL, 0.25g of gelatin and 1000mL of distilled water.
The LB liquid culture medium has the formula: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, 1000mL of distilled water and pH 7.0.
The copper hydroxide water dispersible granule (effective component and content thereof: 46 percent of copper hydroxide) is prepared by the following steps: dupont, USA.
Example 1: isolation preparation and purification culture of a Raylella phage RS-P48 of Solanaceae A source sample of the Raylella phage RS-P48 of Solanaceae of the present invention was collected from soil near Touhua village of Jianning district, nanjing, jiangsu province, 30g of the sample was soaked in 100mL of sterilized 0.9% NaCl, overnight at 4 ℃ in a refrigerator, filtered through double-layer filter paper, centrifuged at 4000rpm at normal temperature to obtain a supernatant, and the supernatant was filtered through a 0.22 μm filter membrane.
Separation of phage:
(1) Taking 10mL of filtered supernatant, adding into 10mL of 2-time TSB liquid culture medium, simultaneously adding 1mL of phage host bacterium, namely, ralstonia solanacearum RS-20 log-phase bacterium liquid, and placing at 30 ℃ for overnight culture;
(2) Centrifuging the above culture at 8000rpm for 10min, and filtering the supernatant with 0.22 μm filter membrane;
(3) 0.5mL of phage host bacterium Ralstonia solanacearum RS-20 logarithmic phase bacterial liquid is added into 5mL of TSB semisolid agar medium at 40 ℃ and mixed evenly, and poured on a TSA flat plate to prepare a double-layer flat plate containing the host bacterium;
(4) Taking 10 mu L of the prepared supernatant, dripping the supernatant on a solidified double-layer plate, air-drying the plate under the aseptic condition, and then placing the plate at 30 ℃ for overnight culture to form phage spot spots.
And (3) purifying the phage:
(1) Picking the bacteriophage spot with a toothpick, transferring to 1mL of SM liquid, and shaking for 1min;
(2) Performing 10-fold gradient dilution, and taking 10 2 、10 4 And 10 6 Adding 0.5mL of phage host bacteria log-phase bacterial liquid into the diluent respectively, and mixing uniformly;
(3) Standing for 15min, adding the mixed solution into 5mL of TSB semisolid agar culture medium at 40 ℃, pouring the mixed solution onto a TSA plate immediately after mixing uniformly, shaking and flatly placing for 5min, observing after solidifying and placing the plate in a 30 ℃ incubator overnight culture, and obtaining a double-layer plate containing single plaques;
(4) Picking up a single plaque, transferring the plaque into 1mL of SM solution, purifying the plaque for at least more than 3 times according to the method, and finally forming the plaque with consistent shape and size on a flat plate;
(5) Picking single plaques with consistent shapes and sizes by using toothpicks, placing the single plaques in 50mL of TSB liquid culture medium containing 1mL of logarithmic phase host bacterium liquid, and shaking at 180rpm at 30 ℃ overnight;
(6) Centrifuging the culture at 8000rpm for 10min, and filtering the supernatant with 0.22 μm filter membrane to obtain purified phage solution, i.e. Ralstonia solanacearum phage RS-P48. The Ralstonia solanacearum phage RS-P48 (Ralstonia solanacearum phase RS-P48) produced a single circular plaque on the Ralstonia solanacearum lawn, see FIG. 1. The solanaceae Ralstonia solanacearum phage RS-P48 (Ralstonia solanacearum phase RS-P48) has a preservation number of CCTCC NO: M2020748.
Example 2: electron microscope observation of solanaceae ralstonia phage RS-P48
The phage RS-P48 solution prepared in example 1 was used for electron microscopy: and (3) dropping a 20 mu L sample on a copper mesh, naturally precipitating for 15min, absorbing the redundant liquid from the side by using filter paper, adding 1 drop of 2% phosphotungstic acid on the copper mesh, dyeing for 10min, absorbing the dye solution from the side by using the filter paper, drying, and observing by using an electron microscope.
As a result, as shown in FIG. 2, it was found that the form of the phage RS-P48 of Ralstonia solanacearum observed under an electron microscope had a polyhedral stereosymmetric head and a long tail, the head diameter was 70 to 80nm, the tail length was 35 to 40nm, and the tail diameter was 10 to 16nm. The Ralstonia solanacearum bacteriophage RS-P48 belongs to the family of Autographiviridae.
Example 3: preparation of solanaceae ralstonia phage RS-P48 particles and extraction and sequencing of genome
(1) Taking 100mL of the phage RS-P48 solution prepared in the embodiment 1, sequentially adding 20 muL of DNaseI and 20 muL of RNaseA with the concentration of 5mg/mL, incubating at 37 ℃ for 60min, adding 5.84g of NaCl, and placing in an ice bath for 1h after dissolving;
(2) Centrifuging at 11000rpm for 10min at 4 ℃, transferring the centrifuged supernatant into a new centrifuge tube, adding solid PEG8000 to make the final concentration 10% (w/v), and carrying out ice bath for 1h after the PEG8000 is completely dissolved;
centrifuging at 11000rpm for 20min at 4 deg.C, adding 1mLSM solution to resuspend and precipitate to obtain phage particle concentrated solution, and storing at 4 deg.C.
And extracting phage nucleic acid by using a lambda phage genome DNA kit and sequencing. Through nucleotide sequencing, the solanaceae Ralstonia solanacearum phase RS-P48 has a nucleotide sequence shown in SEQ ID No. 1.
The sequence of the solanaceae Ralstonia phage RS-P48 is aligned on NCBI website to obtain the phage belonging to the autograpliidae family.
Example 4: SDS-PAGE analysis of Ralstonia solanacearum phage RS-P48 (Ralstonia solanacearum phase RS-P48)
Taking the bacteriophage RS-P48 solution prepared in the example 1, and mixing the solution according to the mass-to-volume ratio of 1:10 adding PEG8000, dissolving, ice-cooling for 3h, centrifuging at 11000rpm at 4 ℃ for 20min, discarding supernatant, and resuspending the precipitate with proper amount of sterile water to obtain the bacteriophage RS-P48 concentrated solution. Uniformly mixing 15 mu L of concentrated liquid containing phage particles with 3 mu L of 6 times of loading buffer solution, carrying out water bath at 100 ℃ for 5min to denature protein, adding the treated sample and a protein Marker into a gel loading hole, marking, carrying out electrophoresis on the sample to be detected to a junction of concentrated gel and separation gel after 90V about 100min after the power supply is switched on, then increasing the voltage to 150V, continuing the electrophoresis to the lower edge of the separation gel, stopping the electrophoresis, placing the whole piece of separation gel into a glass plate, dyeing by using a conventional Coomassie brilliant blue dyeing method, taking out and scanning after a protein band develops color, and obtaining a result shown in figure 3.
The results show that: the molecular weight standard of the protein Marker is 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp in sequence; at least 6 protein bands can be observed from the bacteriophage RS-P48, which indicates that the bacteriophage RS-P48 particle at least contains 6 structural proteins, and the main structural proteins are all proteins with molecular weight of more than about 2000 bp.
Example 5: determination of titer of solanaceae ralstonia phage RS-P48
Using SM solution as diluent, 10 times gradient of stock solution of Ralstonia solanacearum bacteriophage RS-P48 solution (prepared from example 1) was diluted to l0 8 And (4) doubling. Respectively take l0 5 、l0 6 、l0 7 And l0 8 The diluted phage culture solution (L000 μ L) was mixed with the host bacterial solution (300 μ L) and allowed to stand for 15min to allow the mixture to be fully bound to the receptors on the bacterial surface. And adding the mixed solution into 4mL of TSB semisolid agar culture medium cooled to 50 ℃, uniformly mixing, immediately paving on a solidified TSA plate, and carrying out inverted culture at 30 ℃ for 6-8 h after agar is solidified. Three replicates of each dilution were taken and counted as the average of the three replicates of that dilution. Here, phage titer (PFU/mL) = average plaque number × dilution factor.
TABLE 1 titer of Ralstonia solanacearum bacteriophage RS-P48 after 24h cultivation
As can be seen from Table 1, the R.solanacearum phage RS-P48 has high affinity and cleavage ability, and the R.solanacearum phage RS-P48 has 10 hours after being cultured for 24 hours 10 Titers above PFU/mL.
The solanaceae ralstonia phage RS-P48 can specifically and partially or completely inactivate the solanaceae ralstonia, can complete mass multiplication only by a small amount of initial phage, and provides a high-quality phage strain source for industrial production of phage bactericides.
Example 6: virulence gene or poor gene deletion detection assay for the ralstonia solanacearum bacteriophage RS-P48 this example selects 103 virulence genes identified as originating from lysogenic bacteriophages derived from pathogenic bacteria as shown in table 2, and determines whether they contain the following virulence genes by measuring the whole genome of the ralstonia solanacearum bacteriophage RS-P48 solution (prepared from example 1) and performing bioinformatics analysis on it. The results show that the solanaceae ralstonia phage RS-P48 does not contain the following virulence genes or undesirable genes, and therefore cannot encode proteins that may pose potential health risks, and there is no possibility of carrying lysogenic genes, so the solanaceae ralstonia phage RS-P48 does not affect the health of the human or animal body.
TABLE 2 major known virulence genes of lysogenic phages in pathogenic bacteria
Example 7: toxicological experiments
The experimental mice are divided into two groups (phage group and control group) at random after 20 mice with half male and female are bred adaptively for three days, each group comprises 10 mice (5 mice for male and female), and the dose of the phage group is 10 10 PFU/kg of a Ralstonia solanacearum bacteriophage RS-P48,the control group was given an equal amount of physiological saline for 15d continuously, and the experimental mice were sacrificed by neck-off and examined for visceral status.
The results of the experiment show that this dose of the Ralstonia solanacearum bacteriophage RS-P48 solution (prepared from example 1) has no effect on the daily behavior of the mice. The viscera were examined by dissection without abnormality. The solanaceae ralstonia phage RS-P48 has biological safety and can be used as a crop disease control preparation.
Example 8: determination of the optimal infection Multiplicity (MOI) of the Ralstonia solanacearum by the Ralstonia solanacearum bacteriophage RS-P48 on the Ralstonia solanacearum, a single colony of the host bacterium Ralstonia solanacearum RS-20 is picked up, inoculated into a test tube containing 3mL of TSB liquid culture medium, and subjected to shaking culture overnight under the condition of 180rpm in a shaking table at the temperature of 30 ℃ to obtain a host bacterium suspension. The host bacterial suspension is transferred to 10mL of TSB liquid culture medium according to the proportion of 1. Phage RS-P48 solution (prepared from example 1) and phage host bacteria (MOI = purified phage solution titer/phage host bacteria concentration) were added at ratios of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001, 0.00001, 0.000001, 0.0000001, 0.00000001 for MOI, respectively, and TSB liquid medium was added to make the total volume of each tube the same. Shaking and culturing at 180rpm in a shaker at 30 ℃ for 24h. Centrifuging at 10000g for 10min after the culture is finished, collecting the supernatant culture solution to obtain a bacteriophage RS-P48 pure culture solution, and measuring the titer of each treated bacteriophage by adopting a double-layer plate method. Each point was subjected to duplicate multi-tube culture and averaged to obtain the MOI producing the highest phage titer as the optimal multiplicity of infection. The experiment was repeated 3 times.
TABLE 3 potency of the Ralstonia solanacearum bacteriophage RS-P48 at different multiplicity of infection
As can be seen from Table 3, the titer of the bacteriophage RS-P48 reaches the highest 9.6X 10 under the condition of 24h culture 10 PFU/mL, its MOI =0.00000001. Indicating that a large number of proliferations can be accomplished with only a small amount of initial Ralstonia solanacearum phage. The bacteriophage RS-P48 is industrializedThe production of the phage bactericide provides a high-quality phage strain source.
Example 9: determination of pH and temperature stability of Ralstonia solanacearum bacteriophage RS-P48
9-1: stability of Ralstonia solanacearum bacteriophage RS-P48 under different pH conditions
Adding 900 μ L of TSB liquid culture medium with pH = 1-14 into sterile EP tube, placing the EP tube in thermostatic water bath at 25 deg.C, adding 00 μ L of bacteriophage RS-P48 pure culture solution (prepared in example 8) after temperature balance to make initial titer of 2.6 × 10 10 PFU/mL, standing at room temperature. Samples were taken at 1h, 4h, 8h, 24h and 96h of reaction, and the phage titer was determined by a double-layer plate method after appropriate dilution of each treated sample. The experiment was repeated 3 times.
TABLE 4 stability and alkali resistance of bacteriophage RS-P48 under different pH conditions
The results are shown in table 4, the titer of the bacteriophage RS-P48 has not changed significantly between pH =6 and 11, indicating that it has better stability under neutral, slightly acidic and alkaline conditions.
The titer of the bacteriophage RS-P48 was somewhat reduced under acidic conditions at pH =3 and basic conditions at pH =12, but the titer was reduced by about 3 to 4 orders of magnitude compared to pH =7, indicating better tolerance under acidic and basic conditions. Phage RS-P48 still had 10 after 4 hours of treatment under alkaline conditions of pH =13 4 Titer of PFU/mL. Under very basic conditions of pH =14, the titer of phage RS-P48 dropped to 0 after 4 hours of treatment. Under very acidic conditions of pH =2, the titer of phage RS-P48 then dropped to 0 after 1 hour of treatment.
The solanaceae Ralstonia phage RS-P48 (Ralstonia solanacearum phase RS-P48) is tolerant at pH = 3-12, and the titer does not decrease by more than 4 orders of magnitude within 96 h.
9-2: stability of Ralstonia solanacearum bacteriophage RS-P48 under different temperature conditions
Each of the obtained solutions had a titer of 3.7X 10 of 00. Mu.L 8 PFU/mL phage RS-P48 pure culture medium (prepared in example 8) was filled in a sterile EP tube and placed in a water bath at 55 deg.C, 65 deg.C, and 75 deg.C for 2h, 24h, and 48h, respectively. After the action time is over, the sample tube is taken out and immediately placed in an ice bath for cooling, and the phage titer is measured by adopting a double-layer plate method after the sample tube is properly diluted. The experiment was repeated 3 times.
TABLE 5 potency of Ralstonia solanacearum phage RS-P48 at different temperatures
As shown in Table 5, the titer of the panel treated with the Ralstonia solanacearum bacteriophage RS-P48 at 55 ℃ for 48 hours was unchanged. The Ralstonia solanacearum bacteriophage RS-P48 has good high temperature resistance, and the titer is 10 at the temperature of 55-75 DEG C 6 PFU/mL or more; compared with the control, the survival rate of the water bath at 65 ℃ for 2h is still more than 80%, and the survival rate of the water bath at 75 ℃ for 2h is still 10 6 PFU/mL titer.
Example 10: test of splitting capacity of solanaceous Ralstonia phage RS-P48 on solanaceous Ralstonia of different crop disease sources
The lysis spectrum of the phage was determined by double-layer plate titration. Respectively selecting 132 single colonies of the ralstonia solanacearum separated from bacterial wilt focuses of crops such as pumpkins, ginseng fruits, cablin potchouli herb, balsam pears, swamp cabbage, strawberries, chrysanthemum, eucalyptus, sunflowers, plukenetia volubilis and sesames, inoculating the single colonies into a test tube containing 3mL of TSB liquid culture medium, and culturing at 30 ℃ and 180rpm overnight to obtain bacterial liquids of the strains. mu.L of the bacterial suspension was mixed with TSB semi-solid agar medium and spread on a common agar plate, 5. Mu.L of bacteriophage RS-P48 solution (prepared in example 1) was dropped on the plate, air-dried naturally and cultured overnight at 30 ℃ to observe the results.
TABLE 6 lysis results of Ralstonia solanacearum bacteriophage RS-P48 on Ralstonia solanacearum from different crop disease sources
Note: "+ + + +" is completely clear; "+ +" is medium and clear; "+" is slightly clear; uncracked is "-".
The results are shown in Table 6, the bacteriophage RS-P48 has strong cracking capability on the solanaceae Ralstonia with different crop disease sources, and the cracking rate can reach 94.7%.
The Ralstonia solanacearum phage RS-P48 is a strict virulent phage, has high specificity and schizolysis to host bacteria, and has a wide host range, and the Ralstonia solanacearum phage RS-P48 has a wide schizolysis spectrum.
Example 11: in a splitting test of the solanaceae ralstonia phage RS-P48 on nonpathogenic beneficial bacteria, 8 nonpathogenic rhizobia, 8 nonpathogenic bacillus subtilis, 7 nonpathogenic bacillus megaterium and 7 nonpathogenic bacillus licheniformis are respectively inoculated into a test tube containing 3mL of TSB liquid culture medium, and are cultured for 8 hours at 30 ℃ and 180rpm to prepare bacterial liquid of each strain. 300 μ L of each bacterial suspension was mixed with TSB semi-solid agar medium and plated on common agar plates. mu.L of each phage RS-P48 solution (prepared in example 1) was dropped on the plate, naturally dried, and then cultured at 30 ℃ for 24 hours, and the results were observed.
TABLE 7 lysis test of the Ralstonia solanacearum bacteriophage RS-P48 against non-pathogenic, beneficial bacteria
Note: "+ + + +" is completely clear; "+ +" is medium and clear; "+" is slightly clear; the non-cleaved is "-".
As shown in Table 7, in this example, none of the 30 non-pathogenic bacteria were recognized by the bacteriophage RS-P48. The test phage has extremely strong host specificity and has no damage to microbial communities.
The interaction of the solanaceae Ralstonia phage RS-P48 and the non-host pathogenic bacteria can not identify any one of 30 tested non-host pathogenic bacteria, and the specificity is good.
Example 12: the sterilizing effect of the Ralstonia solanacearum bacteriophage RS-P48 in the liquid is used for culturing the Ralstonia solanacearum RS-20 to the logarithmic phase, the Ralstonia solanacearum RS-20 is subpackaged into different test tubes, the TSB liquid culture medium with the same volume is used for diluting the bacterial liquid until the final concentration of the Ralstonia solanacearum RS-20 is 2.3 multiplied by 10 3 cfu/mL. Respectively inoculating to the cells with final concentration of 1 × 10 2 PFU/mL、1×10 3 PFU/mL，1×10 4 PFU/mL、1×10 5 PFU/mL、1×10 6 PFU/mL phage RS-P48 pure medium (prepared in example 8). Setting a control group and a blank group at the same time, wherein the control group is given a final concentration of 2.3 × 10 3 cfu/mL of Ralstonia solanacearum RS-20; the blank group was given an equal amount of physiological saline. The treatments were cultured with shaking at 150rpm at 30 ℃ for 4 hours, and the residual amount of the Ralstonia solanacearum was measured. The detection method comprises the following steps: each treated sample was diluted with sterile water, 100. Mu.L of the diluted solution was applied to a TSA plate, and the number of colonies on the plate was counted after incubation at 30 ℃ for 24 hours. Number of ralstonia solanacearum = number of colonies on TSA plate × dilution multiple × 10.
TABLE 8 Sterilization Effect of Ralstonia solanacearum bacteriophage RS-P48 in liquids at different concentrations
As is clear from Table 8, the final concentration of the Ralstonia solanacearum phage RS-P48 was 1X 10 2 Under the condition of PFU/mL, the growth of the Ralstonia solanacearum RS-20 in the liquid culture medium can be well controlled; the concentration of the Ralstonia solanacearum bacteriophage RS-P48 is 10 3 PFU/mL of the Ralstonia solanacearum has good killing capacity; when the concentration of the Ralstonia solanacearum bacteriophage RS-P48 is more than or equal to 1 multiplied by 10 3 When PFU/mL, the killing rate of the compound to the ralstonia solanacearum reaches more than 94 percent, and the compound has no antagonism to other combined substances; when the final concentration of the Ralstonia solanacearum phage RS-P48 is more than or equal to 1 x 10 4 When PFU/mL, the killing rate of the compound can reach more than 99 percent to the Ralstonia solanacearum.
Example 13: preparation of composition of Ralstonia solanacearum bacteriophage RS-P48
Preparation of stock solution of Ralstonia solanacearum phage GP1 (Ralstonia solanacearum phase GP 1): inoculating Ralstonia solanacearum phage GP1 host bacteria into a conical flask containing 500mL LB liquid culture medium, shaking and culturing at 150rpm at 30 ℃ until OD value is 0.1, adding 1mL of 1 × 10 titer 4 PFU/mL Ralstonia solanacearum phage GP1 (Ralstonia solanacearum phase GP 1) was shake cultured at 30 ℃ for 24h at 200 rpm. Centrifuging the fermentation liquid at 8000rpm for 15min, and filtering the supernatant with 0.22 μm filter membrane to obtain stock solution of bacteriophage GP1 of Ralstonia solanacearum.
Preparation of stock solution of Ralstonia solanacearum phage GP2 (Ralstonia solanacearum phage GP 2): inoculating Ralstonia solanacearum phage GP2 host bacteria into a conical flask containing 500mLLB liquid culture medium, performing shake culture at 30 ℃ and 150rpm until OD value is 0.1, adding 1mL of 1 × 10 titer 4 PFU/mL Ralstonia solanacearum phage GP2 (Ralstonia)solanacerum phase GP 2), incubated at 30 ℃ for 24h with shaking at 200 rpm. Centrifuging the fermentation liquid at 8000rpm for 15min, and filtering the supernatant with 0.22 μm filter membrane to obtain stock solution of bacteriophage GP2 of Ralstonia solanacearum.
Preparation of stock solution of Ralstonia solanacearum phage GP3 (Ralstonia solanacearum phage GP 3): inoculating Ralstonia solanacearum phage GP3 host bacteria into a conical flask containing 500mL of LB liquid culture medium, performing shake culture at 150rpm at 30 ℃ until OD value is 0.1, and adding 1mL of host bacteria with titer of 1 × 10 4 PFU/mL Ralstonia solanacearum phage GP3 (Ralstonia solanacearum phase GP 3) was shake cultured at 30 ℃ for 24h at 200 rpm. Centrifuging the fermentation liquid at 8000rpm for 15min, and filtering the supernatant with 0.22 μm filter membrane to obtain stock solution of bacteriophage GP3 of Ralstonia solanacearum.
Respectively taking out the titer of 1 × 10 9 PFU/mL of a purified culture solution of a Ralstonia solanacearum phage RS-P48 (prepared in example 8) and a stock solution of a Ralstonia solanacearum phage GP1 (prepared in example 1) were mixed uniformly in equal volumes with 2 phage to prepare a composition of a Ralstonia solanacearum phage RS-P48 (Ralstonia solanacearum phage RS-P48) and a Ralstonia solanacearum phage GP1 (Ralstonia solanacearum phage GP 1) (composition 1).
Respectively taking out the titer of 1 × 10 9 PFU/mL of a purified culture solution of a Ralstonia solanacearum phage RS-P48 (prepared in example 8) and a stock solution of a Ralstonia solanacearum phage GP2 (prepared in example 2) were mixed uniformly in equal volumes to prepare a composition of a Ralstonia solanacearum phage RS-P48 (Ralstonia solanacearum phage RS-P48) and a Ralstonia solanacearum phage GP2 (Ralstonia solanacearum phage GP 2) (composition 2).
Respectively taking the titer of 1 multiplied by 10 9 PFU/mL pure culture solution of Ralstonia solanacearum phage RS-P48 (obtained in example 8) and stock solution of Ralstonia solanacearum phage GP3 (obtained in example 3) were mixed uniformly in equal volumes to prepare Ralstonia solanacearum phage RS-P48 (Ralstonia solanacearum phage RS-P48) and Ralstonia solanacearum phage GP3 (obtained in example 3)phase GP 3) (composition 3).
Respectively taking out the titer of 1 × 10 9 PFU/mL of a pure culture solution of a Ralstonia solanacearum phage RS-P48 (produced in example 8), stock solutions of a Ralstonia solanacearum phage GP1 (RAlstonia solanacearum phage GP 1) and a Ralstonia solanacearum phage GP2 (Ralstonia solanacearum phage GP 2) were uniformly mixed in equal volumes to prepare a composition (composition 4).
Respectively taking out the titer of 1 × 10 9 PFU/mL of a pure culture solution of a Ralstonia solanacearum phage RS-P48 (produced in example 8) and stock solutions of Ralstonia solanacearum phage GP1 (RAlstonia solanacearum phage GP 1) and Ralstonia solanacearum phage GP3 (Ralstonia solanacearum phage GP 3) were uniformly mixed in equal volumes to prepare a composition (composition 5).
Respectively taking out the titer of 1 × 10 9 PFU/mL of a pure culture solution of a Ralstonia solanacearum phage RS-P48 (prepared in example 8), stock solutions of a Ralstonia solanacearum phage GP1 (Ralstonia solanacearum phage GP 1), a Ralstonia solanacearum phage GP2 (Ralstonia solanacearum phage GP 2) and a Ralstonia solanacearum phage GP3 (Ralstonia solanacearum phage GP 3), and 4 phages were uniformly mixed in equal volumes to prepare a composition (composition 6).
Respectively taking the titer of 1 multiplied by 10 9 PFU/mL pure culture solution of Ralstonia solanacearum phage RS-P48 (prepared in example 8) and copper hydroxide water dispersible granules, 2 of the pure culture solutions and the copper hydroxide water dispersible granules are uniformly mixed in equal volumes to prepare a composition, and the final application concentration of the copper hydroxide water dispersible granules in the composition is 1200 times of the solution (composition 7).
The combination of the embodiment is not limited to 1200 times of the copper hydroxide water dispersible granule, and may also be chemical bactericides such as polyoxin, flumorph, dimethomorph, prochloraz, difenoconazole, flusilazole, myclobutanil, mancozeb, thiophanate-methyl, carbendazim, chlorothalonil, fructosan, and amobam 700 times of the liquid. The embodiment can also be used together with a chemical disinfectant to achieve the effect of preventing and killing.
Example 14: the bactericidal effect of the composition containing the bacteriophage RS-P48 of Ralstonia solanacearum in the liquid the compositions 1 to 7 containing the bacteriophage RS-P48 of Ralstonia solanacearum, and the preparation method thereof is shown in example 13.
Culturing Ralstonia solanacearum RS-20 to logarithmic phase, subpackaging into different test tubes, diluting with TSB liquid culture medium to make the final concentration of Ralstonia solanacearum RS-20 be 2 × 10 3 cfu/mL, to which the final concentration of 1X 10 prepared in example 13 was respectively added 6 PFU/mL of compositions 1 to 7, and the titer of each phage was equal in compositions 1 to 7. A control group and a blank group (CK) were simultaneously prepared, and the control group was administered at a final concentration of 2X 10 3 cfu/mL of Ralstonia solanacearum RS-20; the blank group was given an equal amount of physiological saline. The treatments were cultured with shaking at 150rpm at 30 ℃ for 4 hours, and the residual amount of the Ralstonia solanacearum was measured. The detection method comprises the following steps: each treated sample was diluted with sterile water, 100. Mu.L of the diluted solution was applied to a TSA plate, and the number of colonies on the plate was counted after incubation at 30 ℃ for 24 hours. Number of ralstonia solanacearum = number of colonies on TSA plate × dilution multiple × 10.
TABLE 9 Bactericidal Effect of compositions containing Ralstonia solanacearum bacteriophage RS-P48 in liquid
As is clear from Table 9, the final concentration of the composition containing the Ralstonia solanacearum phage RS-P48 was 1X 10 6 When PFU/mL, the various compositions have good killing effect on the Ralstonia solanacearum. The result shows that the solanaceae ralstonia phage RS-P48 can be used together with other substances for preventing and treating bacteria, and has no antagonism on other substances.
Example 15: controlling eucalyptus seed for bacterial wilt of eucalyptus by using Ralstonia solanacearum bacteriophage RS-P48 and its composition by 0.1% 2 The surface is sterilized for 5 minutes and washed clean with sterile water. Uniformly sowing the seeds on sterile soil contained in a large sterile culture dish, covering sterilized river sand with the thickness of about 3mm on the seeds, pouring sterile distilled water until the soil is thoroughly wetted,and (3) covering a culture dish cover, keeping the relative humidity above 90%, and illuminating for 12 hours every day, wherein the temperature in the day and at night are respectively about 30 ℃ and 25 ℃. And after the seeds germinate, uncovering the culture dish cover, and placing the culture dish cover in an environment with the relative humidity of 70 percent and the temperature of 25-28 ℃ and 20-23 ℃ in the daytime and at night respectively for continuous culture.
When the nursery stock grows to 5-6 cm, the substrate is washed by sterile water, the nursery stock is cultivated in water at room temperature for 3-5 d, 420 tissue culture seedlings with good growth vigor are taken and randomly divided into 7 groups (a phage 3 group, a composition 6, a composition 7, a control group and a blank group), and 60 seedlings in each group are slightly wounded with roots by using a sterile scalpel.
Placing the roots of 6 eucalyptus groups except the blank group at a final concentration of 1 × 10 5 Soaking cfu/mL pseudomonas solanacearum GDMCC 1.79 in the bacterial liquid for 30 minutes; the roots of the blank group of eucalyptus trees are soaked in the same amount of sterile distilled water for 30 minutes.
Each phage group: the phage RS-P48 pure culture solution (prepared in example 8) was added to sterile soil to make the soil contain phage concentrations 1X 10 4 PFU/g、1×10 5 PFU/g、1×10 6 PFU/g；
Composition 6 group: the liquid composition 6 (prepared in example 13) was added to sterile soil to give a soil phage concentration of 1X 10 6 PFU/g；
Composition 7 group: the liquid composition 7 (prepared in example 13) was added to sterile soil to give a soil phage concentration of 1X 10 6 PFU/g。
After the soil and the phage liquid are uniformly mixed, the mixture is loaded into small aseptic bowls according to the amount of 90g per bowl, 10mL of sterile distilled water is added into a chassis of each small bowl, and after the sterile distilled water is completely infiltrated into the soil, the groups of soaked seedlings are transplanted into the small bowls.
And (3) filling sterile soil into small sterile pots according to the amount of 90g per pot, adding 10mL of sterile distilled water into a chassis of each small pot, and transplanting the seedlings soaked in the control group and the blank group into the small pots after the sterile distilled water completely permeates into the soil.
The transplanted seedlings of the groups are cultured under the conditions of 30-33 ℃ in the daytime, 25-28 ℃ at night and 90% humidity. Morbidity was recorded at 2, 5, 8, 10, 12, 15, 18, 20, 22, 25, 28, 30d post inoculation, respectively. Incidence = number of diseased plants/total plants × 100%.
TABLE 10 effects of Ralstonia solanacearum bacteriophage RS-P48 and compositions thereof on eucalyptus bacterial wilt
As can be seen from table 10, the incidence of bacterial wilt of eucalyptus was 91% in the control group of eucalyptus plants after 30 days of inoculation. The composition 6 has the best effect of preventing and treating the eucalyptus bacterial wilt, and the incidence rate of the eucalyptus is 19% after 30 days of the experiment; the composition was divided into 7 times. In each experimental group of the phage, the higher the concentration of the phage RS-P48 is, the lower the incidence rate of eucalyptus is; the concentration of the bacteriophage RS-P48 is 10 6 The incidence of eucalyptus is kept within 26% after the experiment for 30d at the PFU/g. The solanaceae ralstonia phage and the composition thereof can be used as a biological bactericide to effectively prevent and treat the eucalyptus bacterial wilt.
Example 16: preparation of reagent or kit of Ralstonia solanacearum phage RS-P48 (Ralstonia solanacearum phage RS-P48) and composition thereof, and content of 5-10 mL of 1 × 10 titer in used reagent or kit 7 PFU/mL of a composition of Salmonella phage, ralstonia solanacearum phase RS-P48, or Ralstonia solanacearum phase RS-P48, 1L of TSB liquid medium, 1L of TSB semi-solid agar medium, 1L of TSA solid medium; among them, the solanaceae Ralstonia solanacearum phage RS-P48 (Ralstonia solanacearum phage RS-P48) is the phage RS-P48 solution prepared in example 1, or is the phage RS-P48 pure culture solution prepared in example 8.
The use method of the reagent or the kit comprises the following steps: taking out the titer of 1 × 10 7 PFU/mL of a Ralstonia solanacearum phage RS-P48 (Ralstonia solanacearum phage RS-P48) phage liquid or a Ralstonia solanacearum phage RS-P48 (Ra)lstonia solanacearum phase RS-P48) composition 1-composition 6 (prepared in example 13) lysis profiles of test phages were determined using a double plate titration method. And (3) selecting a single bacterial colony to be detected, inoculating the single bacterial colony to a target liquid culture medium, and performing shake culture at a target temperature in combination with the growth characteristics of the bacterial strain to be detected to obtain a bacterial liquid of the bacterial strain to be detected. 300 mu L of bacterial suspension of the strain to be detected is mixed with 5mL of TSB semisolid culture medium and is paved on a TSA plate, and 10 mu L of liquid of the solanaceae Ralstonia solanacearum phase RS-P48 or the composition of the solanaceae Ralstonia solanacearum phase RS-P48 is dripped on the plate. And after natural air drying, culturing at a target temperature according to the growth characteristics of the strain to be detected, and observing the result.
The solanaceae ralstonia phage RS-P48 can be used for preparing a composition, a reagent or a kit, is applied to the rapid detection of the solanaceae ralstonia, and comprises but is not limited to the detection of the solanaceae ralstonia in a target sample in the form of the reagent, the kit and the like, or the screening of target pathogenic bacteria in a clinical sample, so that the detection sensitivity is effectively ensured.
The embodiments of the present invention are preferred embodiments of the present invention, and the scope of the present invention is not limited by these embodiments, so: all equivalent changes made according to the structure, shape and principle of the invention are covered by the protection scope of the invention.
Claims (8)
1. A Ralstonia solanacearum bacteriophage characterized by: the solanaceae Ralstonia phage is solanaceae Ralstonia solanacearum phage RS-P48 (Ralstonia solanacearum phage RS-P48), and the preservation number is CCTCC NO: M2020748.
2. The bacteriophage of claim 1, wherein said bacteriophage is selected from the group consisting of: the titer of the Ralstonia solanacearum bacteriophage RS-P48 is more than 106PFU/mL under the condition of 55-75 ℃.
3. A composition comprising the ralstonia solanacearum bacteriophage RS-P48 of claim 1 or 2.
4. The composition of claim 3, wherein: the composition also comprises any one or more of solanaceae Ralstonia phage GP1 (Ralstonia solanacearum phase GP 1), solanaceae Ralstonia phage GP2 (Ralstonia solanacearum phase GP 2) and solanaceae Ralstonia phage GP3 (Ralstonia solanacearum phase GP 3); wherein, the solanaceae Ralstonia solanacearum phage GP1 (Ralstonia solanacearum phase GP 1) has a preservation number of CCTCC NO: M2016633; solanaceae Ralstonia solanacearum phage GP2 (Ralstonia solanacearum phase GP 2) with the preservation number of CCTCC NO: M2016634; the solanaceae Ralstonia solanacearum phage GP3 (Ralstonia solanacearum phase GP 3) with the preservation number of CCTCC NO: M2016635.
5. The composition of claim 3, wherein: the composition also includes a bactericide.
6. A reagent or kit comprising the Ralstonia solanacearum bacteriophage RS-P48 of claim 1 or 2 or a composition comprising the Ralstonia solanacearum bacteriophage RS-P48 of any one of claims 3 to 5.
7. Use of the ralstonia solanacearum bacteriophage RS-P48 according to claim 1 or 2 for killing ralstonia solanacearum, characterized in that: the titer of the Ralstonia solanacearum phage RS-P48 is 103-106 PFU/mL.
8. The application of a Ralstonia solanacearum bacteriophage composition is characterized in that: the composition of the ralstonia solanacearum bacteriophage RS-P48 as set forth in any one of claims 3 to 4, is used as an effective ingredient of disinfectants, medicaments and pesticides for preventing but not limited to bacterial diseases caused by ralstonia solanacearum.
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| CN108676778A (en) * | 2018-04-20 | 2018-10-19 | 南京农业大学 | One plant of prevention soil passes bacteriophage and its application of bacterial wilt |
| CN109136194A (en) * | 2017-06-28 | 2019-01-04 | 菲吉乐科(南京)生物科技有限公司 | Novel Ralstonia solanacearum bacteriophage and combinations thereof and application |
| CN109511681A (en) * | 2018-11-17 | 2019-03-26 | 菲吉乐科(南京)生物科技有限公司 | A kind of bacteriophage aqua preservative and its preparation method and application |
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| CN109136194A (en) * | 2017-06-28 | 2019-01-04 | 菲吉乐科(南京)生物科技有限公司 | Novel Ralstonia solanacearum bacteriophage and combinations thereof and application |
| CN108676778A (en) * | 2018-04-20 | 2018-10-19 | 南京农业大学 | One plant of prevention soil passes bacteriophage and its application of bacterial wilt |
| CN109511681A (en) * | 2018-11-17 | 2019-03-26 | 菲吉乐科(南京)生物科技有限公司 | A kind of bacteriophage aqua preservative and its preparation method and application |
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