CN112359024B - Pseudomonas syringae bacteriophage and composition, kit and application thereof - Google Patents

Abstract

The invention relates to the field of microorganisms, and particularly discloses a pseudomonas syringae bacteriophage, a composition, a kit and an application thereof, wherein the pseudomonas syringae bacteriophage is pseudomonas syringae kiwi pathopoiesia variant bacteriophage PSA-P1 with the preservation number of M2020252; the bacteriophage has higher tolerance to ultraviolet rays and pH, and can play a better role in preventing and treating in different preventing and treating environments; the composition at least comprises one bacteriophage PSA-P1; the kit contains a composition of phage PSA-P1 or phage PSA-P1; phage PSA-P1 composition for use in, but not limited to, killing Pseudomonas syringae at 10¹ PFU/mL~10⁸ Within the titer range of PFU/mL, the bacteriostasis rate of pseudomonas syringae and kiwi fruit pathogenic variant phage PSA-P1 on pseudomonas syringae reaches 54.1-94.9%.

Description

Pseudomonas syringae bacteriophage and composition, kit and application thereof

Technical Field

The invention relates to the field of bacteriophage, in particular to pseudomonas syringae bacteriophage and a composition, a kit and application thereof.

Background

Pseudomonas syringae belongs to the genus Pseudomonas of the family Pseudomonas, and has 57 pathotypes, of which Pseudomonas syringae is a causative variant of Actinidia actinidia (Psa; Pseudomonas syringae pv. actindiae). Psa has a variety of genetic genes with strong adaptability, so that the kiwi fruit tree is easy to invade and cause infection all year round.

At present, in the related technologies, on one hand, chemical prevention and control are mainly adopted, and methods such as field spraying, pus discharge wound smearing, dry injection and the like are adopted for applying the medicine, but the effect is not ideal from the existing prevention and control situation. On the other hand, with the recent national call for resistance reduction and no resistance on crops, domestic and foreign scholars actively research emerging antibacterial biological agents, and as the pseudomonas syringae bacteriophage has the specific and efficient cracking capability on target bacteria (pseudomonas syringae kiwi pathogenic variety), the quantity of pathogenic bacteria in the environment can be obviously reduced, and the occurrence and prevalence of diseases can be controlled or reduced. Therefore, the pseudomonas syringae bacteriophage can be used as an antibacterial infection preparation.

In view of the above-mentioned related art, the inventors believe that the tolerance of the phage differs in different control environments, thereby affecting the titer of the phage.

Disclosure of Invention

In order to solve the technical problem of the tolerance of the phage in different control environments, the application provides the pseudomonas syringae phage, and the composition, the kit and the application thereof.

The invention provides a pseudomonas syringae bacteriophage, and a composition, a kit and an application thereof, and adopts the following technical scheme:

in a first aspect, the invention provides a pseudomonas syringae bacteriophage, which adopts the following technical scheme:

the Pseudomonas syringae phage is Pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1(Pseudomonas syringae pv. Actinidiae phase PSA-P1), and the preservation number is CCTCC NO: m2020252.

By adopting the technical scheme, the pseudomonas syringae kiwi fruit pathopoiesia variety phage PSA-P1 has excellent ultraviolet resistance and pH resistance, is stored in China Center for Type Culture Collection (CCTCC) with the preservation number of M2020252.

Preferably, the Pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1(Pseudomonas syringae pv. actinodiae phage PSA-P1) is a virulent phage and comprises a head part and a telescopic tail part which are polyhedral and three-dimensionally symmetrical, wherein the diameter of the head part is 50-55 nm, the length of the tail part is 15-20 nm, and the diameter of the tail part is 6-10 nm, and the phage belongs to the family Autograpiviridae.

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 Pseudomonas syringae kiwi pathopoiesia variety phage PSA-P1(Pseudomonas syringae pv. Actinidiae phase PSA-P1) has a nucleotide sequence shown in SEQ ID No. 1.

Preferably, the Pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1(Pseudomonas syringae pv. actinodiae phase PSA-P1) is cultured for 24h under the condition that the MOI (multiplicity of infection) is 0.000001, and the titer reaches 7 multiplied by 10¹⁰PFU/mL or more.

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 pseudomonas syringae bacteriophage only needs to be added in a small amount, and can infect pseudomonas syringae to proliferate and obtain a large amount of progeny bacteriophage. The invention provides a high-quality phage strain source for the industrial production of phage bactericides.

Preferably, the Pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1(Pseudomonas syringae pv. actinodiae phase PSA-P1) has tolerance under the condition of pH 3-12, and the titer is reduced by no more than 4 orders of magnitude within 96 h.

By adopting the technical scheme, the pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1 has excellent tolerance under the condition of pH3 or pH12, namely under the acidic or alkaline condition, and can play an effective prevention and treatment role when the prevention and treatment environment is between pH 3-12.

Preferably, the titer of the Pseudomonas syringae kiwi pathopoiesia variety phage PSA-P1(Pseudomonas syringae pv. actinodiae phase PSA-P1) is reduced by no more than 1 order of magnitude after the Pseudomonas syringae kiwi pathopoiesia variety phage is subjected to ultraviolet radiation for 8 hours.

By adopting the technical scheme, when the ultraviolet rays in the control environment are stronger, the pseudomonas syringae kiwifruit pathopoiesia variety phage PSA-P1 has good tolerance and less titer reduction, and can play an effective control role on pathogenic bacteria.

In a second aspect, the invention provides a pseudomonas syringae bacteriophage composition, which adopts the following technical scheme:

a Pseudomonas syringae bacteriophage composition, the composition at least comprises a Pseudomonas syringae kiwi pathopoiesia phage PSA-P1(Pseudomonas syringae pv. Actinidiae phage PSA-P1).

Preferably, the composition includes a chemical germicide.

As one embodiment of the invention, the pseudomonas syringae kiwifruit pathovar phage PSA-P1 and a chemical bactericide are used in combination as a composition. By way of illustration, the proportional relationship between pseudomonas syringae and actinidia pathovar phage PSA-P1 and the 700-fold liquid of amobam can be determined by one skilled in the art in conjunction with the present application and the actual field of application and general knowledge in the art.

In a third aspect, the invention provides a pseudomonas syringae phage kit, which adopts the following technical scheme:

a Pseudomonas syringae phage kit, which contains the Pseudomonas syringae kiwi pathopoiesia phage PSA-P1(Pseudomonas syringae pv. Actinidiae phage PSA-P1) or Pseudomonas syringae kiwi pathopoiesia phage PSA-P1(Pseudomonas syringae pv. Actinidiae phagePSA-P1) composition.

Through the technical scheme, the pseudomonas syringae phage is applied to the rapid detection of pseudomonas syringae, and the detection comprises but is not limited to detection of pseudomonas syringae in the forms of test paper, test paper boxes and the like, or screening of target pathogenic bacteria in clinical samples, so that the detection sensitivity is effectively ensured.

In a fourth aspect, the invention provides an application of pseudomonas syringae bacteriophage, which adopts the following technical scheme:

the application of Pseudomonas syringae phage is that the Pseudomonas syringae kiwi pathopoiesia phage PSA-P1(Pseudomonas syringae pv. Actinidiae phase PSA-P1) is 10¹PFU/mL～10⁸The bacteriostasis rate to pseudomonas syringae reaches 54.1-94.9 percent in the titer range of PFU/mL.

In a fifth aspect, the invention provides an application of a pseudomonas syringae phage composition, which adopts the following technical scheme:

the application of the pseudomonas syringae and kiwi pathopoiesia variant phage PSA-P1 composition is used as an effective component of a biological disinfectant or a biological pesticide to prevent and control but not limit bacterial diseases caused by pseudomonas syringae.

By adopting the technical scheme, the pseudomonas syringae kiwifruit pathovar phage PSA-P1 and the composition thereof can be used for treating and preventing bacterial infection caused by pseudomonas syringae and not limited by pseudomonas syringae, and can be used as a biological agent for preventing and treating diseases caused by pseudomonas syringae and not limited by pseudomonas syringae.

In conclusion, the invention has the following beneficial effects:

1. the pseudomonas syringae kiwifruit pathogenic variety phage PSA-P1 is a virulent phage separated from nature, has higher tolerance to ultraviolet rays and pH, is suitable for different control environments, and can play a better biological control effect on kiwifruit canker;

2. the pseudomonas syringae kiwi fruit pathopoiesia variety phage PSA-P1 is a virulent phage separated from the nature, a test phage does not contain a virulence gene or a bad gene, the DNA of the phage cannot encode protein which can cause potential health risks, and the possibility of carrying a lysogenic gene does not exist;

3. the pseudomonas syringae kiwifruit pathogenic variant phage PSA-P1 has high affinity and cracking capacity, and reaches 10 in 24h of culture¹⁰A titer of PFU/mL or greater; pseudomonas syringae and kiwi fruit pathogenic variant phage PSA-P1 can specifically and partially or completely inactivate Pseudomonas syringae, and can complete massive proliferation by only a small amount of initial phage, thereby providing a high-quality phage strain source for industrial production of phage bactericides; the pseudomonas syringae kiwifruit pathovar phage PSA-P1 or the composition thereof can be prepared into various products for detection, disinfection, plant protection and the like and applied industrially by the technicians in the field according to the description of the application and the common general knowledge in the field;

4. the pseudomonas syringae kiwifruit pathogenic variant phage PSA-P1 is a strict virulent phage, has high specificity and schizolysis to host bacteria, and has a wider host range, and the schizolysis rate to 45 pseudomonas syringae reaches 91.1%; pseudomonas syringae and kiwi pathopoiesia variegate phage PSA-P1 can be used as an effective component of various products for environmental disinfection, for example, the pseudomonas syringae and kiwi pathopoiesia variegate phage PSA-P1 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 an aqueous carrier and the like, and can effectively control the growth and activity of target bacteria; the liquid soaking, spraying forms include but are not limited to detergents, disinfectants, detergents, etc.; the aqueous carrier includes but is not limited to phosphate buffer solution, TSB medium, LB medium, chlorine free water and so on;

5. the pseudomonas syringae kiwifruit pathogenic variant phage PSA-P1 interacts with non-host pathogenic bacteria, any one of 10 tested non-host pathogenic bacteria cannot be identified, and the specificity is good;

6. the pseudomonas syringae kiwifruit pathogenic variety phage PSA-P1 is good in stability, has tolerance under the condition that the pH is 3-12, and has titer reduction within 96h not more than 4 orders of magnitude; after the ultraviolet radiation is carried out for 8 hours, the titer is reduced by no more than 1 magnitude order;

7. the pseudomonas syringae kiwi pathogenic variant phage PSA-P1 can be used for preparing a composition, a reagent or a kit, is applied to the rapid detection of pseudomonas syringae, and comprises but is not limited to detection of pseudomonas syringae in a target sample in the forms of test paper, a kit and the like, or screening of target pathogenic bacteria in a clinical sample, so that the detection sensitivity is effectively ensured;

8. pseudomonas syringae kiwifruit pathovar phage PSA-P1 and its composition with concentration of 10 in liquid culture medium³PFU/mL pseudomonas syringae has good killing capacity; when the concentration of Pseudomonas syringae kiwi fruit pathogenic variant phage PSA-P1 is more than or equal to 1 × 10⁶When PFU/mL, the killing rate of the compound to pseudomonas syringae reaches more than 91.8 percent, and the compound has no antagonism to other combined substances;

9. the Pseudomonas syringae kiwifruit pathopoiesia variant phage PSA-P1(Pseudomonas syringae pv. actinodiae phase PSA-P1) and the composition thereof can be prepared into biological agents which can be applied to control diseases caused by Pseudomonas syringae and are not limited to Pseudomonas syringae by the technicians in the field according to the description of the application and the common knowledge in the field.

Drawings

FIG. 1 is a schematic representation of plaques of the present invention;

FIG. 2 is a schematic diagram of electron microscope results of the bacteriophage of the present invention;

FIG. 3 is a schematic representation of the structure of a plaque sample appearing in a lysogenic assay;

FIG. 4 is a schematic diagram of the structure of a plaque sample that did not appear in the lysogenic assay.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings 1 to 4 and examples.

In the following examples, the reference numbers of the strains are the same as the reference numbers of the same company.

Pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1(Pseudomonas syringae pv. Actinidiae phase PSA-P1), with the preservation number of CCTCC NO: m2020252, the preservation unit is China center for type culture Collection, and the preservation time is 30 days at 06 months in 2020.

The Xanthomonas campestris phage YHC5(Xanthomonas axonopodis phase YHC5) has the preservation number of CCTCC NO of M2018579, the preservation unit is China center for type culture Collection, and the preservation time is 2018, 08 months and 30 days.

Xanthomonas carpi citrus pathovar citri (Xanthomonas axonopodis pv. citri) deposit number ACCC 03526, obtained by contacting the depository with a depository.

Pseudomonas syringae Cucumis sativus var (Pseudomonas syringae pv. lachrymans), deposit No. ATCC7386, available by contact with depository.

Pseudomonas syringae tomato pathogenic variety (Pseudomonas syringae pv. tomato), deposit number ATCC BAA-871D-5, available for purchase by contact with a depository.

Pseudomonas syringae tobacco pathogenic variety (Pseudomonas syringae pv. tabaci), deposit number ATCC13453, 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 a sterile flat plate, and cooling and solidifying the TSA solid culture medium 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: 8.5g of sodium chloride, 2g of magnesium sulfate, 50mL of 1mol/LTris-HCl, 0.25g of gelatin and 1000mL of distilled water.

Example 1: isolation preparation and purification culture of pseudomonas syringae kiwi fruit pathogenic variant phage PSA-P1

The source sample of pseudomonas syringae kiwifruit pathogenic variant phage PSA-P1 is collected from mass-colored farmer market sewage in Jiangning district of Nanjing city, Jiangsu province, filtered by double-layer filter paper, centrifuged at low speed and normal temperature, and the supernatant is filtered by a filter membrane of 0.22 mu m.

Separation of phage:

(1) taking 10mL of filtered supernatant, adding the filtered supernatant into 10mL of TSB liquid culture medium with 2 times of the volume of the TSB liquid culture medium, simultaneously adding 1mL of phage host bacteria Psa-1 log phase bacterial liquid, and placing the mixture at the temperature of 28 ℃ for overnight culture;

(2) centrifuging the above culture at 8000rpm for 10min, and filtering the supernatant with 0.22 μm filter membrane;

(3) taking 0.5mL of phage host bacteria Psa-1 log-phase bacterial liquid, adding 5mL of TSB semisolid agar culture medium at 40 ℃, uniformly mixing, pouring the mixture on a TSA plate, and preparing a double-layer plate containing the host bacteria;

(4) taking 10 mu L of the prepared supernatant, dripping the supernatant on a solidified double-layer plate, air-drying the plate under a sterile condition, and placing the plate at 28 ℃ overnight for 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 1 min;

(2) performing 10-fold gradient dilution, and taking 10²、10⁴And 10⁶Adding 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 medium at 40 ℃, mixing uniformly, pouring the mixed solution onto a TSA plate, shaking uniformly and flatly for 5min, standing in a 28 ℃ incubator overnight after solidification, and observing to obtain a double-layer plate containing single plaques;

(4) picking up a single plaque, transferring the single plaque into 1mL of SM solution, purifying for at least 3 times according to the method, and finally forming plaques with consistent shapes and sizes 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 overnight at the temperature of 28 ℃;

(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. Pseudomonas syringae kiwi pathogenic variant phage PSA-P1(Pseudomonas syringae pv. Actinidiae phase PSA-P1). Pseudomonas syringae Actinidia pathovar kiwii phage PSA-P1(Pseudomonas syringae pv. Actinidiae phase PSA-P1) produced single circular plaques on Pseudomonas syringae lawn, see FIG. 1. Pseudomonas syringae kiwi fruit pathogenic variant phage PSA-P1(Pseudomonas syringae pv. Actinidiae phase PSA-P1), with the preservation number of CCTCC M: 2020252.

Example 2: electron microscopy of Pseudomonas syringae Kiwi fruit pathovar phage PSA-P1 the purified phage solution prepared in example 1 was used for electron microscopy: dropping 20 μ L sample on copper mesh, allowing it to settle naturally for 15min, absorbing the excessive liquid from the side with filter paper, adding 1 drop of 2% phosphotungstic acid on the copper mesh, dyeing for 10min, absorbing the dye solution from the side with filter paper, drying, and observing with electron microscope.

The result is shown in figure 2, and the observation of the form of pseudomonas syringae kiwi fruit pathopoiesia variety phage PSA-P1 under an electron microscope shows that the phage has a polyhedral three-dimensional symmetrical head and a longer tail, and the diameter of the head is about 50-55 nm; the length of the tail part is 15-20 nm, and the diameter of the tail part is 6-10 nm. Pseudomonas syringae kiwi fruit pathogenic variant phage PSA-P1 belongs to the family Autographiviridae.

Example 3: preparation of pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1 particles and extraction and sequencing of genome

(1) Taking 100mL of the purified phage solution prepared in the example 1, sequentially adding 20 μ L of DNaseI 20 μ L, RNaseA 20 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;

(3) 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 Pseudomonas syringae kiwi pathopoiesia variety phage PSA-P1(Pseudomonas syringae pv. Actinidiae phase PSA-P1) has a nucleotide sequence shown in SEQ ID No. 1.

The sequence of Pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1 is aligned on NCBI website to obtain the phage belonging to Autographiviridae family.

Example 4 potency assay of Pseudomonas syringae Kiwi berry pathogenic variants phage PSA-P1 Using SM as diluent, stock solutions of Pseudomonas syringae Kiwi berry pathogenic variants phage PSA-P1 (from example 1) were diluted stepwise in 10-fold gradient to l0⁸And (4) doubling. Respectively taking l0⁵、l0⁶、l0⁷And l0⁸The 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. Adding the mixed solution into 4mL of semi-solid agar culture medium cooled to 50 ℃, uniformly mixing, immediately spreading on a solidified solid agar plate, and performing inverted culture at 28 ℃ for 6-8h after the agar is solidified. Three replicates of each dilution were taken and counted as the average of the three replicates of that dilution. Wherein the phage titer (PFU/mL) is the average plaque number multiplied by the dilution factor as shown in Table 1, and the Pseudomonas syringae kiwifruit pathopoiesia variety phage PSA-P1 has 10 h after culturing for 12h¹⁰Titers above PFU/mL.

TABLE 1 titer of Pseudomonas syringae Kiwi pathovar phage PSA-P1 after 12h of culture

Incubation time	4h	8h	12h
				Phage PSA-P1 titer (PFU/mL)	4.1x109	1.7x1010	3.3x1010

Example 5: virulence gene or undesirable gene deletion detection test of pseudomonas syringae kiwi fruit pathogenic variant phage PSA-P1

The selection of 103 virulence genes identified as being derived from lysogenic phages within pathogenic bacteria in this example is shown in table 2, and the determination of whether they contain the following virulence genes was performed by determining the whole genome of the pseudomonas syringae kiwifruit pathovar phage PSA-P1 and performing bioinformatic analysis on it. The results show that pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1 does not contain the following virulence genes or undesirable genes and therefore does not encode proteins that may pose potential health risks, and therefore pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1 does not affect the health of the human or animal body.

TABLE 2 major known virulence genes of lysogenic phages in pathogenic bacteria

Example 6: 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 10¹⁰PFU/kg Pseudomonas syringae Kiwi pathopoiesia variant phage PSA-P1, control group was given physiological saline in equal amount for 15 days continuously, and the test mice were sacrificed by neck-cutting and examined for visceral status.

The experimental result shows that the pseudomonas syringae kiwifruit pathovar phage PSA-P1 at the dose has no influence on the daily behaviors of the mice. The viscera were examined by dissection without abnormality. Pseudomonas syringae kiwifruit pathopoiesia variant phage PSA-P1 has biological safety, and can be used as crop disease control preparation.

Example 7: determination of Pseudomonas syringae Kiwi pathovar phage PSA-P1 on the optimal multiplicity of infection (MOI) of Pseudomonas syringae

Selecting a single colony of host bacterium pseudomonas syringae Psa-1, inoculating the single colony into a test tube containing 3mL of TSB liquid culture medium, and carrying out shake culture in a shaking table at the temperature of 28 ℃ and overnight at the speed of 180rpm to obtain a host bacterium suspension. The host bacterial suspension is transferred to 10mL of TSB liquid culture medium according to the proportion of 1:100, and the host bacterial suspension is subjected to shaking culture to the prophase of logarithm under the conditions that the temperature is 28 ℃ and the rotating speed is 180 rpm. Phage PSA-P1 purified solution (prepared in example 1) and phage host bacteria (MOI ═ purified phage solution titer/phage host bacteria concentration) were added at ratios of MOI of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001, 0.00001, 0.000001, and 0.0000001, respectively, and TSB liquid medium was added to make the total volume of each tube the same. The culture was carried out in a shaker at 28 ℃ for 24 hours with shaking at 180 rpm. After the culture is finished, 10000g of the culture solution is centrifuged for 10min, and the supernatant culture solution is collected, and the titer of the phage for each treatment is determined by 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 titer of Pseudomonas syringae Kiwi pathovar phage PSA-P1 at different multiplicity of infection

MOI	PSA-P1(PFU/mL)	Host bacterium (cfu/mL)	PSA-P1 potency (PFU/mL)
				100	107	105	1.2×108
10	107	106	3.3×108
				1	107	107	4.6×108
0.1	107	108	1.4×109
				0.01	107	109	5.8×109
0.001	107	1010	7.2×109
				0.0001	106	1010	2.4×1010
0.00001	105	1010	4.5×1010
				0.00000	104	1010	7.4×1010
0.0000001	103	1010	5.2×1010

As can be seen from Table 3, the phage PSA-P1 titer reached a maximum of 7.4X 10 under 24h culture¹⁰PFU/mL, its MOI is 0.000001. Indicating that only a small amount of initial Pseudomonas syringae phage is needed to achieve large scale proliferation. The phage PSA-P1 provides a high-quality phage strain source for the industrial production of phage bactericides.

Example 8: determination of pH and temperature stability of Pseudomonas syringae Kiwi pathovar phage PSA-P1

8-1: stability of Pseudomonas syringae Kiwi pathopoia var phage PSA-P1 under different pH conditions 900. mu.L of TSB liquid medium with pH of 1-14 was added to each sterile EP tube, the EP tubes were placed in a thermostatic water bath at 25 ℃ and after temperature equilibration, 00. mu.L of purified phage solution (prepared in example 7) was added to give an initial titer of 1X10¹⁰PFU/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 of bacteriophage PSA-P1 at different pH conditions

The results are shown in table 4, the titer of the phage PSA-P1 has not changed significantly between pH 5 and 10, indicating that it has good stability under neutral, slightly acidic and slightly alkaline conditions.

Under acidic conditions at pH3 and basic conditions at pH12, the titer of phage PSA-P1 decreased to some extent, but by about 3 orders of magnitude compared to pH 7, indicating better tolerance under acidic and basic conditions. Under both very acidic conditions at pH 2 and very basic conditions at pH 13, the titer of phage PSA-P1 decreased to 0 within 1 hour.

8-2: stability of Pseudomonas syringae Kiwi fruit pathopoiesia variegated phage PSA-P1 under different temperature conditions

The titer is 1.0 multiplied by 10⁷The phage PSA-P1 (prepared in example 7) was subjected to conditions of 4 deg.C, 25 deg.C and 40 deg.C, and the titer was measured by periodic sampling.

TABLE 5 stability of Pseudomonas syringae Kiwi pathovar phage PSA-P1 at 4 deg.C

TABLE 6 stability of Pseudomonas syringae Kiwi pathovar phage PSA-P1 at 25 deg.C

TABLE 7 stability of Pseudomonas syringae Kiwi fruit pathovar phage PSA-P1 at 40 deg.C

As can be seen from tables 5 to 7, the phage PSA-P1 has better stability at 4 ℃, the titer does not significantly decrease after 3 months of storage, and the titer still does not exceed 1 order of magnitude after 12 months of storage; under the condition of 25 ℃, the titer is not obviously reduced after the phage PSA-P1 is stored for 4 weeks; at 40 ℃, the titer of the phage PSA-P1 does not significantly decrease within 24 hours, and after 72 hours, the titer decreases by 1 order of magnitude. Therefore, the phage PSA-P1 has better stability under different temperature conditions.

Example 9: pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1 tolerance to ultraviolet light

Taking 10mL of the product with the titer of 1 × 10⁸PFU/mL of PSA-P1 phage (prepared from example 7) plated on 90mm platesAfter being placed in a bacteria culture dish, the bacteria culture dish is placed in a super clean workbench and is irradiated under an ultraviolet lamp (20w, 20 cm). Sampling at 0min, 20min, 40min, 1h, 2h, 3h, 4h, 5h, 6h, 7h and 8h respectively, placing in the dark for 30min, and determining the titer of the phage by using a double-layer plate method.

TABLE 8 stability of Pseudomonas syringae Kiwi fruit pathovar phage PSA-P1 under UV irradiation

As shown in Table 8, the titer of Pseudomonas syringae Actinidia pathovar phage PSA-P1 was reduced by an order of magnitude at 8h of UV irradiation, and thus the phage of the present invention was more resistant to UV.

Example 10: pseudomonas syringae kiwifruit pathovar phage PSA-P1 lysis ability test on Pseudomonas syringae from different origins

The lysis spectrum of the phage was determined by double-plate titration. Respectively selecting single colonies of 45 pseudomonas syringae separated from 6 provinces such as Shandong, Sichuan, Chongqing, Anhui, Guangdong and Henan, inoculating the single colonies into a test tube containing 3mL of TSB liquid culture medium, and culturing at 28 ℃ and 180rpm overnight to obtain bacterial liquids of the strains. mu.L of each bacterial suspension was mixed with TSB semi-solid agar medium and spread on a common agar plate, 5. mu.L of purified phage PSA-P1 solution (prepared in example 1) was dropped on the plate, air-dried naturally, cultured overnight at 28 ℃ and the results were observed.

TABLE 9 lysis results of Pseudomonas syringae Actinidia var typhimurium phage PSA-P1 on Pseudomonas syringae from different origins

Note: "+ + + +" is completely clear; "+ +" is medium and clear; "+" is slightly clear; the non-cleaved is "-".

The results are shown in Table 9, the phage PSA-P1 has strong lytic capacity to Pseudomonas syringae from different origins, and the lytic rate can reach 91.1%. Pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1 has a broad lysis spectrum.

Example 11: lysis test of Pseudomonas syringae Actinidia var typhimurium phage PSA-P1 against non-pathogenic beneficial bacteria

Selecting 5 nonpathogenic rhizobia and 5 nonpathogenic bacillus subtilis, respectively inoculating the 5 nonpathogenic bacillus subtilis into test tubes containing 3mL of TSB liquid culture medium, and culturing at 30 ℃ and 180rpm for 8h to obtain 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 of the purified phage PSA-P1 solutions (prepared in example 1) was dropped on the plate, allowed to air dry, and then cultured at 30 ℃ for 24 hours, and the results were observed.

TABLE 10 lysis test of Pseudomonas syringae Actinidia pathovar phage PSA-P1 against non-pathogenic beneficial bacteria

Note: "+ + + +" is completely clear; "+ +" is medium and clear; "+" is slightly clear; the non-cleaved is "-".

As shown in Table 10, in this example, none of the 10 non-pathogenic bacteria were recognized by the phage PSA-P1. The test phage has extremely strong host specificity and has no damage to microbial communities.

Example 12: lysogenic characterization of Pseudomonas syringae Actinidia var typhimurium phage PSA-P1 will give 100. mu.L (0PFU/mL, 1.0X 10⁴PFU/mL，1.0×10⁵PFU/mL，1.0×10⁶PFU/mL，1.0×10⁷PFU/mL) of phage PSA-P1 (prepared in example 7) with 100. mu.L of Pseudomonas syringae Psa-1 (1.0X 10)⁸cfu/mL) were mixed and inoculated into a liquid culture containing 10mL of TSBThe medium was shake-cultured in a 50mL centrifuge tube at 28 ℃ for 48 hours. The turbid culture solution obtained was applied by gradient dilution to a TSA plate and cultured in an incubator at 28 ℃ for 48 hours. Picking the central part of 30-50 single colonies on a TSA plate, placing the central part in an EP tube containing 200 mu L of TSB liquid culture medium, and carrying out shake culture at 28 ℃ for 24 h; subsequently, mitomycin C with the final concentration of 0.5 mug/mL is added into the EP tube, and the culture is continued for 12 h; filtering the obtained culture solution with 0.22 μm filter membrane for sterilization, dripping on Psa-1 double-layer plate, and culturing at 28 deg.C; simultaneously adding the titer of 1.0 multiplied by 10⁷PFU/mL phage PSA-P1 (prepared in example 7) was spotted onto the bi-layer plate as a positive control. After 24h the double plate was observed and if plaques appeared, the phage PSA-P1 was confirmed to be a lysogenic phage.

The results showed that all control plates had plaques (FIG. 3) and all test plates had no plaques (FIG. 4), indicating that Pseudomonas syringae Actinidia pathovar phage PSA-P1 was not lysogenic and was a virulent phage.

Example 13: turbidity method for detecting bacteriostasis rate of pseudomonas syringae kiwi fruit pathogenic variant phage PSA-P1 on pseudomonas syringae

Selecting a single colony of the host bacteria Psa-1, inoculating the single colony into a test tube containing 3mL of TSB, and carrying out overnight culture at the temperature of 28 ℃ for 180r/min until the single colony is turbid to obtain a host bacteria liquid; the titer is 2 multiplied by 10¹⁰Phage PSA-P1 (prepared in example 7) in PFU/mL was gradually diluted in a gradient of sterile water to the titer of each treatment group. As shown in Table 11, 100. mu.L of the Psa-1 bacterial suspension and 100. mu.L of each diluted phage PSA-P1 were added to 50mL centrifuge tubes each containing 10mL of TSB broth, and the suspension was incubated at 28 ℃ and 120r/min for 24 hours using 100. mu.L of Psa-1 bacterial suspension mixed with 10mL of TSB broth as a positive control, and the turbidity value of each treatment group was measured with a turbidimeter. The bacteriostasis rate is (positive treatment turbidity-treatment group turbidity)/positive treatment turbidity multiplied by 100 percent

TABLE 11 bacteriostatic effect of Pseudomonas syringae Kiwi fruit pathogenic variant phage PSA-P1 on Pseudomonas syringae

As can be seen from Table 11, about 10¹The bacteriostasis rate of the phage PSA-P1 of PFU/mL to PSA-1 can reach 54.1%, which shows that the pseudomonas syringae kiwifruit pathopoiesia variety phage PSA-P1 can effectively inhibit and kill bacteria under an extremely low dosage.

Example 14: bactericidal effect of pseudomonas syringae kiwi pathopoiesia variety phage PSA-P1 in liquid

Culturing Pseudomonas syringae Psa-1 to logarithmic growth phase, subpackaging into different test tubes, diluting the bacterial liquid with the same volume of TSB liquid culture medium until the final concentration of Pseudomonas syringae Psa-1 is 1 × 10³cfu/mL. Respectively inoculating to the mixture with final concentration of 1 × 10²PFU/mL、1×10³PFU/mL，1×10⁴PFU/mL、1×10⁵PFU/mL、1×10⁶PFU/mL Pseudomonas syringae Actinidia var typhimurium phage PSA-P1(Pseudomonas syringae pv. Actinidiae phase PSA-P1) (prepared in example 1). Setting a control group and a blank group at the same time, wherein the control group is given with a final concentration of 1x10³cfu/mL Pseudomonas syringae Psa-1; the blank group was given an equal amount of physiological saline. Each treatment was cultured with shaking at 28 ℃ and 150rpm for 4 hours, and the residual amount of Pseudomonas syringae was measured. The detection method comprises the following steps: after each treated sample was diluted with sterile water, 100. mu.L of the diluted solution was applied to a TSA solid plate, and the number of colonies on the plate was counted after incubation at 28 ℃ for 24 hours. The number of pseudomonas syringae (number of colonies on TSA plate) x dilution multiple x 10.

TABLE 12 Bactericidal efficacy of Pseudomonas syringae Kiwi pathovar phage PSA-P1 in liquids at different concentrations

As can be seen from Table 12, Pseudomonas syringae Kiwi fruit pathovar phage PSA-P1 was present at a final concentration of1×10²PFU/mL, the growth of the pseudomonas syringae Psa-1 in a liquid culture medium can be well controlled; when the final concentration of pseudomonas syringae kiwifruit pathogenic variety phage PSA-P1 is more than or equal to 1 × 10⁴When PFU/mL, the killing rate of the pseudomonas syringae can reach more than 99 percent.

Example 15: preparation of Pseudomonas syringae Kiwi fruit pathovar phage PSA-P1 composition

1. The final concentrations were 1X10 respectively²PFU/mL、1×10³PFU/mL、1×10⁴PFU/mL、1×10⁵PFU/mL、1×10⁶PFU/mL of the purified phage PSA-P1 solution (prepared in example 1) was mixed with a final concentration of 50% amobam 700-fold solution (chemical bactericide) in equal volumes to prepare 1:1 of composition 1, composition 2, composition 3, composition 4 and composition 5, respectively.

2. The final concentrations were 1X10 respectively²PFU/mL、1×10³PFU/mL、1×10⁴PFU/mL、1×10⁵PFU/mL、1×10⁶PFU/mL of the purified phage PSA-P1 solution (prepared in example 1) was mixed with a final concentration of 1X10⁶PFU/mL of Xanthomonas carpi phage liquid was mixed uniformly in equal volumes to make 1:1 of composition 6, composition 7, composition 8, composition 9 and composition 10.

3. The final concentrations were 1X10 respectively²PFU/mL、1×10³PFU/mL、1×10⁴PFU/mL、1×10⁵PFU/mL、1×10⁶PFU/mL of the purified phage PSA-P1 solution (prepared in example 1) was mixed with 50% final concentration of 700-fold ambam solution to a final concentration of 1X10⁶PFU/mL of Xanthomonas carpi phage liquid was mixed uniformly in equal volumes to make 1:1:1 compositions 11, 12, 13, 14 and 15.

Example 16: bactericidal effect of pseudomonas syringae kiwifruit pathovar phage PSA-P1 composition in liquid

Culturing Pseudomonas syringae Psa-1 to logarithmic growth phase, subpackaging into different test tubes, diluting the bacterial liquid with the same volume of TSB liquid culture medium until the final concentration of Pseudomonas syringae Psa-1 is 1 × 10³cfu/mL, into which realms are respectively accessedThe composition of Pseudomonas syringae Kiwi pathovar phage PSA-P1 prepared in example 15, control group and blank group were set simultaneously, and the control group was given at a final concentration of 1X10³cfu/mL Pseudomonas syringae Psa-1; the blank group was given an equal amount of physiological saline. Carrying out shake culture on the treatments at 28 ℃ and 150rpm, detecting the residual quantity of pseudomonas syringae Psa-1 after 4h, wherein the detection method comprises the following steps: after each treated sample was diluted with sterile water, 100. mu.L of the diluted solution was applied to a TSA solid plate, and the number of colonies on the plate was counted after incubation at 37 ℃ for 24 hours. The number of pseudomonas syringae (number of colonies on TSA plate) x dilution multiple x 10.

TABLE 13 Bactericidal efficacy of Pseudomonas syringae Kiwi pathovar phage PSA-P1 compositions in liquids at different concentrations

As can be seen from Table 13, the Pseudomonas syringae Kiwi fruit pathovar phage PSA-P1 composition at each concentration has a good killing effect on Pseudomonas syringae. The pseudomonas syringae kiwifruit pathogenic variety phage PSA-P1 can be used together with other substances to control bacteria, and has no antagonism to other substances.

The combination of the embodiment is not limited to the 700-fold liquid of amobam, but may also be chemical bactericides such as polyoxin, flumorph, dimethomorph, prochloraz, difenoconazole, flusilazole, myclobutanil, mancozeb, thiophanate-methyl, carbendazim, chlorothalonil, and fructosan. The embodiment can also be used together with a chemical disinfectant to achieve the effect of preventing and killing.

Example 17: pseudomonas syringae kiwi fruit pathogenic variant phage PSA-P1 and its composition for preventing and treating kiwifruit canker

360 kiwi trees are randomly divided into 6 groups (phage 3 group, composition 5, control group and blank group) after adaptive culture for 1 month, each group comprises 60 kiwi trees, and the dosages of phage experiment groups are respectively 1 × 10⁴PFU/mL、1×10⁵PFU/mL、1×10⁶PFU/mL of phage tested (prepared in example 7) and 1X10⁵cfu/mL Pseudomonas syringae Psa-1; control group was given 1X10⁵cfu/mL Pseudomonas syringae Psa-1; the blank group is given with the same amount of normal saline, 1L is inoculated by adopting a stem infusion method, the incidence rate of kiwifruit canker within 15d is counted from the inoculation, and the incidence rate is the incidence plant number/total plant number multiplied by 100%.

TABLE 14 Effect of Pseudomonas syringae Kiwi fruit pathovar phage PSA-P1 and compositions thereof on Kiwi fruit canker

Time

104PFU/mL

105PFU/mL

106PFU/mL

Composition 3

Control group

Blank group

1d

0％

0％

0％

0％

0％

0％

2d

1％

0％

0％

0％

6％

0％

3d

3％

1％

0％

0％

11％

0％

4d

6％

3％

0％

0％

20％

0％

5d

7％

4％

0％

0％

34％

0％

6d

8％

6％

3％

3％

40％

0％

7d

11％

8％

3％

3％

48％

0％

8d

13％

9％

5％

4％

52％

0％

9d

17％

11％

7％

5％

60％

0％

10d

19％

13％

7％

7％

62％

0％

11d

20％

16％

8％

7％

68％

0％

12d

20％

16％

9％

7％

74％

0％

13d

22％

17％

9％

8％

82％

0％

14d

24％

18％

10％

8％

90％

0％

15d

26％

18％

10％

8％

100％

0％

As shown in Table 14, the kiwifruit trees in the control group broke up at 15 days after inoculationThe incidence rate of the ulcer disease reaches 100 percent. In each experimental group of the phage, the higher the concentration of the phage PSA-P1 is, the lower the incidence rate of the kiwi fruit tree is; phage PSA-P1 concentration of 10⁶And when the concentration is PFU/mL, after root irrigation is carried out for 15 days, the incidence rate of the kiwi fruit trees is kept within 10 percent. The pseudomonas syringae bacteriophage and the composition thereof can be used as a biological bactericide to effectively prevent and treat the kiwifruit canker.

Example 18: preparation and application of pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1 and kit of pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1 composition

The kit contains 5-10 mL of the titer of 1 multiplied by 10⁷PFU/mL Pseudomonas syringae Actinidia var typhimurium phage PSA-P1 liquid or Pseudomonas syringae Actinidia var typhimurium phage PSA-P1 composition, 1L TSB semisolid culture medium, 1L TSA culture medium.

The using method of the kit comprises the following steps: taking out the titer of 1 × 10⁷PFU/mL Pseudomonas syringae kiwi fruit pathopoiesia variant phage PSA-P1 liquid or Pseudomonas syringae kiwi fruit pathopoiesia variant phage PSA-P1 composition, using double plate droplet method to determine the test phage lysis spectrum. 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. Mixing 300 μ L of the suspension of the strain to be detected with 5mL of TSB semisolid culture medium, spreading on a TSA plate, and dripping 10 μ L of Pseudomonas syringae Kiwi fruit pathogenic variant phage PSA-P1 liquid or Pseudomonas syringae Kiwi fruit pathogenic variant phage PSA-P1 composition 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.

Example 19:

application of pseudomonas syringae kiwi fruit pathopoiesia variant phage PSA-P1 and kit of composition thereof to citrus canker

The main component of the kit 1 is 5-10 mL with the titer of 3 multiplied by 10⁸PFU/mL Pseudomonas syringae Actinidia pathovar phage PSA-P1 liquid.

The main component of the kit 2 is 5-10 mL with the titer of 3 multiplied by 10⁸PFU/mL of Xanthomonas campestris YHC5 liquid.

The main component of the kit 3 is 5-10 mL of 700 times of ambam solution with the final concentration of 50%.

The main component of the kit 4 is 5-10 mL with the titer of 3 multiplied by 10⁸PFU/mL pseudomonas syringae kiwifruit pathopoiesia variety phage PSA-P1 liquid, 5-10 mL titer is 3 x10⁸PFU/mL of the carpet xanthium gracile bacteriophage YHC5 liquid and 5-10 mL of the ambam 700-fold liquid with the final concentration of 50%.

The test process comprises the following steps: 120 citrus trees are subjected to adaptive culture for 1 month and then are randomly divided into 6 groups (a kit 1 group, a kit 2 group, a kit 3 group, a kit 4 group, a positive control group and a negative control group), and 20 citrus trees are cultured in each group. Respectively adding 1L of the extract into the solution with the final concentration of 1x10 by adopting a stem infusion method³cfu/mL Xanthomonas carpi citrus pathopoiesia variant ACCC 03526(Xanthomonas axonopodis pv. citri) performs toxicity attack treatment on 100 citrus trees in a kit group 1, a kit group 2, a kit group 3, a kit group 4 and a positive control group, and 1L of physiological saline is respectively input into 20 citrus trees in the negative control group by a stem infusion method. After three days of challenge treatment, 5 groups of citrus trees subjected to challenge treatment are respectively inoculated with 1L of each thousand-fold dilution liquid of the kit 1 group, the kit 2 group, the kit 3 group and the kit 4 group by adopting a stem infusion method, and the positive control group and the negative control group are both given with the same amount of physiological saline. The incidence of citrus canker was counted 15 days after inoculation, and the incidence rate was defined as the number of diseased plants/total number of plants × 100%.

TABLE 15 Effect of Pseudomonas syringae Kiwi fruit pathovar phage PSA-P1 and kits of compositions thereof on Citrus canker

As shown in table 15, the citrus incidence of the kit 4 group was lower than that of the kit 2 group, indicating that the kit 4 containing the composition had significant control of citrus canker, and the kit 4 containing the composition had better control of citrus canker than the kit 2 containing only the xanthomonas carpi phage YHC 5.

Example 20:

application of pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1 and kit of composition thereof to cucumber bacterial angular leaf spot

The kit of example 19 was used for each of kit 1, kit 2, kit 3, and kit 4.

The test process comprises the following steps: 120 cucumber vines are subjected to adaptive culture for 1 month and then are randomly divided into 6 groups (a kit 1 group, a kit 2 group, a kit 3 group, a kit 4 group, a positive control group and a negative control group), and 20 plants are respectively adopted. Respectively adopting root irrigation method to make 1L final concentration be 1x10³cfu/mL Pseudomonas syringae cucumber pathogenic variant ATCC7386(Pseudomonas syringae pv. lachrymans) 100 cucumbers in the kit 1 group, the kit 2 group, the kit 3 group, the kit 4 group and the positive control group were subjected to detoxification treatment, and 20 cucumbers in the negative control group were applied with 1L physiological saline by root irrigation. After three days of toxin attacking, 1L of thousands times of dilution liquid of the kit 1 group, the kit 2 group, the kit 3 group and the kit 4 group is respectively inoculated by adopting a root irrigation method, and the positive control group and the negative control group are respectively given with the same amount of physiological saline. The disease rate of cucumber bacterial angular leaf spot within 15 days from inoculation is counted, and the disease rate is the number of diseased plants/total plants multiplied by 100%.

TABLE 16 influence of Pseudomonas syringae Kiwi fruit pathovar phage PSA-P1 and kit of compositions thereof on cucumber bacterial angular leaf spot

As shown in table 16, the cucumber incidence of the kit 4 group was lower than that of the kit 1 group, indicating that the kit 4 containing the composition had a significant effect on controlling cucumber bacterial angular leaf spot, and the kit 4 containing the composition had a better effect on controlling cucumber incidence than the kit 1 containing only pseudomonas syringae kiwi pathovar phage PSA-P1.

Example 20:

application of pseudomonas syringae kiwifruit pathopoiesia variant phage PSA-P1 and kit of composition thereof to bacterial leaf spot of tomato

The kit of example 19 was used for each of kit 1, kit 2, kit 3, and kit 4.

The test process comprises the following steps: 120 tomato vines are randomly divided into 6 groups (a kit 1 group, a kit 2 group, a kit 3 group, a kit 4 group, a positive control group and a negative control group) after being adaptively cultured for 1 month, and each group comprises 20 tomato vines. Respectively adopting root irrigation method to make 1L final concentration be 1x10³cfu/mL Pseudomonas syringae tomato pathogenic variant ATCC BAA-871D-5(Pseudomonas syringae pv. tomato) 100 tomatoes in the kit 1 group, the kit 2 group, the kit 3 group, the kit 4 group and the positive control group are subjected to challenge treatment, and 1L of physiological saline is applied to 20 tomatoes in the negative control group by a root irrigation method respectively. After three days of toxin attacking, 1L of thousands times of dilution liquid of the kit 1 group, the kit 2 group, the kit 3 group and the kit 4 group is respectively inoculated by adopting a root irrigation method, and the positive control group and the negative control group are respectively given with the same amount of physiological saline. The incidence of bacterial leaf spot of tomato within 15 days from inoculation was counted, and the incidence rate was the number of diseased plants/total number of plants × 100%.

TABLE 17 influence of Pseudomonas syringae Kiwi fruit pathovar phage PSA-P1 and kit of compositions thereof on bacterial leaf spot of tomato

As shown in table 17, the tomato incidence of the kit 4 group was lower than that of the kit 1 group, indicating that the kit 4 containing the composition has significant control of bacterial leaf spot of tomato, and the kit 4 containing the composition has better control effect on tomato incidence than the kit 1 containing only pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1.

Example 21:

application of caryophylli pseudomonad kiwi fruit pathovar bacterium bacteriophage PSA-P1 and kit of composition thereof to bean sickness

The kit of example 19 was used for each of kit 1, kit 2, kit 3, and kit 4.

The test process comprises the following steps: 120 kidney beans were randomly divided into 6 groups (kit 1 group, kit 2 group, kit 3 group, kit 4 group, positive control group and negative control group) after adaptive culture for 1 month, and 20 plants were each group. Respectively adopting root irrigation method to make 1L final concentration be 1x10³cfu/mL Pseudomonas syringae Phaseolus vulgaris pathogenic variant ATCC21781(Pseudomonas syringae pv. phaseolicola) 100 Phaseolus vulgaris in the kit 1, the kit 2, the kit 3, the kit 4 and the positive control group were subjected to detoxification treatment, and 20 Phaseolus vulgaris in the negative control group were applied with 1L of physiological saline by root irrigation. After three days of toxin attacking, 1L of thousands times of dilution liquid of the kit 1 group, the kit 2 group, the kit 3 group and the kit 4 group is respectively inoculated by adopting a root irrigation method, and the positive control group and the negative control group are respectively given with the same amount of physiological saline. The incidence of the soybean phytophthora blight within 15 days from inoculation is counted, and the incidence is the number of the affected strains/the total strains multiplied by 100%.

TABLE 18 influence of Pseudomonas syringae Kiwi fruit pathovar phage PSA-P1 and kit of composition thereof on Phaseolus vulgaris blight

As shown in table 18, the incidence of kidney beans in the kit 4 group was lower than that in the kit 1 group, indicating that the kit 4 containing the composition has significant control of kidney bean phytophthora blight, and the kit 4 containing the composition has better control effect on the incidence than the kit 1 containing only pseudomonas syringae kiwi pathopoiesia phage PSA-P1.

Example 22:

application of pseudomonas syringae kiwifruit pathopoiesia variant phage PSA-P1 and kit of composition thereof to tobacco wildfire

The kit of example 19 was used for each of kit 1, kit 2, kit 3, and kit 4.

The test process comprises the following steps: 120 tobacco plants were randomly divided into 6 groups (kit 1 group, kit 2 group, kit 3 group, kit 4 group, positive control group and negative control group) after adaptive culture for 1 month, and 20 plants were each group. Respectively adopting root irrigation method to make 1L final concentration be 1x10³cfu/mL Pseudomonas syringae tobacco pathogenic variant ATCC13453(Pseudomonas syringae pv. tabaci) 100 tobacco strains in the kit 1 group, the kit 2 group, the kit 3 group, the kit 4 group and the positive control group were subjected to detoxification treatment, and 1L of physiological saline was applied to 20 tobacco strains in the negative control group by root irrigation. After three days of toxin attacking, 1L of thousands times of dilution liquid of the kit 1 group, the kit 2 group, the kit 3 group and the kit 4 group is respectively inoculated by adopting a root irrigation method, and the positive control group and the negative control group are respectively given with the same amount of physiological saline. The incidence of tobacco wildfire was counted from inoculation within 15 days, and the incidence rate was defined as the number of diseased plants/total number of plants × 100%.

TABLE 19 influence of Pseudomonas syringae Kiwi fruit pathovar phage PSA-P1 and kit of composition thereof on wildfire of tobacco

As shown in table 19, the incidence of tobacco in the kit 4 group was lower than that in the kit 1 group, indicating that the kit 4 containing the composition has significant control of tobacco wildfire, and the kit 4 containing the composition has better control effect on the incidence than the kit 1 containing only pseudomonas syringae actinidia pathovar phage PSA-P1.

In conclusion, the Pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1(Pseudomonas syringae pv. actinodiace phase PSA-P1) and the composition thereof have high safety, can be used as effective components of a preparation kit and a biological disinfectant or a biological pesticide, and can be used for preventing and treating various bacterial diseases caused by Pseudomonas syringae.

Through examples 19-22, it can be seen that the use of pseudomonas syringae kiwifruit pathovar phage PSA-P1 alone has a killing effect on pseudomonas syringae used in an offensive experiment, and can significantly control diseases of an offensive plant, but the control effect of the composition containing phage PSA-P1 on diseases of the offensive plant is better.

The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.

Sequence listing

<110> Philippinecaceae (Nanjing) Biotech Ltd

<120> pseudomonas syringae bacteriophage and composition, kit and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 40200

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ggtcttgctg aacgactgga agttcgatgc gaaggtctga tccttctcgc cgaagtacat 60

gcccatggtc tgagcgtcca gcgcctgcaa ggtcttgagt tgctgcttga cgtatggcgg 120

gagatccttc tcacccttga agtcatccgg gttgaaactc attgccgcct caagcgagga 180

ggtcaggccg gggagcttca tgcggttcga ccgcgaccat gcgacggact gctgcaacga 240

ccagtcgctg acctgctgtg ggtcttggcc ctgagcgatc atctcctgcg ccttctgagc 300

cagcccagcc tcgaaggtct tggcgatctt gttacgatct tggtcgctgt acgcttggtt 360

caaacctagc gggatcgcac cttggttgta catcgccaac atggagctgc catagccttg 420

gttctccttc tgccgcgcct tggccgcaga gtccatacgg agacgaagac cagcgatggc 480

gtgagcactg taggtgccgg ggaattcctt gttgatctgc tcaatgcgct tgagggtgga 540

accccacgac gcctgaccgt tgaccgctgc cagctccatc ttacccatct cgtcgccgag 600

cataggtgcc atctcgcggg cgtgccactg gtcgtactga tcctgcgcgg tcttgaggag 660

ctggctcccc ttcgaccagt ctgtgacgtt gagctgatcc agcagtcgct tgtcaccctg 720

tgctgccgtg gtggccgctg cctgcatcag catcttcttc atgctgaact cagacacgcc 780

catcatcttg gcacgcgctg ggatctcgtt catgacgatg tgttcagccg aagcattcgg 840

gtcacccagc aggtcttgga tgctggtgtt cagcgcctcc tcccgcttgg caacacgata 900

gtcgcctcgg atcttctcac ccacaccgac cagcgcttgc tggctttcct gaatgttcat 960

ggaggtgtca cggagcgtcc acttgtccac gccgtacttg tccagcagag gttggtattc 1020

cttctgctgt gccttcatga agtcctcgtt ggacatctca gggttttctt gcatcaactg 1080

cacgaggcgg ttgtttgcct cctgcacgtc gtgcttggag acgatggtgt tgtaggccat 1140

acgacctgca acggtggcgt cttgggtgat gccctgacgc tcgttctctg ccttccagat 1200

gtcctgtgca gcgatggtgc tctgcttgat ccggtcttct tcaacctgcc gctcgacctc 1260

ggtttgcgcc tgctccgaca gggtgttggc gaagttcgcc agtccaccca tcacgtcggc 1320

gaggccgttg gatacatacc cagcggcccc tgcgcggttg gtctgtgcag cctgttggaa 1380

gaaagcccgg ttgcgttgtg cgccaccgac gccaagttct acggcttcac gttctgccat 1440

gttaacctcc tgcgtttacc ttctgtgcct gctcgaactt ctggctacct tggtaggcac 1500

cgccaaacgc actgagagca gacccgccaa ttttgagcgc cccggacgcc cacgatgggc 1560

gactgatagg cgagtagtcc atgcctgctt gaccctgaga tcggatctgc ttggcttggt 1620

tcgttacgtt gtccagcttg ctctgacgag tagcttggat gttgtcgagg ttgtgctgac 1680

gatcacgcgc cagcccttgc tcttgcatca gaaccgattg cccagcagta ccgctcgccg 1740

ctgccatggt gttcacccgg ccacgcgctt gcatgtactg cttctgcatc tcggtgttct 1800

gatccgcttc ctccagcagc gcatcctgct caacctgcga caggtcgcca tacgattgct 1860

cgatggcttt ggcctgctcg cggttgtgga tctcctgctg ttcggctgcg gcgtccgctt 1920

ggttcttgct gtcttgcaca gagacgacgg tagcagcgac cgaagccgct gctgctgcgt 1980

acatcataat cgggatagct acggccatta caacctccgc ccagtttgct ggtagctgcc 2040

ctcccactcc aacccacgta tctggagcgg caggtggcta tcagagaata cttcgatggt 2100

agcctcgtgg gtgaacagcc ggacaggaac cggcagcgtg ccactgatta gtggtgcgaa 2160

gcctacccgg ttgttgtggc tacccatgcg ccgaccatag aacggatact tccactggcg 2220

accttgtggg tcagatacca gcacagtgaa gctgcccgtc cggtcgtagt tcacggacat 2280

gcgacccatg gtgatgcggt caatggccat gacgttgcca ttctggtcac gcaggaatgg 2340

ctgggttgga atgtagcggc taccgtagcg gcgacccatc aggatcgtca ccgactgcgt 2400

gacagtctcg tctgccagcg aggtgctggt gtagaacgaa cggaggccca gacctgcttg 2460

gaactcgaac tcggtgccaa tgtcggacgg ccagcacccg gttgcatgca cagccaacca 2520

gcggtcgatg tccgcagtct gcacgtcgaa cggcatcgtc cagcgccaga ccttggccac 2580

gtcatcccac gtcgcggtgg tagtcacctt gcggtcgagg cggcagggga agtccatgcc 2640

tgcatcgtcg tcgtcgttgg tgagagccat gtgctcaagg tacacgttgc catcggcgtg 2700

caggaagatg aagaacacgt cgtcctctac gaactgaaca tgcagcaact tgctctgcga 2760

gtcgaacgtc cagcggtgcc atgcagcctg caccttgtcc gctccctgcc acagccagtt 2820

gtacacatag gccacatgct cctcagcatc ggtgcgcact accagcacgt tggtgttcgg 2880

gttgctgctg agctggcgaa cctgcccctt aatcagcttc tcgacgtgct cggtgattgg 2940

ccgagcctgc ttggtgtcct tgtccgagtc ggtgaacatc tcgcggatgc ccgaccagtt 3000

gcccgcgtca tacgcgaaca tgatcgactc accagtgatg gcaggcttgg cgaagatgtt 3060

catcgggaac gaagtcacct gcttgaacac cacgttggct gccgtcaccg gcttgctgcc 3120

ctcgatcatg aactgcccgt tgctggcgaa gaagatggcg tcaccatcca ccgtcacgga 3180

gttcttgatg atgttgatct tctcactgtc ggagaaggta tcaatcgggt cgctgtccac 3240

ttccgtctga ctcgactcct tgaagaagtt gaagaagtca ttcgagcgag tgaacaccgc 3300

cgactcatcg ctggtgaaga acagtcggtt ctggaacgta cccacggact gcacgtagtt 3360

attggtgaac gacgggaact tcacagtcgt gtcattacca accttgcggt cttcccatgc 3420

accggcccgc agctcgaagt gaggcacgcc agcattgatg gtggtacgga tcagggtgtg 3480

aggcatcgtc cggttgtcga agcccaactt ggccccgccc tccagtgact cctcccactt 3540

gaccttcgag ccatcggtgc ccactgcctt gagccagtag ctgttacgct tcacgccctc 3600

ggtgttctga atctcaacaa tccagttaag tggtgcatac ggtggcaggt aggccggaga 3660

cttcacgcgg ttctgtactg cgatcaggtc gccgccgttg gcaccgtcca ccgtggtgat 3720

ggtgttgatc ggctgaccat ttcggtagat gaagatcacg ttgttgttct gcgtgacgtt 3780

gaactgctcc tgcgcaccgg atgccgagaa cgtatagttc cccgcaccgt cgccagttgc 3840

cgtggtgata ccctcgcgca gcatgtgtgc gaacacctca gcgatgcggt tggtcttcac 3900

gttgttcgac gcacggtcgg cagactcagc gccaccatcc ccggtgtacg aagctcgcgg 3960

ggtgttgatg gtaaccacta cgccgccctg tccgttcatg tccgggttga tgcggatgat 4020

gtacgagcgg ctataggttg cgtactggca gtagaccagc gccgcgttgg acgggttcgg 4080

tgcctgtacg tcaccacggg ccagcacctg caccttgttg ttggcaagga aggtgtagtc 4140

gccgatggtg ctgaatgcca tgttgcgcat cgggtcatcg gtggtcatgt agtcccacgt 4200

agactgcggc gcatccacgg tacacttgac gcccttggtg ttgaagatcg aaggcgtggc 4260

ggtgtccggt tcaacgaaga tgaaatactt ctcctcgtcg cctcggtcat agaagtgaat 4320

ggtagtacgc gggtcgtatg gcttgccgct ggtcatcacc agacgggcca cgttcgacgt 4380

accgattcgc ttgtacaggg agtccagcac agacgggatc atgttctctt gcaacgtgca 4440

ttgcccgttg attcgatccc ggtcagcctg ctggctcatg ccttggatag gtcgccccat 4500

cccagcttga actcttgcca tgtggcctcc ttagttgttg ttcatgaacc caccgatgct 4560

ggcgatgggg tgacgcaggt tcgggttctg gaatgcgttc cgctttcggt aggtcgcatc 4620

ctcctgctcc agcgcgatga agctacgctg cgcctgctgg ttcagggctg taaacttcgc 4680

ctgatccacg tccttgtcgt ggacgtacca gaagcgggca gcatccacca ccgcatcctt 4740

ggcggtctgc ggcaggtcgt cgtagtccac gaaggccacg atgtccagcg ggatgtactt 4800

gagcttcgcg tccaccagcg ccgacaggtc gaagccatac tccagcgtgt ccatcaggtg 4860

gttgcctcgc ttgacgaggt tcacttcacg gagcgagtgg gtacgcttga gccgcacggc 4920

cagacagttg ttcggcagca cgactcggcc agtgtcggga gcagggtaga gcttgtggaa 4980

gccctcttgg ttgaaccagt aacccttgcc cttgttggtc tggatggtgg cgctgatatc 5040

atccagcacc cggttgatgt cgccagcgtc aacgtgatag tcgatctcgt tgagcgtggt 5100

gataccggcc tgacccatcg catcgagcac gcggttgatg gcatgcagct tggtcagttc 5160

cggcatgaag ccagctactt tcatgcgtcc tcctagtctc acactgtgag acaaaaataa 5220

gccccaaggc caacccgaag gttagccaag gggcatgggt gttactcgcc ggtttgcagc 5280

gcggcaggct tgaccagtgc agacacagcg gcctgtactg ccagcgcgaa cgcttcgacg 5340

ttctggacgc ccggcccggt tgggtcggca ccctcgaagg aatcgacctg catacgctta 5400

cgcttgacgc gggtggtcag gcgagcggtc acgtcttcca gcgaggcgtc ggttgcgttg 5460

gcaccgaagc cagccatcag gtgttcccag cgatcaggga tagcgccctc ggcgaggtac 5520

gagtcaacgt accaagtctt gttcgctttg ttccaccaga tctcgccggt cagggcgatg 5580

gagcggccaa ccagcagagc ttcgttgccg aagatcacga acatgcaggc ttccatgtcg 5640

gcggtcacgt cgtagcggaa accgttcgac gccttggaca gcaggtggtg gtcgttcact 5700

tcaccgtcga tgtcgatgtg gtcgcggttc ttgttcggga agtggttggt cgggacaact 5760

gggatgttgt acgacttgag cacgaacccg gtaacaccag cggtgccgta ggtgttgtag 5820

cgagcatcgc agacgcgctc ggcgtcacgc aggctgttga acaccggcca tggcatggcg 5880

atggtcagct tggacagatc cagatcttgc aggactgcct gctcgatcac catctcaacg 5940

aacgacagga tgcgagtcgg gtcgtccatg ttgaagttct tgtcctgtgc agcgatggag 6000

aagccgtggc ccttcacacg cggcacgccg ttgcgggcag tgctgttgtt aatcagcgca 6060

ccgtagatgc actgttgcag cagcatgcga tcttccatct tcttgatggc ggtcacctgc 6120

tcctcggcca gcttgccctt gaccatgatg tcgtcctgca cgtcgtcgag catgccgact 6180

acgttacgag cgatcacggt ggtgtcgatc accagcgagt ttttgtcgaa ctcggcttga 6240

gcgccgcgca cgtctttacc cggagccagc gcctgcactt cggtggtgcc gaggtacttg 6300

ttgctgacgg tgttggtgcc ctgaacctgt tgcaggtcga agtaggtcat catgcccatc 6360

aggttcacgt aggcgcgctt gatctggccg gtgaacttct cgaaggccag agtggctgct 6420

tcgccggatt tggagatctt ggggtcaacc aagttgttaa cgtcggacat gtaatacctc 6480

tcttgtcaaa tgggagattc gagcagcgga cttgccgact gctctatcta tactgcaact 6540

taattactgg atgccgcgac ggatcgactg gctgcgcatg gaatcgacct gacgttggta 6600

gtcgcggtcg ttccagtatt tgtcgctgtc catgatcttg ttgtactcgg caccagtgag 6660

ggtggacttg ccggtcaggt cgccaccagc aggcttgccg tcagcagtgc cgtcgcccaa 6720

cagttccacc gagcggtcgc ccttggtgat cttgtcgtgc tgcgccatct gctgcttggc 6780

catctggatg accatgcgct gggcttccca gctatcgccg cccatgatcg cgttgtagct 6840

ggcgatctgc ttgtcgtcga agttggcgag gatgaaggac tcgatcttct cgatgccttc 6900

cttgccacca accagctcgg cgtactctgc ctgcacagct tcctgcgcct tggcagcgtc 6960

ggcctgcgtg cgggcatgct ctgcgatggt ctgttggttc aggccacggt acaggttcag 7020

gtagccgtca accatgacct tgccgaactt ctcgtccagc ttggcgcggg tgtcggcaga 7080

cagctcgaac ttgccacctt cggcaaacag ctcggtgagc agggccttct cgtcaacgcc 7140

agcatcagcc agagctgccg agatttcaga aggcacctcg acttcaactt gctgatcgcc 7200

gaagtgatag acaggcgcat cgccttcctc gcccttcttg tcgccctctt cttcgccgcc 7260

tttcttgcca tcgccttcgc cagcatcgtc gcctgcgttc tggacatcat cagcattcgc 7320

tccttcttca ccagtgttgc cagtggtttc ggattcggta gtctgcgtct ggtcaccatc 7380

gccgggttcc tctgcgaagt tggtagggcc ggtcgagccg gtcaggtcga ggacgccatc 7440

cttcggcgag ttgtcgatta cttctacttc ggttgccatt tacatacctc cagcattatt 7500

catgagattg tcagcgatgg ttgggacggc cttctgcaag ccctcttgga tcatcgcctg 7560

ttgctgctgc tcttgacgct gcgcctgcgc ttccgcgaac tggtcgtcag tcatcatgaa 7620

cggcagagtg aggttgagct ggttgctgat gtagctggag taagcacccc aatccacacg 7680

ctcctgcatt ggctgaggcc acgctgctgg catgctcacc atctcggtga actgctggat 7740

gcgatccaac tcggacatct tgctcagtgc ctcgatgccg gtcatcaggg tgatcttgac 7800

gtgctccttg ccaagctcca tgccgatgcg gttaagcaac aggcgagcat acggcttctg 7860

caacgtcggg gccagcaggg tgtagttgcc acccagcgcc tgttcgttcg actggctgtc 7920

acggcggatc tcgtaggcag tcacgcgctc ggcgttacgc tggatctgcg agtccatcat 7980

gaaggcttgt cccacgcggc gctcgtactt gtcgagcact gcactgatgg gcgtgaagtc 8040

cgcgtacttc tccaactgga ggacaccgat gtcgtcgatg ttgccgtaca cgtactcacc 8100

cgtggggctg ttaatcaggt ggtcaacgtc ggtggtagca cccggcttaa ccagatactt 8160

gacgtcggcc atcaggatca tgcccttggc gattgcctcg ctcaggaact gaatcatgtg 8220

caggtcacct gcatgcagct ccaccttgga gcgcccatag tcctcgccgt agttcgcctc 8280

ccaacggagt acgatgaacg ggaaccgatc ctcatgcacg cggtactcgg tgcccacgat 8340

gtgaccctca gcttcctgtc ggatgatgaa gaagtcaccg tcgcgcttgc actcggtgta 8400

cagctcgatg ttggtgtcag agtcacgacc gccggggctg gcccggttgg cgcgaatgac 8460

agcttggatc tctggcggga atgtgtcgag cgccttgtgc tccagcagga tcagcttgag 8520

caacgtgccg gacttgtcac gcttgaccac gtagcggttg agcgggtagt tcacggcgtc 8580

tcccttcttg ggagtgtaga ggcaggtgtt gccgatagcc agcaagtggc ggatggcttg 8640

cccgatggca gaccgtccac tgattcgctc gtgctccagc attgcctcct tgacggcagc 8700

agccagcagg gactgagcct taacgactgc gcccgcttcc tgctgcaatt ggatcagagt 8760

tgcctgctcc agctccgtgc cgaagaacgg agagtgtggc gggaacatgg tcatcaccag 8820

tcggttctcc agatgcgtca gtgccgaagc cccgaagctc tgccagccag tggtgttcat 8880

gctgtcgcca tagtcaccca tgcctacgtg gtcgaagtca accagcaggt gtgggatggt 8940

gtagcgactg aatcgcttgc cgcgctccac gaaagcgttg cgcattggct tgagcttttc 9000

gtatgcctgc ttgatggtct tagacttctg accaccggcc tgcttgacgt tggcgcgacc 9060

gcgaagggtc aggtcgatct gccccgtctg gtgtacgata ggtcggacat cgcggttcat 9120

agcacctcct tagacggcga ggccagtgcc tgctaccgac ttcggacgga cgagctgacg 9180

cttgccttga tctgcgccgg tatcgacatc gccgtcaccg agggttacgt cttccggttg 9240

gacatccacc atccgctcag ggcggtcagc ggtgaaagtc tctttcggct tcttgggttt 9300

gctgctcatg ttacctcctt gcggtactca gtacctacgt gggaatatcc caagcgctgg 9360

tagaactcgg cagtctgctc ctcatggata ccagaggcta ctgagagctg cacatgcgtg 9420

gctcccttct ccttggccca tgcctcccag ccctgcacga gcttgtaagc agctcggctc 9480

ccccggtatt caggctcgac ataaaggatc gtgtccatcg ccatcttggc cgggttccaa 9540

ggcagcgggc aacaaaagcc ccacagaaag cccactagct tgttgtcgtc gaacaccagc 9600

aagaagcagc cgtcctccat cataatggtt tgggctgctg attgcagagc gtagtcctgt 9660

tggactgggt tgctcttggt gtccgcctcc tcagcgtagc gaactgacag tggggcgatt 9720

agtagcatgt cgagcaaggt tgcttgacga gtgtggatca tggcttgaat gttgcctcga 9780

tctggcgctt gagcgcgcgc ttcgtgctat tggctacgaa cgtcccgagg ggcgacgtag 9840

gcgattctgg atcttctgcc aagaatcgca tcaaagtttc gtaagcctcc cggccaatgc 9900

gttgcacctg tggagcaccc cgcaagctgg aaggctcatg ggattgtccg gtgtttcgca 9960

tcgacgggga gaggtggctg tcattcattc gaggtcgtcc tctcctgttg cggttgatag 10020

agatgccacc gtagatacaa tggcatcgac ccgtaggcca tggtgttctg cctgctcatg 10080

caggtcttct ggaatctccg acccagcctg agccagatcg aagattgcct tgaaggtttc 10140

gtattgcgtc atgcaaactc cttcttctat agtgcaactt aaaagtcgcc gacggttttg 10200

tcgtggcgtt cttccatgac tttcggcagg cggatcttgc ccttcgagct gtcggacagt 10260

ccgcgcacgc ggtagacctt gttgaccgga ttgtcaacgt ggccgactgg cttggtgcag 10320

aagccgtgga acaggcgctc ggcgtcttcg tgagtccagc ccttgccgag catggccttg 10380

acgaggttgt cgccttccca cttgaagatc aggttcgcca ccttgttggt gtacttgccc 10440

gtgccttcct cgatgccgac gcacagcagg tcgtagttga tctcccgcac ccacttcatc 10500

atgcgccagc ccttgtggcc agcttcccag ccggtctgtg gcttgaacac ggcaccttcc 10560

tcgccgagag cgatcagctc cagagcgaag gcttccacct cgttctcgtt ccacagcggg 10620

cgcatcggca actgagtggc ccggacaggc ggggtcagca tcaggttgcg gaacatgcgg 10680

ttaacccggt cggtgtacac cacgtcggac ttgcccttga tgaactcctc gatggtgagg 10740

tggtcatgca ggtacaggtg caggtcaggc agcagcgcct cctgctcagc cgacagcggc 10800

ttggtgcggt tggggttcac gatgcccgac aggacttcga ggctgcactt gctgttgcac 10860

agttcggcga tgtagatacc gacaggccac agggccgatt gcagttcctg ctccagcgct 10920

tgcacgttgg tcatcaggcg gctggtgcgg ttgaagatca gcacgcggtt gggcaggctg 10980

aggacgagcg cgtatacgcc atccttcttg gtctgcccat acaccggcca cgtcacattc 11040

ttgtgcatct tgccgttgtt aacttcctgc cagtgcttga gcttcatgac agtcttttct 11100

tcgccacggt tagcggtggc acctacgaag ttccagatac gttcgaccat tggtcacctc 11160

agatcttgat gatacggagt ttacgcaacg tccagatgac gaagcggagg aaccagctac 11220

ccttcggcgg gcgtgggttg gtgctgttcg gcaacgccca tgcgtagtcg tatgccttgc 11280

ggtacttgaa ccggcaatca accggcacct tctgcaacat cagcggcagg cggtgcaggc 11340

cacgttggaa gccgaggttc tcgtagtaag cgtgctgaga cattagcgca cctccaagaa 11400

tgcacggttg tagagttcag ggttcagcca ctggcggcgg tcgagcatgg ccggggtgtc 11460

accgttctta ccgtcaacca gcttgcacag gttggggatg aagcggcgca gctcgtgggc 11520

gaagctcgcg tccatacggt tctcgatgga catgtggcgc aggtgctcag gcaggcgagt 11580

ctcggtcgcg gtgccctcga agcgggtgcc ggggttctgg acttccaaca cgaagtcaca 11640

ggcacgggcc tcgttctgcg ggcgcacgtc ggtgacgacg gccagcaggt tcaggttggc 11700

acctgccacc tcgttcagct cctcggccag acgacgaacc caaaagtcag ggtcgatgcg 11760

gcgagcggct tgcccgagga taatcagcag gtcacggcca gagcactgga tgatgctgcc 11820

ttccttggtg tggtcgccgt acactacgat gcggttggcg ctgttgcgag cttgcaggcc 11880

cagctcgtgg cctattgcgt cgagccattc gttcatgttg ctgtgcggaa ggccacgttg 11940

gttcagccac acttcgagca tgaacgggtt gaacttgaac tcacccggca cgtctttcag 12000

ctcgtcgatg aagcggcgct cacaggccgt gacgaacacg gtcatgtcac gcaacgcctt 12060

ggcgaagctg gtacgctgga tggtgtagcc acgtcggtgc atctcgttga tgatgacgtc 12120

tgctgcgtag tctttaccgg agcgcttggc tccattcatt cctagtacaa acatttgatg 12180

atacctcgtt gggtttggtt gggggcaccc tcacgcaagg cgagggctaa ggttcacttg 12240

agaacgttct ggatctcagg taggaagtca gcaaggctga cacctcggtc aacgatcacc 12300

gggaagtaga acgacccgat gcggttgcac agttgtggga cgacggcatc cagataccga 12360

ccttgctcgc gcagcatacc gagacgaaca acgtcgtcct ctggtaggcg tcggcggcgg 12420

acggcaatgt gctgtcggtg cggcgaggta cggaggtgca acacaatggt cggcgggttt 12480

gcccgcagct cctccatgcc cttgtcgatc agcgacatgc gaatggccag ctcctcgaag 12540

tcgctgtcca ccacttggcg catctcgtcg gctacggtct ggacggtgcc catgtcaacg 12600

acacggacgg actcacccag caccttcacc aattgctcag cgatggtagt acgaccggag 12660

gcccggccac ctaccacaac gattaccttg tccattacag actcccagtc cagcgaccct 12720

tggcgtcgag acgcatcggg atcagttgag gttggctgtc gatgatgact gcacaaccga 12780

ggatcggctt gccatggtag ttctccccgt aggcgaaggc cgggtggtca gggtcgatca 12840

ggcagccggt aatcatggcc cagtattgct tgccggtgga ctgagcatat tccagttgga 12900

acttgccatg ctcatgacct acgacgaggt tctggttcag gtgggctgcc acgcccagct 12960

tgttgctgcc ggcctgatga cggaagttcg tcacctgacc attcggcaac tggatctgcc 13020

actcgaagtt ccagctccag ccctgaccgc caccatcagg gaacagcacc tcacggtagg 13080

ttcgcaggta ctcgacaggg atgccgaagt gcttggcctt gcggtacagc agggagccgt 13140

ggttcgagtg gcacagcagc atgcgcggga agatcttctc cagcttgtgc aggaagacac 13200

gcgcttcacg cagctccttg ccagcgctgt cgaggttcgg gtcgctgtcg tggaacgaca 13260

tggcgtggcc gtcagtctcg tcaccttcgt tgataaccag cgtcggcttg tagtgcgcgg 13320

cgacggcgat caggaagtcg agggcgtcag ggtgatggta cggtgcatgc aggtcaggga 13380

tgacgaggat gcgggacgag tcggcctcga agtcatagcc gaggaagtcc tcgggctttg 13440

gctctttcag ctcacgcatg ttcttgatga cgcggttagc cttcgtcaag gtcatgtagt 13500

gctcgccgtt cttcttcgtc tcgtcgaact gacgcgacca gtaccgggcc agctcacgcg 13560

acacgatggt gccgtgggtg ttggcggtca agacatctgc catcttcttg aagttgacag 13620

cgttggtttc cgggtcggtg gcacgcaggc gagctgccag tacctcctcg tcggtgaagc 13680

gttggcgaat gcgttgagac attgagcctc ctatttcggg agggaagcct tcacagtgtt 13740

ggccgtcttc ttgcgagcgg cggcgttctt cttgttccgg gcctccgttg gtgtgaggaa 13800

cgtgggatgc acgtagttgg tctgtggcac cttgtgggtt tcgtagtacg ccaccagccc 13860

cttgaggatg gcgagcactt cgttaggcgt cttggcaccg ccccacctga tgcacaggtt 13920

tcggatcttg ccctccatgc cgttgatggc acggggcaac gccgcccgga tgattccggt 13980

ttggtggttg tgatccacca cgacgttcga gctggtcatt gcacgcaggt cgcgcccggt 14040

gatcgggcac ttgttgcctt gcttggccat cagctctttc ttgattgctg gtaggtcagc 14100

ggcctttact ttcactcgtt ccatgcggta tcactcccca tcggacagtg gctgcttgct 14160

cgccagaggt cacccttctt ggtctgcatc cacgcgagac ggccctgctc cagcatgcgc 14220

tggtaggcgg tcagttgcat ggtgccacca cggaagttca gagcagggtg agtacccggc 14280

ccgtagtatt ccttgtaggc attgagcacg gcgtagtaca gttccttctc ggagttgcac 14340

ttgtccagca gctcgtaggc atacgacggc cccttgcctt tcaggccaga gtagttgtcg 14400

gcgtcgtcgc ccatgatgat ctgagcgtag aagaacttga ggccgcagcc tttcaagtca 14460

gttacgctca cggactcctt ccgacctacg catacgcgct tggtcttctg ttgtcctgcc 14520

ttcgcaccac gcagccacgt atcatacggg ccgggatgca ccttcggatt gacaggctcg 14580

ccaccgacca gcggccagtg ctcgtagtcg ttgacctcgt taaccttggt cttcggccag 14640

agttcgccga gcttgtcgtt gaagcgcttg acctgcttgc gaggatcgta gtgccagcca 14700

cctgtgatac cgctgtcctt gtcggaggag cagaccacag tgttgccgaa ctcacgatgc 14760

tcaggcgagc caacctcgac gcccagcgca gccgctgcgt catacacggc gatgctgatt 14820

aggtcgtcgg cttcttctcc gtccgacatg atggcaccca gtagacgttg cacgtcttcc 14880

ttgagttcat agaagtacgg aggtttgtca gggttacgtt gtcccttgta gtcctcggtg 14940

aaggcgatgt ccagacggaa gttctttgcg ctgtcagtca tgaacagctt ggcagcgtca 15000

cagccagcag ccgtgatcca tgcgttgagt tccttacaca acatctccca cgcatcttca 15060

tactgcgggg tgtccttgag cgacttggcg tgcccttcct ccacgaggta gagcgcctgc 15120

gtatagctca ggctgtagtg tgctgccact gcgtacccga tctggtacgg caacaggtcg 15180

gcgtcaatga gcgcaaccat gttgccctcc gtgggccaca gcttatagtg aatgtcgcgc 15240

tcggcaatct cgccgccgta gttgaaacca tcggtcatca ttaacctcca ataatgagga 15300

catgataatg atgataatgg tgtggacaaa ataaagcccc accctagttt agggtgaggc 15360

tactggcgca ggccggagaa ttaaccctcg aaggattctt cttcggcgac cggggcttcg 15420

gcttcgggtt cagcggctgc ttcttccttg gccgggacac cgacttcctt gtacagcagg 15480

taggcggcga ccacgttggc cttggcgatc aggcccttgt cggtcgaacc ttcggcggcg 15540

gtggcgcgag cgaacgacag accttcggcg aagaacttca gggtgcccag ctcggcttgc 15600

gcttcggctt cggtcttgtg ggctttctta ccaaccagct caccggactg ctcgtcgatg 15660

acgacgaact ggtctacgaa tttgccagca acaacggtgg ccagagcttt gacgatcaga 15720

ttaacttggg acatgagaca cctctctttc atttgggatg ggcgagcaac attgctctat 15780

ctatactgca acttaaagct ggccctgcca ccgctggtta tctaccagtg ggccttagca 15840

cttgctttat ctatactgca acttaaagca gcacggtgta gcaccctgcc cggttcgacc 15900

ttgctcaatg agttcggttc gaccatgggg tgctatcctt gatgccttgt gcttaggtcg 15960

gtgcaactct tggctcaggc ttaccgcaag gggaatgagc tgtggttgac cacggacttg 16020

ggcagactgt ctcacagtgt gagactacta cggactgaca ctatgcgacg ttatgcgcgg 16080

tcagttaagc gatgcgtttc acacgcacga ctgagtgctc ggggaagtcg tagttgactc 16140

gatccccggc agcgtcacgg aagttcacgc agacgaagcc gtcacggtag acgacatgca 16200

ggtcttcggt gccgacgcct tccacgaagg ttgggccggt gtacgaatcc gcctgctcga 16260

cggtggtaac ttccgcgtcg tcagccttgg ccagacgtgc gtggccagcg cggatagcgc 16320

cagccgggtt caggtggatc tggacgagac gagctttcat tcgccgaact cctgttcttc 16380

atcgagctgc tcgaccttgc ccgctggctg ctgaggcttc tgctcaccgg agtcatcacc 16440

ggactcgtcg tccgacttct tctccttgcg agtgtagcgc tccttgtccg cgtcgaagat 16500

cgactggatc agctcctgcg acgggtgagt accggccttg aactcctcgg tttccatgac 16560

ggtttcacgg aactcgcgga cagggtgcag gcgctccagc acttccttgg tcagctcgcc 16620

ttccatcagg aagccgggcg caccttcgag cggggcgaag gccggggcgt tctgcaacag 16680

ctccagcgtg tcttcgccga tctcggccat gttggagaag ttgatgaact tcggcgtgcc 16740

atcttcgttg gtttccttgc cgcccttgag cgacagagac acgagctggt tgcccatggt 16800

ggtgaagcct ttgtgcttgg ccatgccacc catcgccggg atgaaggtct tgtgcaggaa 16860

cgacttgtcg cccttcttga gcgggaagcc cttcacgaag aacatcgggc tgccgtcgtc 16920

cagcttgtct tccttaccca gcaggtggaa gatagcgaac gcatacggag ctggggcttt 16980

cacttcaccc ttgaagactt cgctgaacga gccaacacgg atcagtgccc acaggcgggc 17040

gttgcgggtg cccactttgg gctgcttgaa ctggctcacc gtttcggcaa cggcaccacc 17100

atagtcaaat gcgtcggaca tgtaacctcc aatgatgttg agagcgaact tgctctatct 17160

atactgcaac ttaaaactgt gagactaagc cacctagggt gtgagacttt tcctaggtgg 17220

ctcgctcatt agtgcgtatc gtgccacgac ttaccgatct tatattcccc ggccagcggg 17280

atgttcagct tgaacaactc gccagtcttg gtcatcgtct cggcaaggat ctgacctgcc 17340

ttgtggtact tgcgggagca cacgatgatg ccgtccttcg cagacacctt gctggcagca 17400

gaccacatgc gaccgtcaac gtctacatgc acacgcttct cctcgacatc gaagacagcc 17460

ttgatggcct tcttctcgtc ctgcccttcg gccacggtgt aagggagttc gtactcgatg 17520

ctgagcactt cctcctctgg cacctccatc tgcacctcgt catgcacgtt ggcaatgaag 17580

cgaggatggc ccagcttgtc gagtgcaatg ccctcggact tcatctggtt ctcagcgagg 17640

cacagaccgt acttcatggt cagggagccg gtcatctgga gcaacacgtt caacacggtg 17700

tgaaccttga tcttattacc ggagcggcgg atgcggcccc agcgtccgtc gatggcatgc 17760

aggtagccgt actggttacc ctctgcttcg aggcggtcga tcagtgcagc cagcgttggc 17820

agctcacgac ggaagcgagc aacacgctgc tccatctcgg cttcgctgat accacacacg 17880

cgggcaaggt tctttatgcc agagccatac aggaatgcgt agatgaatgt cttcgcagtg 17940

tcacgcttga gcaaaccagc gagctgttgg ttgtggctgt ggatgtcacc gtggagcacg 18000

acctcgatgt acgtcgggtc attcatgaag tgcgccaaca tgcgcagctc cagacccgag 18060

ccgtcgcagc ccagcaccat catgcccttg cctgcaatga acaggtggcg caatgggtgc 18120

aggccgcgag acgggatgtt cacgacgtac ttgtgtcgca tgcggaacgt gttggtgccg 18180

atggagaatg cggcggcagg tacgcgccac tcctcgtcag cacttaccgg ccactcaccc 18240

atgcgggcga agtagtcctg agcctcgcag ctatgctcgc ggttgaatgc tcgggccatc 18300

aggccacgac actcacgctt gccgttcgct tgcttcggcc actccttgtg ctcggcgaag 18360

tattccatat cgccgacgtt caggatctgg ctccgacggg agcgcaggac gtaccagctt 18420

accagccctt gcaggaactc ggggacggag ccatcacgct cggcccacgc cttcaacgac 18480

gcttcgtcaa tcttgccaga ccatggcttc ggtggctcgc cagacgggtt ctcgtccgcg 18540

ttctccatcc actcctcgtc accatcagag aagttaacac cgcgccaccc gcgaggatag 18600

agcacgttct ctttcatgta ttcgaggttg ccgaggccga tctgctcgta cacgataggc 18660

gtgaacgggc cagcgaccag cgggtctttg gtgtcgttga cgttgccgac catctcgggg 18720

aacaccttct tgaggttggc cgagtagtcg ccggacttgg ttacgatggc ccacattgtt 18780

gctcgcttgc cgatccgttg ttcggtgcgg gtaaggagct gggtcagggc ttgacgtagc 18840

cacgccggat cgtcgttgaa ggctttctgg taagcgttgc aggtggcggt tatgtgagcc 18900

atcttcatcg gctcggtcac cagacggtct gggatcgctg ggcggatctt gacagcgatg 18960

tcttccatct gcttgccgag catcttccag tcttcccacg cttggtgcat gtccagacgg 19020

aaaccacgca gcgcctgacg agtgatcgcc agtgccacct gagtctccat cctaagggca 19080

gaggagatgc cgaagcgggt gtccttgttg acgccacgct tgacagcctc ggcccagtcg 19140

ccgtgcatca gccagaagaa catgtccttg ccgatgatcg tatcctctac gcatcggtgg 19200

atcatgtggt cggtcaggtg cgaccagtcc tcgttctctg gcttgaagcg gccaatgcgg 19260

atgccgtgcg cagcaatgct gtgcggcccc acgttgccca cgcccatggc aaacgcctgc 19320

ggtggcggct tgcggtcagg gttggtgagc tgggagatca gcatggtgtc catcatgcgc 19380

atcggaaagt acgcttgatg cgggcggtct ttgccacgac gctcaaggta attgtacttc 19440

cagacgtggg gccacacgta ctccagaagg aacacgtcat agcccacgcc gttctggaac 19500

accagcgact ccgcttccat catggcatgc agggcatctt cgaggtagcc gtcttgctgg 19560

tcctcttgat ccagcttcac ccgtgcagcg ttgcgcttct cgtagggatc gaagaacacg 19620

aacgtttcgc cagtgaaggc gtcggtcagt acgatctcat gcacgcacga tgggtcgttg 19680

taacgaaggt cgggcagcaa gccctcggct tcactatcca cgatccagaa gcggccttcg 19740

ccgctgatct tacgctggcc ctttggccac ttgctcgggt agaactgcaa tgcgtccatg 19800

atggttactc ctgcgggaag atgtcgtctt gctcgggctt cggttgcggg ttgattgcat 19860

cccacttggc ctgcatgtag tcgatgcggc cttgggccag accttcgcgg gttacgcgca 19920

ggccgtgctt gtcaggatgg aacatcccga ggaacaccgg ggtggtgccg cacatgacga 19980

actgattcag gttgcaatca tgctgcgcga cgaggttccc ggcgtcatgt tccgtgtcat 20040

aaaacacgat gtcgagccga agccggggaa gctgtacgac cagtcggacg cagtgatctt 20100

cttcggtagg ctcggacggc gcacaggcgg cggcgcgggg atcgtcgagg tagtcgttgt 20160

cgctcgttgc ttttgggtgc ggctcgccag tccagtacga cttgacgacg tgcgcatctt 20220

cgagacgggc gacgagatca gcgaggtcgc tgatgctgta ctcttgcaca ccgacggcga 20280

tgtcgatgtc cttggcgtta cggtcgtagt ggacatcacg ggcgaagcct ccgcagatga 20340

caccttgaaa gccacactgg ctgaggatgt cgaggattgg gtaagcacct gcaagcaggg 20400

cacggttgtg gtgattcatg ggggcacctc actaggttgg gttgttacct tttaagcccc 20460

acctccgagg agatggggca gggtgttaca gctcggggcc ttcgacttcg atgacgccta 20520

cgtcgaggta ggggtaggct tcctgcaact gctcgcggat ctggttggcg agcttgtcac 20580

agtcggcgtc gtcttcgagg tgatcgactt cgagggtcag ggtcatgcgg gccatggggc 20640

ctccttaacg aatggagatg gacaggtcgt actgggtacg ggatttgtag cacacggatg 20700

cggcgacgga cagcacgagg aactcgtcgt ccgggtgctg cttggccaga cggccagctt 20760

cctcggtcgc ctcttgcagg gtcaggtgca gcttggtggg caggccaccg ttcttgcgcc 20820

acacgtagaa ctcaccgaga tccgacacgt cgctggccgg tggcaggctg agctgctggg 20880

cctcggtgag cagaccacgg ttgaacgtca tgttgcacgc gcaggtgtag acgtgggcgt 20940

tggccttcca gtgcgcaggc aggaacatgc tgcggaagta cggcaggctg tactgctggg 21000

tcacgccgtt gcggaactcg atgaatgcac cgttcatggt gacggtcttc accttgccga 21060

tccacatgtg ccgggtggca ccctcgcggt aatagatgac ttccttgccc tccagcgaca 21120

ggacagcgcg gtcgtcgtgg cgggtgatgg tgtcggcgat ggtcttatgg gtcatggtca 21180

atcctcatta gttggggggc gaagcgagac tgcttcccag tagtctcacg ctgtgagact 21240

accagtgaaa ctagctctgt tgcatggact gggtgacagc caccttatcc agaacccact 21300

tggtcagctt gcggcactga tccgcatcgc ggtcttgcag cacgcactcc agagggaagg 21360

cgtagccatt catcccagca ggcaagggcg tcaccgcaga taggtagatg cccagcgggc 21420

tagtccacga ccagttgatg cgccactcga tgtacttgcc gaggacacgc accgcatcgg 21480

ctggacggat gccagccttg gtcagtacct cgaacgcacc gcccttcacc ttggacacga 21540

aggtcgaaag catggcgctg tggatgtcgc gggcagtgtg cgtcacgtcg ttgaagttac 21600

gtgcgctgat ggtcaccgcc tccagagcct cgtcagtgcc gatggcttgc aggtctgcga 21660

tggatacctt gcccatcagc aggtcggtca ggtgttcgcg tactttgtaa gtgttcatct 21720

tgattcctcg tagttggggg cgagggcacg aacgtaccct ctatctatac agcaacttaa 21780

agtctcacag cgtgagacta ataggcttgt ggttcgtcca gatcaaacgg gacatcaccg 21840

gggtctacga ccggctcggt cagtggtact ggtgcggtgc gattgccacg gctttcacgg 21900

gtgcgctgtg ggtcatcacc ttggtcgaag ttgtcacggg cacgctgctc tggtggcttg 21960

ttgtctcctt tgcggcctac ctccggcagt tcgtatacac cctccagctc ggtgtacttg 22020

ccagtcctga tgtccttctc tgccaccacc acgctgccta ccatgtggcc gatgcctcgg 22080

ttcttgaggt tacggtacag ggtgatgcac ttgtggcgga agacttcggc catggtgtta 22140

cgctcgattc cccacacggc attggcccaa aaggtaatgg agccagcacc acggaagtca 22200

ccctcgtaca cctcgccacc ttgagtgtgg ccgatgcgtc cgttgccccc gcctaccttg 22260

ataaggtgag acagcaggaa gatgttaacg gcgttctcgt ccttgaatgt cccgatgcgc 22320

ttcatcgttt cgtcaatcgc ctgcacgcca gtggccactt ggcccttgtc gttcttgtgc 22380

tcgaaggcag tcaggttgtc gatgacgaag tatttgtagc ccatggacag cgcgtcctgt 22440

agcacctcca tcaccgcatc cacgtccttg ctaccgccga ggtcagcgat cagcaatcgg 22500

tcttggttgt cgagcatgtc gagtgcgtcg tccaagtcct gttgcgtgta gtcgcgggcc 22560

gggttgtact cagcgcccag ctctacctcc tcgggagttt gcggcgggga gttgaagtcc 22620

ttgccgacca gcatgccagc gaaggtacga gccacctcct ctttctggtt ctccagatag 22680

acaacgacga catcttcgcc catgtccatg aggttggcga cgtgcgccat ggtggtgtcg 22740

gtcttgccca caccagtgcc agcgccccac acggcgaggt agtgcagccg cacgccaaag 22800

gtgatcttgt tgaatccctt gagccagtac gacttgccca tctccaccat ctggcgggcc 22860

ttggccttga tgtcacccac acgcttgagt ttgcccttga cctgatactc cgccgcgttg 22920

aataccgcac tcacaaactc ggagtcgcgg ccattcttga ggcagtcgtt cgggtctttg 22980

ttgccgtgtg gcatggtcag cttcttgagc gtcgggccgt tgcgcaggag cttggacacc 23040

tcgcgggcca gcttctcgcc cgtgtcgtcg tcatcaaagg cgaacaccac ggtcttgaac 23100

gagcacagaa actccttgtg cttgatgaac tcctccagcg cctgctcacc cttgttcggg 23160

ccgtacacat ggaacagctt gaggccatcc agattggtgc gggagccgag gccatcgttc 23220

aggccgttca gttccttaac catcatctgc tgggttgctg ccacgtcgca ctcaccgcca 23280

acgaggacgc agatgccacg gcgttggccg ctgtctgcga tgcgcttggt tgcttgcatg 23340

ccgaacaggt cttgatcccc gaacagcttg ccgagatgcc caaaggagaa gtccttcggc 23400

agggtgcggc acttggcacc gaccagctcc ccttcctcat agcgaggata gtagtggcgg 23460

ttaaccttgc ctgcttcgtc gtgccccacg cggataccat acagcgcagc gattgcacca 23520

tggatgccac gggtcacaag gtgagaacgt ttcaggtcgc cgaaccattc gacttcccga 23580

tcccattcca cctgcttctc gtcgcgctca gcttcggtca tcacttccca gcgatcagcc 23640

atgcgcatgc cgcccaatgc caaggcacgg agcagcgggt ctttcagctt gccttcgtgt 23700

tccatctgac ggaactcgcc ggggctgtac ttgatgtcgc cagtgattgg caactggtcg 23760

attggggtgg cctcacccgc agcctcgtag tagggcttgc ctgtcttgtg gaagtgacca 23820

cgagaacagt agccgccacc gtccttgaag cggataagat ggttgcccgt cttgtcgtgt 23880

ccgcactcgc ggcacttgac acacggttcg ttcttgagga tctggctcat gcgtgtttct 23940

ccaccgacag ccagccgacc cgcttcatct tccgttgagc tgcgtaacgt tcctttcggg 24000

cgtcaggctc ttggttgcgg cggtctttct tacggctgat cttttcgtct actcgggaca 24060

tttctgtcac tcctaaaggg ttggttatct atgctgctac ttaatagaag tcatcaggct 24120

ctacgtcgaa cagagtctca cactgtgaga caagatcgtc atagctgatg tcgtcgtcga 24180

actcgtggcc gcaacggcac tcacctgaga tgcgcacgtc gtgctcctta gatgcctcgc 24240

gcacggctgt ctcgatgttg tacgttactt gggtcttgcc aagccagtta gtgtacccat 24300

aggcaacgac catattctta tgacggcaga tgtttcttct catgactgct attccttcta 24360

gatgatgata agtggagtat ggtagccact gtaactacta gacctactcc ctactactaa 24420

ccctaatggt cactactaag gactactggt cactactggt tgctactggt ggattctcct 24480

atactgctac ttaattaatt ctcacagaaa agtctcacag tgtgagacag aacgaagaaa 24540

gcccgctcag atttctccga acgggcgata ataaatcagg cgaaaatgta cttggactct 24600

cgtacagtct ccagatccag cgtaccagtg gccgggagat ccagctcaag gtcagccatc 24660

actcggtcgc actgagcttc gtagaaatcc ttaacaacgt cgtgcttctg gtacatgtca 24720

acgaaggaat ctacaagaat cttgcgcagt ttggcggtgt tgccagcgtg cgtaccgaac 24780

gagtcgtgga taactgcgat gctgtggata ccgttgttgc tgaaaccttc ggtcgccagc 24840

accagatggc ttgcgtccat gctgtgaacg aagttaggag cagccgacga cttcatggca 24900

cgagccgaaa gctgtgggtt ctcctcgcgg atggtgaaga acgtgtcgcc catcatctgc 24960

gtcttgacac ggcgagcggt gctgtcgtag atagcctgat gcacgatgaa cccggtagga 25020

gtcacatgct caagcggtac gttgcggatc gaaacttcgt gggtcacctt cttgatgaac 25080

ttcatcgccg cacgagctgc aacaacgacg tgaccgatac cttcccaagt cagaccagac 25140

gcgaacagct cggcttgcat ccggctcatg ctacccttgc catcgttgaa attatgcacg 25200

gcagtaggct tgcggaactg ggcacgggcc ttgtcgttct cgtccttctg gagcgagtcc 25260

agatgctgag ccatggactc acggcaggtc atctgagacg aaccatacgg cagggtcatt 25320

actggcttct tgcacaggct acgggtcacg ccgatgcgca gccactcggt agcgatcatc 25380

tgcgactcaa gcgcgttcaa ggtgaacggt ggagccatgg cagaatccca cacgccgcca 25440

tcttctacga ttgccttgtg gcgggcagcg gccttgctca gcatgcgctc gaacgactcg 25500

cacggaacgt ggccgtccac gatgtcttcc atccagcggc gcacgatctt ggctacagcg 25560

ccgtagatgt cctgtggtgt agtacccggc acgaggttaa cttccttgcc gcctacttcg 25620

tcgcgcagca tggcgctgta gtgctggata ccggagcagg aaccatccat tgccaccgca 25680

acgcggctca ggaacttctc aggtgcctca gtctcacagt gtgagacaaa atcggcgtac 25740

tcaaggcacc atgcgaggaa ctgccacggc ttgtcggcct gagtccagtc agtgaaggtc 25800

agcgggtcgg cggcgatgtc gaggcacagg tcaatgaacg agtcttcact gaccagcgcc 25860

acgcgctcgt caaactcctt cttgtcccat ccccacacgt tcgcgccgtg aaccttgaac 25920

caatattcgc cttcgtctcc cagcgccatc gcgttagcga actggcacag cgctttctgc 25980

aagtcgccgc cctgtggcga gatcagcgag gattgcgcat agacacgacc acggaagtcg 26040

caggtataca cgaagtgcat acgctcaaag tcttgatact gcaagccttg gtcaatggtg 26100

gccgatgcct cacggtactt ggccttacgc tcggcctcgt cgttgtaggt cttggccgct 26160

tccatcttcc acgccttgaa agcttcttgt tgctcaggct caaggacgtc catcagctcc 26220

ttgccgcgaa gctcggagaa catcgacgga accgggcacg gaggcgttgc cagcttctca 26280

cgcgcaggca gggccaacgg caggccgcgc agacgcgcct cgtttgcaac ctgcaagata 26340

cgcttgttaa cgcgccactc tacgccctgc aatgcgttca cagccgcata gacgctcggc 26400

atttgcttgc gggtcaggcg cgaaagcacc ttgtggtcac ggcacttaac gagcggcatg 26460

ccacgttgca tcttgggcgt atgccagcca cctgagcgcg ggccagtcca atcaagcgga 26520

cgaacgacac aaggcgcata agcaggcgac atttcaccca ctacggcctt gtactcctca 26580

atccattgtt ccattgcctc tgtggcttca atgactacca cttcatcacg aacaccccga 26640

tggatgttac gcttttggat aacaggcacg ccattgagca acatgttgtt ggcaaacagc 26700

tcaatgagct tagagccaag gttgaaaata tcgttgtcgc tccagcccac aaaacgctca 26760

aggtctaccc cgaacttgtc tttgcgggtc tggtcttcac tcagcagctt ttcagcatgc 26820

accatcacgt cgtggccgtg gccgtacttc tgagacgatt ggcgcttgag ggattctttg 26880

attgcctgaa tgtacttagg cgcggcgttc tccagcttgg taaaacgcac ctcgtcttca 26940

atacggcgac cgatagtctc ggccagattc tgcgcggtct ggttaggatt catcagacca 27000

tcaaagatgc acttgattgc aatgtagcta ctcacctcag ttggcatgca acggagatgc 27060

acgaggcttg cggacggacg cccgcgacgg ccttcgtagt agtctagata ggcttggatg 27120

ccatcggcca tcggcttgac gaagttgcga gtcagacgac ggaaccaatc ggtgtcagaa 27180

gcggcaccag cggcaatggc gcggtcgttg ttgcgaatga atcgctccat acctgcgccg 27240

tgcattttgg cctctagggc cagttgtgtg tcaagcatag tcattcctta cttggattcg 27300

gcgcgtaccc cgtttgctat gcaatagatg gtatcgcgct cggtattggt caagcagccc 27360

tgatagagcc acttataata caggttctta tccaatgagc gcaagcgtgc ggcgaactgc 27420

gcccgctgtt tgttcaacgg gtcagtgtag attgccatca cgcgcttctc aagctcggcg 27480

aagtccagtt gcagcgaggt cggccagctt tcgtctagac tcagttgagt ttcgtcgtgg 27540

tactggcgcg acttcaatgc accaaacagg ctaggcccga tcgaattagc gtccattgaa 27600

tgaacccaat ttgcggcaat gctatgttgc ttcatggcgt gatgttcctg ttattcgacc 27660

aatgttagtc atagatgcca ccattctaga tggcatctag tctaccactg tgagacttat 27720

tcagcggcca gcaagttgtc gcgggctgct accagaagat caaaggcttc gtgaccgttg 27780

ccatagccag ccttgaccag accacggaaa gcggccttga tcttgctttc cgacttcgcg 27840

tcatcctcgg agataccgag cacgacagcc gggtttttgt gcttgaactg tggcagtacc 27900

ggagcactag cgcccttgtt gtactgagtt tgcgcgtctt gcatctcctt gatttgagcc 27960

agaagctcgg agatttgctt gcgcaggtcg gcgttatcgg ccagcaattg cttgtcagcc 28020

tcgccgcccg ctacaggtgc gacaggagcc gaaggctctt gcgacccgcc gctaggcgcg 28080

ctattttcgc ccgcttcacc ttctacgccc ggcgcttgtg gcagttgttg cagcgagttt 28140

tcaccgtctt gcttgctggt gtcaatctgc tgctgcttag gcgcgatagg ctcgggattg 28200

agcaagagat tcagcgtggt agtggtcagc gagccattgc tagccagctc ggcggccttc 28260

tctaacactt cgtcgtctgc ctgagtagcc agcgcataca gtacgcgcat gctaaccccg 28320

gtaaagcggt cgtcattacc gaacgtctcg gcaaccttca tcagcttgta agcctgcgct 28380

ttcttgatgg agaagtgagc gaatgcccaa tccaagaagg ccgtagcctt ctcgccttgc 28440

tccttgaact cgcaattagc ttcgatcaga agtttgccaa ccttgaccga ttcaagctgg 28500

atattgtcaa gagattggtt aatctcaatg acaatttctt ccatgcgagt tacggccagt 28560

tctacgttgg tgttttcgat agtcatgatg ttagttcctt gggtattgtt cggccaatat 28620

tagccaccga tgcacccgcc tacgtggtgc atctagtcta ctactggttt gttgggagcc 28680

agatacagcg gcagttcatc agtaccgcta ggctgtaggt tccggctgct acttgacagt 28740

tacggcaatg gcgaaagcgc ttcatggttt gagcaccatg tagacgacat cgggttgccc 28800

ttctgtccag ttcggttcgc gcttgtactc agtgaatccg tgccgcttgt agaagtcagg 28860

caggaagcca tcgaagcaat ccagcgtttt accgccttct tgaatcgcct tctgtacaag 28920

ccagctaccc gagacgccca agctcacaag agcctgaatc tcgccgtcaa tgacagcaaa 28980

catgccggta agacgctggc cgtccttcac gagaaacagc tttgcgtagt gcttatagaa 29040

catctcttga atgtccttgt gtggcgtgtt tgggtagcgt tctttaaact tgaggaagcg 29100

ctgagccagt gcgctaacga agtcgtcagc cttggtcagt gcctcaccat gtttatgcac 29160

tcggcgagcg tgtccaagag cgtagtagaa cttggtaacg tctgccatct gaatgataat 29220

gctagccatg gttgttgctc ctatgatggt ttgagtgttt cggaaagccc tctgttacca 29280

aagggcagac ctgaaacaaa caatccaagg aacatcacta atccagattg ttaaagagcg 29340

tatcggctag ctagttgcta gctgatgtcg gtcattctac gtagtgctta gctacctgtc 29400

aacaactatt ttcaatcttt ttcgttgtct tgtgcgagcc agtgagacgc cgtatctagc 29460

gcgtcctcct tgtcgcttgt gtggtagtcc tctgatgggt tgcggtagac acggccaaag 29520

gctgagatgt tctcaaacca taccgcgtac tcctcccatt catcgttcca ccatacccgc 29580

acgcggcgag tgagtgttac cagtgtgcta ggcgcttctt gcgagtgtac tagcttcatg 29640

ctgcaacccc gaggataacc agcgaggcga taactacagg cgttagtagt gccacgatcc 29700

cgcttgctgt aagcaacgag tagaacaagg cagcatagcc gtatctaaac aggtagtagg 29760

tcatagcgta gtctcccatt gtgagacaag tagctaagca ctacgtagag cgaccgagtt 29820

gttaaagagc gtatcaagta gctagtggct aggccgtgtc gcgtcttgat ggttgccatt 29880

ctacagcctt cctacatggt gtcaactacc tttcgcaatt attttctaca tgactgcaac 29940

ttaattccct accgagtagc gcccgagtta gctaccgagc cgcaacccta ggccgctggc 30000

catcgccctg acctaccctt acaccttcct actaccgagt agagcaccta ccagacgcta 30060

ccagcggcta ccgagtaagg aaccgagtta gcacccgaga tgcacccgag gcagctacca 30120

gcggctacca gtggctacca tggtggaacc ttgtaagcat gcgagatgca acaaacagat 30180

gccgcccggt gcgtctggtg tcaactgctt tcggctgatt atttcttgtc tcacactgtg 30240

agactgccag cctgtcacca gtcctacccg attagctacc tgtctgcttg cgtatcagcc 30300

tcacagtgcg cccgattgag cgcggtagcc atcgtctaac ccttaccctt gcatctcccc 30360

tactgcctag gcaggccgct agcagctacc gaggcagcta ccgaggtggc acccggtcac 30420

cttccaagag gcaagggcac ccccttcgag ccaccgagtc gcccgagggt ggcacggggg 30480

aatccggcgg cggctaccgg cgggaaaccc ctcacacgta caaaccaatt ttgattctgg 30540

tctgctagcg agaagccacc gaggtagcca ccgagatcac tctcttaatc ttccttgtca 30600

tacccgttca gggcattaca caggtctact gcttccgagt agtctaccag tccctgcgtc 30660

aagccaccta ctgacgatgc gtcaacaagc ggatgctggc agcgaactgg ttcacttaca 30720

acggatgact gagcacagcc cagtacggac aggctcaggc acaggagtat cgcggaacgc 30780

aggagcttca tcaagcacct ccttcacctt gagtctatct gtctgtgcct tggtagccac 30840

cttgggcacc tgagtgcgca gggacttgac cttggcttcg gactgctcca gcttctcctt 30900

gtagtcatca cgttccttgc ggtaggtgat ggctctatcc cattgagaat aggcaagaag 30960

ggctaggagc aggatggtag cgatcgtcag gagcttctgt ctcatagacc actcctgcac 31020

aatgctgcat cgcttgctcg tctgttgccc agtcccttgg acggatagcc accaatggta 31080

gccttatagc cgggagccat cccgtacttg cccttccatg gggcttcgat tgcatcgcag 31140

gcagccttcc agttgcgagc acgtaggtgc tgcatgtaag gcgcttccac gtagctgttg 31200

gtgaactgcg agccacgcca cccggacttc ccgaggttga tagcggtgct ggtgaaacca 31260

gccactaccg tgtcgggagc gcccttcggc atgtacttca agacagcttg catgtactcg 31320

ttcgtggtac ggagcaagtc cttgtcgcac tccagttggg tgtactgcat cttagggata 31380

ccctgagtct gcccgtaaca ccacgtctta accccgccct gatcgaggta cggggtgagg 31440

gataggccct ccttctcggc aacatggacg ctagctgttg ctagcgccac cgtcgcaccg 31500

agggccagaa gtcgttgctt taacacgatc cttcctccat gcggcgaact tcattactaa 31560

gaagacagtc gagagcagga agtagatccc gctcactgct gccacgatgt ctggtaaact 31620

ccatcccatt atgctcacgc ccacaacagc agcagaggcg gtgctgttgc cgatagtacc 31680

ggcatcgtcc gccagttgct gcaagattgt cattgtgcct cctccgtagt tctttggttc 31740

agtaaggaac ccgaaggcgg tgctggaggc gagcgcttcg cgctacaagg gactctcagt 31800

gccttcgagt ggttatggtg ctgcggggcg gtcgagcacc cacaggttgc cgttgttcag 31860

catgttgacc atgcggccag caggaacagc agcggcccag tcaccttcgc agataaactg 31920

ggtggcagcg ccttgggtca cggtcaatgc tgcaacgcca gcgacagcgc actggatgct 31980

cttggtcatc gcagcaaagc tgatcttctg gtcggtgcca ttgcacacgt acagcttgcc 32040

atcagacgca tcggagatgg tcagggtggc atcggagaag tacgtcagtt cgagcttgtt 32100

gctcacgttg atgttgccag agccatcagg gccgttaccg ttgaccgtag taactacgtt 32160

cagggtcagg gcaccttcgg cgtttggctc aacaccattg atcgaggtca ccgtaccttt 32220

cgggatgacc agctcgatgt tgccagcttc gtcaggctca acgccattga cggacttgac 32280

agtaccctca ccgccaccac caccttgacc catcgctacc cacgcatccg atgcacgatg 32340

accgagggcc atgtacaggg agttgtcgtc gagcaggatg gtagcaccac gggtcttgcc 32400

cgagatgctg gtgatgttga tcgggtgctt agggtcgctc aggttcgcaa gaacaatcgg 32460

agcgcccttc acagccttgg cgaaagggac ggtcaacgct ggagtgtcct tgtccttcgg 32520

gtactcaccc gtgactggag cttgtgccat gttgacctcc tacttaggcc ggggttacgt 32580

cagtgccacc cttggtgaca gcgatccact tgtcggtcgg tgcttcacca gccgcttgga 32640

acaggactgg agcaccggag acaacgacgg tcaccagcga gccaacacgc ttgccggaga 32700

tcacgcggtc gttcagtacc gacttggcat cgctcagcag agcagcagcc acgactggca 32760

tgttgtgcag ggtgacgccg tacttctgga cgtctttgtg acctttctgg tcgccgatga 32820

tgtttggagc ttgggccatt gggcacctcc tagattctat ataagggaac cctctctctt 32880

agaaggagag agggagaata actactggag ccactgggag ctactaagtg ctaccatcac 32940

ccataggggc tactgggcac tcctttgcta cctgtggctt ctcctatact gcaacttaaa 33000

tacccatcgg gctacaggcc acggaatacg cggcctccag cggttttggt catttaatgt 33060

acagcagtct cacactaaat cactgtgaga ctaatgaccg gattgctcaa aatagtcaca 33120

gtctcacagc gttagactag ccccacgagc gccggggaag cccaccacga gtcgattggg 33180

cagaccctga gaagcggttg cccagcttct gaaagcccct gtcaacggcc atggcggcct 33240

ctgcggcgat gccgagcgtc atgttctgca tgtactcgac catctgtttc gggttgcgca 33300

tgatctcgaa gaaccggccc tgctgcttga tctgccgctg caccacgacc ttgagctggt 33360

cgtagtcgag gtctgcaacc acgatggcga cggctgctgc cacggcttcc aatcggtcgt 33420

cgtgtcgcaa gcagtccttc tctcgggtga ggttcgcaag ctggtagaac aggctgtagg 33480

acatgcgagt ctctgccggg tacttctgga tcgagtccca gtctgcctgc actacttcct 33540

gccgcaccac gaagcggtgg gtggacagca caggttcgag gttgtctatg atgcgaagtt 33600

ccttctggcc gacgctgtga acagcttcga tgaccacagg ccactcgcgc tcgaagtacg 33660

gcttgagtgc cgcgatgtgc gcaccgttac cgtagttgtc ctcgatgaag acttccttgc 33720

actcggcacg cttggcgatc tgcacaagct gcatcagctc ggactcgttg tagccaccct 33780

tcacgccgcc agcttcccac aggtagaccg tggtgccaat gagcttgatg atggcgtaac 33840

cagtttcgtc cccgttctta ccgccgcctg ccgggtcaat gaacatgaca gtacgctcga 33900

acttgccgag gtcgtagctg acctgcattg gcaggaagaa gcggtcggag ttacgggagc 33960

cgaacttcgg tgcggtctgc cagtgagtgg cagacgtgtt gcaccagatc ggcatgatcg 34020

ggccttgatg cagcgagaag ttcgccacga cacaatcgct gatacggagc gggtaacggt 34080

cggcgtcgga cagtccggtg ttgagcatga actgcaacat gaacttggcc ttgccttggg 34140

tcagctcctt ctcgttgagg atgtcgttcg ggtacatctc agggcaggtc ggttgcccaa 34200

gggagccatc caagccaccg ccgtattgca gggcagggtt gtcctcgatg tctcgcagga 34260

ggatcggtgc gagcttgtcg ccgtagtcct ccatttgagc cgctgttgga taccgccctg 34320

tccagatgcg gacttggtag ccacgcgacg gcaaccagtt gtagatggac tcgaccgact 34380

gtggcgtgcc caagtagatg atggaaccga actggttgat ggactcgaac tcaagcgagc 34440

tgtccatgag gatctgtcga cccccggctg ttcggctgtt ctgcaacgac tcgatgtcgt 34500

ccgcgataat gaggtcagca cgcgcaccct gcgcacccga ttcaatcgag tagcaggaca 34560

cggacggcga cttgtcacta ccgcgtaagc accagtggat gtcgaacgct tcaatggagc 34620

tgcggtcacc cgcgaacttg tcagggcgca agcactccaa gatgggcatg gcgtagaaga 34680

tcttgatgat ccagcctgcg atctccttgg ctcgcttcgc gttctgcgag aagatcacga 34740

tgcgatagtg cgggttgtgg attaacataa agacagcata gatcgcggtc agggttgtct 34800

tcgcctgacc acgctgtgcc atcaccattc ggtatttgtt acccatgagc aagaacgaca 34860

agatgtcggc ttgaatccgg ttgaggttcg gcttacccac gatcaacgtg ttgatgatga 34920

cttgcgccag atagcacaag cccatcaccg tgtaagggaa cgcctgctgg agttctacca 34980

acttcttgtt gatctctacc tgttgctcaa tggagagatt gcggcccgac attacgcctg 35040

acgctcgtgg ccgtcagtga acggcacaac gttggcgaac ccactgccgg actgctgctg 35100

cttgatctct gccaagcgct tggccagttc ggtttcctcg ctggcctctg gtgccgcgca 35160

ggtgatgccg ttcttgtcgg atacccacac accagcgtcc ttgatgatct tgtcgtcgaa 35220

caccatctcg atgtccgcgc cttcattcag caagtccagc atgcgctgca agcgcttggt 35280

gtacagggtg gtggtgagct ggtggataag acccagctcg tcttcggttg cagcggtctt 35340

gttgctgcgt cgttcaatag ccattagtcg ccctctactt tgataagcca gtggagagcg 35400

tagaacggtg tcatgcactt atgaatgtac gtgatggtgt aggagtgcga gtgctcgtct 35460

acgcccccgc cgaagttgcc ccaacgaacg tcgttggtgt cggcattctc cgtgcggtag 35520

ttcagtggcc gataccgagc gctgtgctct ggcaccgact tccagttgcc gtggtcactc 35580

tccacgcgca ccccgtaggc agtgaactgc gtttcgtagg tgtgctggtg aggtggaagg 35640

tgtgctgcaa acagcatggt cttctcctcg gcggcagacc cgtcagacgt cttggtgtag 35700

cgaacgtcaa tggttccgcc catgctgtgg atcgggtagt taccgcccgc acccacgacc 35760

atgcgatctc ggaagtcagg ccgaccattc gtaccatcca gaatccgcca gcctttcggg 35820

actgtgcggg agtcgcccca ccaaggggcg attgttccgg ttggcaggat ggctcttgcg 35880

agcgcctgaa tctcagcggc tgtgtacatg tcgcaatctg ctgctgtcaa atccgcgagc 35940

cgcttggtaa cccgagtgcg gtttgacata ctaaaattta gatcgccctc gtcgccatcg 36000

tcggcccctg ctgtgcgcat gatccaatac ttggtgacat aaggggcaat cgcctcgtgc 36060

ttgaaccggg cggagaagcc gtggctgtga ggctcagcaa ggccagtcgc ctgattgccg 36120

aactccatgc tggacttgta ccgcttggtc tgcttgttgg ctgcgtcctt cttcttctta 36180

cccgtccgct cagaggagcc gaggatgttg gaagtgtagc cgtgggtgtg aggtggcacc 36240

tccttggttg tcagggcatg accgtcagtc ttcccgtcga taatcaagtc gaagttgtac 36300

ttgccaccag tgctgttcag tgggaagttc ccaccggcac ctacaaccgt gcggttggtc 36360

aggttaggcg tgcgaccgcc caatccgttg cagatgctgt agccttcggg aacgtccgtg 36420

gcgtccccgt tccagacgaa gcacatgccc accggaatgg tggcggacac gagccgtcgg 36480

atctccccgc gcgtatagct gcccaagtcg tgggcagtca gcgcagagac ggacttgcga 36540

atgcgagtag tcatcctgtg ataacctcca gtcttacgcc catgttgtcg tagaatcgga 36600

caagctccgc aacctttgca ggcgtcggaa catccccggt cgattgttgg ctgagtacca 36660

agcggcagtc cactcggcga cggtaggcag ctcgtgccct ctcctcgctg aaatagagca 36720

gcttgtcaat cagagcatcc agctcagccg tggtcatgtc gttgaatgcg aagtccagct 36780

cggtggcctc tggcacgatg ccgaagtcta cgtccttgac cccgccacga cggaaccagc 36840

aggaggtgta gtgctgacca tcgcgtggca tgataagcgg cgagaacgcg cctgtcatgt 36900

tgtagcggtg gtacgtgcaa tccatctcca cctcaacctc cttgtcctct cccgttatcg 36960

ggtcaggtac aatggcaatc gggtactcag ggccggtgag gttgtagcct cccatgtcct 37020

ccaagatacc ctcctgaatc tgcgaaccgt cgccccagtc gatgctggtg gtggacactg 37080

gttcatcgtc ctcctcctcg gagtccaaag agtagtcgaa gacggcagac aggtggcggt 37140

ctggtcggcc ttccagcggg gagccgggag ggtagacagt ccactcaggc tggggtgcca 37200

gcatgttgca gaatgccagc gggtagtacg cggtgctccc cttacggcca atggcctgac 37260

gggtggcgaa cgctacagac ctacgagaca cgacagggtt gccatgggcc tcgatctcct 37320

ccgggttctc cgggttgacc aagtaccact gctccgtctt ctcgtccacg taggtcatcc 37380

gctgattgaa cactgtgcct tccgacacgc tgtactcaga tggcaaccag aacttgtcct 37440

gacgcgagat ctcgtgccag tcctcaacca gccgcagctc aaggttccag atcaccttgt 37500

ctttcggcaa ggcaccacca tcgttcttga acaccacgct gtcacggttc acctgagtcg 37560

aggcccaaat gagcgactgc atggggcggt tggctgcgtc gagcttggtg ttgctccagc 37620

tcaactggtg tgcgatgttg cgccacacac gtcctgtcgg gtctttgttt ggtgcgaatc 37680

caccaaggta gttcgtcttc tccactgggt aacctagctt gtcgaaagcg tagtcgcagt 37740

aggagttggt gaactcaagg tcagaccaga tgatggtgtc gtaggcttcg tactggaaga 37800

actcaaagtt cgggtacttc caatgctgga acgggaagtt gatgatcgga gtgatctcgg 37860

acttcgtggt gaagatctgc gggatgaacg tcagcgccga gaactcagcg tccgggtgtg 37920

cagcctgcgt ctcctccttg gcccgctggg tgtaaacccc taggctgtca cgtagccagt 37980

tggccgcgtc cagttcctgc tgcgttgcct tctgcttggt gaaggtagtg atgtccttcc 38040

acacaggcag gtcgataccg tcagcgagag ctgctcgctt ggtcggttca tcatacacga 38100

acagtcgccc gttctgatac gagccgtccc agtaccatgg ctcacctact tggaacaact 38160

tcttgaggcc cgctggcacg atgtccgcca gctccttgaa gcatcgaccc acatacgagc 38220

ggcctgcctc ctcctcgatg cgcgtcccga agaacgtgct cggagggttg taaccagtaa 38280

gtgctggctt gcccacggag tagtcgccgt accaaccacg gaagcgcttg tccagcttga 38340

gctgggtctt gtcctcgttg ggtacgtcgt tcacctcaag caacttgtac accgtctcct 38400

tgccgccgtc ctcggggtgt gcgaaccgga tgtcggctgc ccactccttg ttcgggttga 38460

acaagccctg atgctcccaa cctcctagga aggggttctc agtggatgac caagggtgct 38520

tgccctcgat cttgatgtac tcgtcgtcaa cctccatcac ttggaagccc tgacagagct 38580

gcatccagtg gcggtggttg tacttcacgt aatcgtagaa cacctccatg gacaccgcaa 38640

tgatgcactc gaagttgaag cgctggcact gttgcaggaa gtcctcgtgc cagaggcgaa 38700

ccacctcgtc caagcactcc tcgcgggaga gctggaactg ctgcgtgtcc tcgtagaaca 38760

ccagcttcgg gtagttactg atgcccatgt agtggttgat gacgccacgg tagcccagct 38820

tgtacatgtc ctcaaccacg cggcgcgggt tggtgttggc aatgtcgtcg aagctggtgg 38880

cgataccgag gtcggtctgg tacggcgtac cgtccgggtg catcggtcgt ccacgggcgt 38940

gggtctttgg tgcgccatcg ctcttgcgga agcagcggat gttgctgacc actacgtcgc 39000

cgagacgagg ctgcttgatt ggtgcccgag gcaagtcctc gttctccagc ccttcctcgt 39060

cgtaaccgcc gattgcgtag aagtccttgt ccaccacgtt gatcgacagc ctgcgcaggt 39120

cggtgactgg caccttctgc ttgaatgggt cgttcgggcc gaagccggtg tacaggttgt 39180

cgaagtccag caccacgtcc atcttgctcg gagtgctggt cagtgggtcg gcgtactgcc 39240

agaggcggat gtaccactcc ttgccgtctg ccgtggtgaa ggtcagggac gtaccgtagg 39300

ccatgtcagc agcacgatag gtgtctggtg tcatgtccat ggtgaacgtc atcttgtagc 39360

cggtgaagtc ccgctcctgt ggctcaacgt agctcttgag cttgttgtgc cgtggcaggt 39420

attcctcgcc gtccatgttg aacaccgcct gcgcgatgtg gtcgttggag aacagcttgg 39480

tagtcagggt gaacccgtcg tccatgctgt tgcggatcgc ggacttggag gggcgctgga 39540

acacggcaaa ccacagttgg ccgtagaaca taggcacccg ctcccactca tggttctctg 39600

gctccgtctc gatgtcaacg gttcggaagc cagagtcata agggcgttcc gcgtacatca 39660

gctcgtaatg gccgtcacgc gaaatcatgt cacctcctta ttcagcgacg tagttaagtg 39720

cgatgccgag gaagaacgca tcctcgctgg agtttgcgag cgtgtcctcg ggatgctgtg 39780

gaacccggct cacacgcagg tacagcactt ggctgatgtc tgctgccggg atggccgcta 39840

ctgcacggta gtgagtaacg tcctgtggtc gtaccgaaac ggtgaactcg atagggttcc 39900

cgaagtcagc ttggaactca ccggggcctt gcaaggcagt ctcgatgcgc caccgggtgt 39960

tgccagcgtt ctcgccggac accatgaagc gcatctccac gccagtagcc acgggcatat 40020

ctggaggagt aataaccacg gcctgacacg aacgggtctt gtcagcgaag aacgcatgag 40080

caggggactc gatcttgcct tgtgggcggt gggtgacgat ggtgctgcca tctgtcagtt 40140

ttgggcggaa gccgttagca ggccaccaca cacgggcgta gctcgacatg ctgtacgcac 40200

Claims (11)

1. The pseudomonas syringae phage is pseudomonas syringae kiwi pathopoiesia phage PSA-P1 (PSA-P1)Pseudomonas syringae pv. Actinidiae phase PSA-P1), and the preservation number is CCTCC NO: m2020252.

2. The Pseudomonas syringae bacteriophage of claim 1, wherein: the pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1(Pseudomonas syringae pv. Actinidiae phage PSA-P1) is a virulent phage, which has a polyhedral and three-dimensionally symmetrical head and a telescopic tail, wherein the diameter of the head is 50-55 nm, the length of the tail is 15-20 nm, and the diameter of the tail is 6-10 nm, and the phage belongs to the family Autograpiviridae.

3. The Pseudomonas syringae bacteriophage of claim 1, wherein: the pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1(Pseudomonas syringae pv. Actinidiae The genome sequence of the phase PSA-P1) is shown as SEQ ID No. 1.

4. The Pseudomonas syringae bacteriophage of claim 1, wherein: the pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1(Pseudomonas syringae pv. Actinidiae phase PSA-P1) under the condition of MOI =0.000001 for 24h, the titer reaches 7 × 10¹⁰ PFU/mL or more.

5. The Pseudomonas syringae bacteriophage of claim 1, wherein: the pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1(Pseudomonas syringae pv. Actinidiae phase PSA-P1) is tolerant at pH = 3-12, with titers decreasing by no more than 4 orders of magnitude within 96 h.

6. The Pseudomonas syringae bacteriophage of claim 1, wherein: the pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1(Pseudomonas syringae pv. Actinidiae phase PSA-P1) after 8h of ultraviolet radiation, the titer does not decrease by more than 1 order of magnitude.

7. A composition comprising the Pseudomonas syringae bacteriophage of any one of claims 1 to 6, wherein: the composition at least comprises one strain of pseudomonas syringae kiwi pathopoiesia variety phage PSA-P1 (A)Pseudomonas syringae pv. Actinidiae phage PSA-P1）。

8. The composition of Pseudomonas syringae phage of claim 7, wherein: the composition includes a chemical germicide.

9. A pseudomonas syringae bacteriophage kit is characterized in that: the kit contains Pseudomonas syringae Kiwi fruit var infestans phage PSA-P1 (PSA-P1) of claim 1Pseudomonas syringae pv. Actinidiae phage PSA-P1) or a bacterium comprising Pseudomonas syringae Actinidia var venetian phage PSA-P1 (see claim 7)Pseudomonas syringae pv. Actinidiae phage PSA-P1).

10. The application of the pseudomonas syringae bacteriophage is characterized in that: the Pseudomonas syringae Actinidia pathovar catarrhalis phage PSA-P1(Pseudomonas syringae pv. Actinidiae P) of claim 1hage PSA-P1) with a preservation number of CCTCC NO: m2020252, at 10¹ PFU/mL~10⁸ PFU/mL, used for controlling Pseudomonas syringae.

11. The application of a pseudomonas syringae phage composition is characterized in that: the composition of Pseudomonas syringae and Actinidia chinensis var pathopoiesia phage PSA-P1 as claimed in any one of claims 7 to 8, used as an effective component of biological disinfectant or biological pesticide for preventing and treating bacterial diseases caused by Pseudomonas syringae.

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