WO2022099837A1 - 一种丁香假单胞菌噬菌体及其组合物、试剂盒和应用 - Google Patents

一种丁香假单胞菌噬菌体及其组合物、试剂盒和应用 Download PDF

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WO2022099837A1
WO2022099837A1 PCT/CN2020/134391 CN2020134391W WO2022099837A1 WO 2022099837 A1 WO2022099837 A1 WO 2022099837A1 CN 2020134391 W CN2020134391 W CN 2020134391W WO 2022099837 A1 WO2022099837 A1 WO 2022099837A1
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phage
psa
pseudomonas syringae
pseudomonas
pathogenic
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PCT/CN2020/134391
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English (en)
French (fr)
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许文建
徐天舜
丛郁
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菲吉乐科(南京)生物科技有限公司
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Priority to KR1020217033436A priority Critical patent/KR20210142680A/ko
Publication of WO2022099837A1 publication Critical patent/WO2022099837A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/40—Viruses, e.g. bacteriophages
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00031—Uses of virus other than therapeutic or vaccine, e.g. disinfectant

Definitions

  • the present invention relates to the field of bacteriophage, more particularly, it relates to a Pseudomonas syringae bacteriophage and its composition, kit and application.
  • Pseudomonas syringae belongs to the genus Pseudomonas of the family Pseudomonas and has 57 pathogenic types. Pseudomonas syringae pv.actinidiae is one of the pathogenic types. Psa has a variety of genetic genes that are highly adaptable, so it is prone to invading kiwifruit trees and causing infection all year round.
  • the prevention and control technology for kiwifruit canker in the related art is mainly based on chemical prevention and control, and uses methods such as field spraying, smearing of pus wounds, and drying injection.
  • the effect is Neither is ideal.
  • the specific and efficient lysis ability of pathogenic varieties can significantly reduce the number of pathogenic bacteria in the environment, and can control or reduce the occurrence and prevalence of diseases. Therefore, Pseudomonas syringae phage can be used as an antibacterial infection agent.
  • the present application provides a Pseudomonas syringae phage and its composition, kit and application.
  • a kind of Pseudomonas syringae bacteriophage and its composition, kit and application provided by the invention adopts the following technical scheme:
  • the present invention provides a kind of Pseudomonas syringae phage, adopts the following technical scheme:
  • a Pseudomonas syringae phage the Pseudomonas syringae phage is Pseudomonas syringae kiwifruit pathogenic variety phage PSA-P1Pseudomonas syringae pv.Actinidiae phage PSA-P1), the deposit number is CCTCC NO: M 2020252.
  • the present application provides a phage PSA-P1 of a pathogenic variant of Pseudomonas syringae kiwifruit, which has excellent UV resistance and pH resistance, and has been deposited in the China Type Culture Collection for preservation. , the deposit number is CCTCC NO: M 2020252.
  • the Pseudomonas syringae pv. Actinidiae phage PSA-P1 is a potent phage with a polyhedral three-dimensional symmetrical head and a retractable tail, and the diameter of the head is It is 50 to 55 nm, the length of the tail is 15 to 20 nm, and the diameter of the tail is 6 to 10 nm. It belongs to the Autographiviridae family of phages.
  • the morphology of the phage is observed from the electron microscope, and it can be seen that the phage has a polyhedral three-dimensional symmetrical head and a stretchable tail, which is helpful for the nucleic acid of the head to be injected into the host bacteria, and can also effectively identify the surface of the host bacteria. special receptors.
  • the Pseudomonas syringae phage PSA-P1 (Pseudomonas syringae pv. Actinidiae phage PSA-P1) has the nucleotide sequence shown in SEQ ID No.1.
  • the multiplicity of infection is the ratio of the number of phage to the number of bacteria, and is an important basis for studying the dose-effect relationship between phage infection of bacteria and production of phage progeny.
  • the Pseudomonas syringae phage of the present invention only needs to add a small amount to infect the Pseudomonas syringae and proliferate to obtain a large number of progeny phages.
  • the invention provides a source of high-quality phage strains for industrial production of phage bactericides.
  • the titer of the Pseudomonas syringae pv. Actinidiae phage PSA-P1 (Pseudomonas syringae pv. Actinidiae phage PSA-P1) after being irradiated by ultraviolet light for 8 hours does not decrease by more than one order of magnitude.
  • the phage PSA-P1 of the Pseudomonas syringae kiwifruit pathogenic variant of the present invention has good tolerance, the titer decreases less, and can play a role in the pathogenic bacteria. effective control.
  • the present invention provides a composition of Pseudomonas syringae bacteriophage, which adopts the following technical scheme:
  • a composition of Pseudomonas syringae bacteriophage contains at least one strain of Pseudomonas syringae pv. Actinidiae phage PSA-P1.
  • the composition includes a chemical bactericide.
  • the Pseudomonas syringae kiwifruit pathogenic variety phage PSA-P1 and a chemical bactericide are used in combination as a composition.
  • the proportional relationship between Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 and 700-fold solution of Dysenium can be determined by those skilled in the art in combination with the present application and the actual application field and common sense in the art .
  • the present invention provides a test kit for Pseudomonas syringae phage, which adopts the following technical scheme:
  • kits of Pseudomonas syringae bacteriophage the kit is provided with the Pseudomonas syringae kiwifruit pathogenic variety phage PSA-P1 (Pseudomonas syringae pv. Actinidiae phage PSA-P1) or Pseudomonas syringae phage PSA-P1 Composition of bacteria Actinidiae phagePSA-P1 (Pseudomonas syringae pv. Actinidiae phagePSA-P1).
  • the Pseudomonas syringae phage of the present invention is applied to the rapid detection of Pseudomonas syringae, including but not limited to the detection of Pseudomonas syringae in the form of test paper, test paper box, etc., or the detection of clinical samples.
  • the target pathogens are screened to effectively ensure the sensitivity of the detection.
  • the fourth aspect provides a kind of application of Pseudomonas syringae bacteriophage, adopts following technical scheme:
  • Pseudomonas syringae phage A kind of application of Pseudomonas syringae phage, said Pseudomonas syringae pv. Actinidiae phage PSA-P1 in 10 1 PFU/mL ⁇ 10 8 PFU/mL Within the titer range, the bacteriostatic rate against Pseudomonas syringae reached 54.1-94.9%.
  • the fifth aspect provides a kind of application of Pseudomonas syringae bacteriophage composition, adopts following technical scheme:
  • the composition of the Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 is used as an active ingredient of a biological disinfectant or a biological pesticide, preventing and controlling but not Limited to bacterial diseases caused by Pseudomonas syringae.
  • the Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 and its composition can be used for the treatment and prevention of bacterial infections caused by Pseudomonas syringae, but not limited to, by Pseudomonas syringae, It is used as a biological agent for the control of diseases caused by, but not limited to, Pseudomonas syringae.
  • the present invention has the following beneficial effects:
  • the Pseudomonas syringae kiwifruit pathogenic variant phage PSA-P1 of the present invention is a potent phage isolated from nature, has high tolerance to ultraviolet rays and pH, and is suitable for different prevention and control environments. Ulcer disease has a better biological control effect;
  • the Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 of the present invention is a virulent phage isolated from nature, the test phage does not contain virulence genes or bad genes, and the DNA of the phage cannot be encoded, which may cause potential health problems Risk protein, there is no possibility of carrying lysogenic genes;
  • the Pseudomonas syringae kiwifruit pathogenic variant phage PSA-P1 of the present invention has high affinity and lysis ability, and reaches a titer of more than 10 10 PFU/mL within 24 hours of culture; Pseudomonas syringae kiwifruit causes disease
  • the variant phage PSA-P1 can specifically partially or completely inactivate Pseudomonas syringae, and only a small amount of initial phage can be used to complete a large number of proliferation, providing a source of high-quality phage strains for industrial production of phage fungicides; those skilled in the art can Records of the application and common knowledge in the art
  • the Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 or its composition described in the application is prepared into various products applied in detection, disinfection and plant protection, etc. and industrially applied ;
  • the Pseudomonas syringae kiwifruit pathogenic variant phage PSA-P1 of the present invention is a strict and potent phage, has high specificity and lysis to the host bacteria, and has a wide host range, and is suitable for 45 strains of Pseudomonas syringae.
  • the lysis rate of Pseudomonas is as high as 91.1%; Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 can be used as an active ingredient in various products used in environmental disinfection, such as but not limited to liquid immersion, spray, and water-based
  • environmental disinfection such as but not limited to liquid immersion, spray, and water-based
  • the combined use of carriers to disinfect and decontaminate water distribution systems, irrigation facilities, aquaculture facilities, public and private facilities or other environmental surfaces can effectively control the growth and activity of target bacteria;
  • the liquid soaking and spraying forms include but are not limited to Detergents, disinfectants, detergents, etc.;
  • the aqueous carrier includes but is not limited to phosphate buffer, TSB medium, LB medium, chlorine free water, etc.;
  • the Pseudomonas syringae kiwifruit pathogenic variant phage PSA-P1 of the present invention can be used to prepare compositions, reagents or kits for rapid detection of Pseudomonas syringae, including but not limited to test strips, test kits Detect Pseudomonas syringae in target samples in other forms, or screen target pathogenic bacteria in clinical samples to effectively ensure the sensitivity of the detection;
  • Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 and its composition have good killing ability to Pseudomonas syringae with a concentration of 10 3 PFU/mL in liquid medium;
  • concentration of pathogenic phage PSA-P1 of kiwifruit is ⁇ 1 ⁇ 10 6 PFU/mL, the killing rate of Pseudomonas syringae is more than 91.8%, and it has no antagonistic effect on other combined substances;
  • the Pseudomonas syringae pv. Actinidiae phage PSA-P1 of the present invention and the composition thereof can be prepared by those skilled in the art according to the description of the present application and common sense in the art.
  • FIG. 1 is a schematic diagram of a plaque of the present invention
  • Fig. 2 is the electron microscope result schematic diagram of phage of the present invention.
  • Fig. 3 is the structural representation of the plaque sample that appears in the lysogenicity test
  • Figure 4 is a schematic diagram of the structure of a sample without plaque in the lysogenicity test.
  • Pseudomonas syringae kiwifruit pathogenic variety phage PSA-P1 (Pseudomonassyringae pv.Actinidiae phage PSA-P1), the deposit number is CCTCC NO: M 2020252, the deposit unit is the China Center for Type Culture Collection, and the deposit time is June 2020 30 days.
  • Carpetgrass Xanthomonas phage YHC5 (Xanthomonas axonopodisphage YHC5), the deposit number is CCTCCNO: M2018579, the deposit unit is the China Center for Type Culture Collection, and the deposit time is August 30, 2018.
  • Xanthomonas rugosa citrus pathogenic varietal (Xanthomonasaxonopodisv.citri), the deposit number is ACCC 03526, which can be purchased from the depository.
  • Pseudomonas syringae cucumber pathogenic varietal (Pseudomonassyringae pv. lachrymans), the deposit number is ATCC7386, which can be purchased by contacting the depository.
  • Pseudomonas syringae tomato pathogenic varietal (Pseudomonassyringae pv.tomato), the deposit number is ATCC BAA-871D-5, can be purchased by contacting the depository.
  • Pseudomonas syringae tobacco pathogenic variety (Pseudomonassyringae pv. tabaci), the deposit number is ATCC13453, can be purchased by contacting the depository.
  • TSB liquid medium tryptone 15g, soybean peptone 5g, sodium chloride 5g, distilled water 1000mL;
  • TSA solid medium tryptone 15g, soy peptone 5g, sodium chloride 5g, agar 15g, distilled water 1000mL;
  • TSA plate sterilize the TSA solid medium and pour it onto a sterile plate, cool and solidify to make a TSA plate;
  • TSB semi-solid agar medium tryptone 15g, soy peptone 5g, sodium chloride 5g, agar 7g, distilled water 1000mL;
  • SM liquid formula sodium chloride 5.8g, magnesium sulfate 2g, 1mol/LTris-HCl 50mL, gelatin 0.25g, distilled water 1000mL.
  • Example 1 Isolation, preparation, purification and culture of Pseudomonas syringae kiwifruit pathogenic phage PSA-P1
  • the source sample of Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 in the present invention was collected from the sewage of Zhongcai farmers market in Jiangning District, Nanjing City, Jiangsu province, filtered with double-layer filter paper, centrifuged at low speed and normal temperature, and then filtered with a 0.22 ⁇ m filter membrane supernatant.
  • Pseudomonas syringae kiwifruit pathogenic variety phage PSA-P1 (Pseudomonassyringae pv. Actinidiae phagePSA-P1), the deposit number is CCTCC M: 2020252.
  • Example 2 Electron Microscopic Observation of Pseudomonas syringae kiwifruit Pathogenic Variety Phage PSA-P1
  • the purified phage solution prepared in Example 1 was taken for electron microscope observation: 20 ⁇ L of the sample was dropped on a copper mesh, and it was allowed to settle naturally for 15 minutes. Absorb excess liquid from the side, add 1 drop of 2% phosphotungstic acid on the copper mesh, dye for 10 minutes, absorb the dye solution from the side with filter paper, and observe by electron microscope after drying.
  • Example 3 Preparation of Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 particles and genome extraction and sequencing
  • Phage nucleic acid was extracted and sequenced using ⁇ phage genomic DNA kit. After nucleotide sequencing, the Pseudomonas syringae phage PSA-P1 (Pseudomonas syringae pv. Actinidiae phage PSA-P1) has the nucleotide sequence shown in SEQ ID No.1.
  • the Pseudomonas syringae kiwifruit pathogenic variant phage PSA-P1 has a titer of more than 10 10 PFU/mL after culturing for 12 h.
  • Table 1 The titer of Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 after culturing for 12h
  • Example 5 Deletion test of virulence gene or defective gene of Pseudomonas syringae kiwifruit pathogenic phage PSA-P1
  • 103 virulence genes identified as originating from lysogenic phages in pathogenic bacteria were selected as shown in Table 2. Informatics analysis to determine whether it contains the following virulence genes. The results show that the Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 does not contain the following virulence genes or bad genes, so it cannot encode proteins that may cause potential health risks. Therefore, the Pseudomonas syringae kiwifruit pathogenic phage PSA- P1 does not affect human or animal health.
  • mice half male and half female, were randomly divided into two groups (phage group, control group) after three days of adaptive feeding, 10 mice in each group (5 males and 5 females), and the dose of phage group was 10 10 PFU/kg Pseudomonas syringae kiwifruit pathogenic variant phage PSA-P1, the control group was given the same amount of normal saline for 15 days, the experimental mice were sacrificed by neck dislocation, and the internal organs were examined.
  • Example 7 Determination of the optimal multiplicity of infection (MOI) of Pseudomonas syringae by phage PSA-P1
  • phage PSA-P1 purified solution prepared from Example 1
  • TSB liquid medium a solution titer/phage host bacterial concentration
  • PSA-P1 Host bacteria (cfu/mL) PSA-P1 titer (PFU/mL) 100 10 7 10 5 1.2 ⁇ 10 8 10 10 7 10 6 3.3 ⁇ 10 8 1 10 7 10 7 4.6 ⁇ 10 8 0.1 10 7 10 8 1.4 ⁇ 10 9 0.01 10 7 10 9 5.8 ⁇ 10 9 0.001 10 7 10 10 7.2 ⁇ 10 9 0.0001 10 6 10 10 2.4 ⁇ 10 10 0.00001 10 5 10 10 4.5 ⁇ 10 10 0.00000 10 4 10 10 7.4 ⁇ 10 10 0.0000001 10 3 10 10 5.2 ⁇ 10 10
  • Phage PSA-P1 provides a source of high-quality phage strains for the industrial production of phage fungicides.
  • Example 8 Determination of pH and temperature stability of Pseudomonas syringae kiwifruit pathogenic variant phage PSA-P1
  • the phage PSA-P1 (prepared in Example 7) with a titer of 1.0 ⁇ 10 7 PFU/mL was placed at 4° C., 25° C. and 40° C. respectively, and the titers were periodically sampled to detect the titer.
  • the phage PSA-P1 has good stability. After 3 months of storage, the titer does not decrease significantly, and after 12 months of storage, the titer decline still does not exceed. 1 order of magnitude; under the condition of 25°C, the titer of bacteriophage PSA-P1 did not decrease significantly after 4 weeks of storage; under the condition of 40°C, the titer of bacteriophage PSA-P1 did not decrease significantly within 24 hours, and after 72 hours, the titer decreased by 1 Magnitude. This shows that the phage PSA-P1 has good stability under different temperature conditions.
  • Example 9 Tolerance test of Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 to ultraviolet light
  • PSA-P1 bacteriophage made by Example 7 with a titer of 1 ⁇ 10 8 PFU/mL and spread it in a 90 mm sterile petri dish, put it into an ultra-clean workbench, and place it in an ultraviolet lamp ( 20w, 20cm) under irradiation. Samples were taken at 0min, 20min, 40min, 1h, 2h, 3h, 4h, 5h, 6h, 7h, and 8h respectively and placed in the dark for 30min, and then the phage titer was determined by double-layer plate method.
  • Example 10 Test of lysis ability of Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 on Pseudomonas syringae from different origins
  • the lysis profile of the phage was determined by double-layer plate drop method.
  • the single colonies of 45 strains of Pseudomonas syringae isolated from 6 provinces including Shandong, Sichuan, Chongqing, Anhui, Guangdong and Henan were picked respectively, inoculated in a test tube filled with 3 mL of TSB liquid medium, and incubated at 28°C. Cultivate overnight at 180 rpm to obtain bacterial broth of each strain.
  • the results are shown in Table 9.
  • the phage PSA-P1 has a strong ability to lyse Pseudomonas syringae from different origins, and its lysis rate can reach 91.1%.
  • Pseudomonas syringae kiwifruit pathogenic variant phage PSA-P1 has a broad lytic spectrum.
  • Example 11 Lysis test of non-pathogenic beneficial bacteria by Pseudomonas syringae kiwifruit pathogenic phage PSA-P1
  • Example 12 Identification of lysogenicity of Pseudomonas syringae kiwifruit pathogenic phage PSA - P1 mL, 1.0 ⁇ 10 7 PFU/mL) of bacteriophage PSA-P1 (prepared in Example 7) and 100 ⁇ L of Pseudomonas syringae Psa-1 (1.0 ⁇ 10 8 cfu/mL) were mixed and inoculated into a liquid culture containing 10 mL of TSB In a 50 mL centrifuge tube, the cells were incubated with shaking at 28 °C for 48 h.
  • the obtained turbid culture medium was diluted and spread on a TSA plate and placed in a 28°C incubator for 48 hours. Pick the central part of 30-50 single colonies on the TSA plate, put them in EP tubes containing 200 ⁇ L TSB liquid medium, and shake them at 28 °C for 24 h; then add the final concentration of 0.5 ⁇ g/ mL of mitomycin C, and continued to cultivate for 12 hours; the obtained culture solution was filtered and sterilized with a 0.22 ⁇ m filter membrane, and then dripped onto a Psa-1 double-layer plate, and cultured at 28 °C; at the same time, the titer was 1.0 ⁇ 10 7 PFU/mL of phage PSA-P1 (prepared in Example 7) was spotted on a double-layer plate as a positive control. After 24 hours, the double-layer plate was observed. If plaques appeared, it was proved that the phage PSA-P1 was a lysogenic phage.
  • Example 13 Detecting the bacteriostatic rate of Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 against Pseudomonas syringae by turbidimetric method
  • Table 11 The antibacterial effect of Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 on Pseudomonas syringae
  • the bacteriostatic rate of about 10 1 PFU/mL of phage PSA-P1 on Psa-1 can reach 54.1%, which indicates that the phage PSA-P1 of Pseudomonas syringae kiwifruit pathogenic variant phage is at a very low dose It can effectively inhibit bacteria and sterilize.
  • Example 14 Bactericidal effect of Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 in liquid
  • ActinidiaephagePSA-P1 (prepared in Example 1). A control group and a blank group were set at the same time. The control group was given Pseudomonas syringae Psa-1 with a final concentration of 1 ⁇ 10 3 cfu/mL; the blank group was given the same amount of normal saline. Each treatment was shaken and cultured at 150 rpm at 28°C, and the residual amount of Pseudomonas syringae was detected after 4 h.
  • the detection method is as follows: after diluting each treated sample with sterile water, take 100 ⁇ L of the dilution solution and spread it on a TSA solid plate, and count the number of colonies on the plate after culturing at 28°C for 24 hours.
  • the number of Pseudomonas syringae the number of colonies on the TSA plate ⁇ the dilution factor ⁇ 10.
  • Example 15 Preparation of the composition of Pseudomonas syringae kiwifruit pathogenic variety phage PSA-P1
  • phage PSA Purified phage PSA with final concentrations of 1 ⁇ 10 2 PFU/mL, 1 ⁇ 10 3 PFU/mL, 1 ⁇ 10 4 PFU/mL, 1 ⁇ 10 5 PFU/mL, and 1 ⁇ 10 6 PFU/mL, respectively.
  • -P1 solution prepared in Example 1 was evenly mixed with equal volume of 700 times of Dysenium solution (chemical bactericide) with a final concentration of 50% to prepare 1:1 composition 1, composition 2, composition 3. Composition 4 and Composition 5.
  • Purified phage PSA with final concentrations of 1 ⁇ 10 2 PFU/mL, 1 ⁇ 10 3 PFU/mL, 1 ⁇ 10 4 PFU/mL, 1 ⁇ 10 5 PFU/mL, and 1 ⁇ 10 6 PFU/mL, respectively -P1 solution was uniformly mixed with equal volume of Xanthomonas rugosa phage liquid with a final concentration of 1 ⁇ 10 6 PFU/mL to prepare 1:1 composition 6, composition 7, and combination Composition 8, Composition 9 and Composition 10.
  • Purified phage PSA with final concentrations of 1 ⁇ 10 2 PFU/mL, 1 ⁇ 10 3 PFU/mL, 1 ⁇ 10 4 PFU/mL, 1 ⁇ 10 5 PFU/mL and 1 ⁇ 10 6 PFU/mL respectively -P1 solution was uniformly mixed with an equal volume of 700-fold dysenium solution with a final concentration of 50% and a final concentration of 1 ⁇ 10 6 PFU/mL of Xanthomonas carpetgrass phage to make 1 : 1:1 Composition 11, Composition 12, Composition 13, Composition 14, and Composition 15.
  • Example 16 Bactericidal effect of Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 composition in liquid
  • the combination of this embodiment is not limited to 700 times of dysenium solution, but also can be polydoxomycin, flumorpholine, dimethomorph, prochloraz, difenoconazole, flusilazole, myclobutanil, Mancozeb, thiophanate-methyl, carbendazim, chlorothalonil, polysaccharide and other chemical fungicides.
  • This embodiment can also be used in combination with a chemical disinfectant to play an anti-kill effect.
  • Example 17 Prevention and treatment of kiwifruit canker disease by Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 and its composition
  • 360 kiwifruit trees were randomly divided into 6 groups (phage 3 group, composition 5, control group and blank group) after 1 month of adaptive culture, 60 trees in each group, and the phage experimental group was given a dose of 1 ⁇ 10 4 PFU/mL, 1 ⁇ 10 5 PFU/mL, 1 ⁇ 10 6 PFU/mL of the test phage (prepared in Example 7) and 1 ⁇ 10 5 cfu/mL of Pseudomonas syringae Psa-1; control group 1 ⁇ 10 5 cfu/mL of Pseudomonas syringae Psa-1 was given; the blank group was given the same amount of normal saline, and 1 L was inoculated by stem infusion method.
  • Example 18 Preparation and use of a kit for Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 and its composition
  • the kit contains 5 to 10 mL of Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 liquid or a composition of Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 with a titer of 1 ⁇ 10 7 PFU/mL , 1L TSB semi-solid medium, 1L TSA medium.
  • the use method of the kit is as follows: taking a composition with a titer of 1 ⁇ 10 7 PFU/mL of Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 liquid or Pseudomonas syringae kiwifruit pathogenic phage PSA-P1, The lysis profile of the test phage was determined by the double-layer plate drop method. Picking a single colony to be detected, inoculating it into a target liquid medium, and performing shaking culture at a target temperature in combination with the growth characteristics of the strain to be detected, to obtain a bacterial liquid of the strain to be detected.
  • the main component of the kit 1 is 5-10 mL of phage PSA-P1 liquid with a titer of 3 ⁇ 10 8 PFU/mL of Pseudomonas syringae kiwifruit pathogenic variety.
  • the main component of the kit 2 is 5-10 mL of 3 ⁇ 10 8 PFU/mL phage Xanthomonas rugosa phage YHC5 liquid.
  • the main component of the kit 3 is 5-10 mL of a 700-fold solution of dysenium with a final concentration of 50%.
  • kit 4 The main components of kit 4 are 5-10 mL of Pseudomonas syringae kiwifruit pathogenic variant phage PSA-P1 liquid with a titer of 3 ⁇ 10 8 PFU/mL, and 5-10 mL of a titer of 3 ⁇ 10 8 PFU/mL mL of Xanthomonas rugosa phage YHC5 liquid, and 5 to 10 mL of 700-fold dysenium solution with a final concentration of 50%. [0082]
  • Test process 120 citrus trees 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 cultivation for 1 month. , 20 in each group.
  • 1 L of Xanthomonas rugosa citrus pathogenic varietal ACCC03526 (Xanthomonasaxonopodispv.citri) with a final concentration of 1 ⁇ 10 3 cfu/mL was applied to Kit 1, Kit 2, Kit 3 and Reagent by stem infusion method.
  • a total of 100 citrus trees in Box 4 and the positive control group were challenged, and 20 citrus trees in the negative control group were injected with 1 L of normal saline by stem infusion method.
  • Kit 2 of Xanthomonas rugosa phage YHC5 was more effective in controlling the incidence of citrus.
  • Kit 1, kit 2, kit 3, and kit 4 all use the kits of Example 19.
  • Table 16 The effect of the kit of Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 and its composition on cucumber bacterial keratosis
  • the incidence of cucumber in the kit 4 group is lower than that in the kit 1 group, indicating that the kit 4 containing the composition has a significant control of cucumber bacterial angular spot, and the kit 4 containing the composition Compared with the kit 1 containing only Pseudomonas syringae kiwifruit pathogenic phage PSA-P1, the effect of controlling the incidence of cucumber is better.
  • Kit 1, kit 2, kit 3, and kit 4 all use the kits of Example 19.
  • Test process 120 tomato vines 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 cultivation for 1 month. , 20 plants in each group. 1 L of Pseudomonas syringae tomato pathogenic varietal ATCBAA-871D-5 (Pseudomonassyringaepv.tomato) with a final concentration of 1x10 3 cfu/mL was applied to kit 1, kit 2 and kit 3 by root irrigation method. A total of 100 tomato plants in the kit 4 group and the positive control group were challenged, and 20 tomato plants in the negative control group were injected with 1 L of normal saline by root irrigation.
  • kit 1, kit 2, kit 3, and kit 4 were inoculated with 1 L of each thousand-fold dilution by root irrigation, and the positive control group and the negative control group were given the same amount of physiological brine.
  • the incidence of tomato in the kit 4 group is lower than that in the kit 1 group, indicating that the kit 4 containing the composition has a significant control of tomato bacterial leaf spot, and the kit 4 containing the composition is more Kit 1 containing only Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 was more effective in controlling tomato morbidity.
  • Kit 1, kit 2, kit 3, and kit 4 all use the kits of Example 19.
  • Test process 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 cultivation for 1 month. 20 plants in each group. Using the root irrigation method, 1L of Pseudomonas syringae bean pathogenic variety ATCC21781 (Pseudomonassyringae pv.phaseolicola) with a final concentration of 1x10 3 cfu/mL was added to Kit 1, Kit 2, Kit 3 and Kit A total of 100 common bean plants in the 4 groups and the positive control group were challenged, and 20 common bean plants in the negative control group were injected with 1 L of normal saline by root irrigation.
  • Pseudomonas syringae bean pathogenic variety ATCC21781 Pseudomonassyringae pv.phaseolicola
  • kit 1, kit 2, kit 3, and kit 4 were inoculated with 1 L of each thousand-fold dilution by root irrigation, and the positive control group and the negative control group were given the same amount of physiological brine.
  • Table 18 The effect of the kit of Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 and its composition on kidney bean sickle disease
  • Kit 1 of Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 was more effective in controlling morbidity.
  • Kit 1, kit 2, kit 3, and kit 4 all use the kits of Example 19.
  • Test process 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 cultivation for 1 month. 20 plants in each group. 1L of Pseudomonas syringae pv.tabaci (Pseudomonas syringae pv.tabaci) with a final concentration of 1x10 3 cfu/mL was used to treat Kit 1, Kit 2, Kit 3, Reagent A total of 100 tobacco plants in Box 4 and the positive control group were challenged, and 20 tobacco plants in the negative control group were treated with 1 L of normal saline by root irrigation.
  • Pseudomonas syringae pv.tabaci Pseudomonas syringae pv.tabaci
  • kit 1, kit 2, kit 3, and kit 4 were inoculated with 1 L of each thousand-fold dilution by root irrigation, and the positive control group and the negative control group were given the same amount of physiological brine.
  • Table 19 The effect of the kit of Pseudomonas syringae kiwifruit pathogenic phage PSA-P1 and its composition on tobacco wildfire
  • the incidence of tobacco in the kit 4 group is lower than that in the kit 1 group, indicating that the kit 4 containing the composition has a significant effect on the control of tobacco wildfire, and the kit 4 with the composition is lower than that containing only cloves Kit 1 of Pseudomonas kiwifruit pathogenic phage PSA-P1 was more effective in controlling the incidence.
  • the Pseudomonas syringae pv. Actinidiae phage PSA-P1 of the present application and the composition thereof have high safety, and can be used as preparation kits and biological disinfectants or biological It is an active ingredient of similar pesticides to prevent but not limited to various bacterial diseases caused by Pseudomonas syringae.

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Abstract

提供了一种保藏编号为CCTCC NO:M 2020252的丁香假单胞菌猕猴桃致病变种噬菌体（ Pseudomonas syringaepv .Actinidiae phage）PSA-P1，该噬菌体对紫外线、pH具有较高的耐性，在10 1 PFU/mL-10 8 PFU/mL的效价范围内，丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1对丁香假单胞菌的抑菌率达到54.1-94.9％。还提供了包含该噬菌体的组合物、试剂盒及其应用。

Description

一种丁香假单胞菌噬菌体及其组合物、试剂盒和应用 技术领域
本发明涉及噬菌体领域，更具体地说，它涉及一种丁香假单胞菌噬菌体及其组合物、试剂盒和应用。
背景技术
丁香假单胞菌属于假单胞菌科假单胞菌属，有57个致病型，丁香假单胞杆菌猕猴桃致病变种(Psa；Pseudomonas syringae pv.actinidiae)是其中的一个致病型。Psa具有适应能力强的多种遗传基因，因此常年易于入侵猕猴桃树并引起感染。
当前，相关技术中对于猕猴桃溃疡病的防控技术，一方面是以化学防治为主，采用田间喷雾、流脓伤口涂抹及注干等方法施药，但从现有的防治情况来看，效果均不理想。另一方面，随着近年来国家在农作物上减抗、无抗的号召，国内外学者积极研究新兴的抗细菌生物制剂，由于丁香假单胞菌噬菌体具备对目标细菌(丁香假单胞菌猕猴桃致病变种)的专一高效裂解能力，能显著降低环境中致病菌的数量，可控制或减少疾病的发生和流行。因此，丁香假单胞菌噬菌体可作为抗细菌感染制剂使用。
目前，关于丁香假单胞菌噬菌体的研究较少，因此，开发寻找针对丁香假单胞菌的强裂解性噬菌体是本技术领域对于由丁香假单胞菌引起的各类植物病害防治中急需解决的问题。
发明内容
为了解决噬菌体在不同防治环境中耐受性的技术难题，本申请提供一种丁香假单胞菌噬菌体及其组合物、试剂盒和应用。
本发明提供的一种丁香假单胞菌噬菌体及其组合物、试剂盒和应用，采用如下的技术方案：
第一方面，本发明提供一种丁香假单胞菌噬菌体，采用如下的技术方案：
一种丁香假单胞菌噬菌体，所述丁香假单胞菌噬菌体为丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1Pseudomonas syringae pv.Actinidiae phage PSA-P1)，保藏编号为CCTCC NO：M 2020252。
通过采用上述技术方案，本申请提供一种丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1，该噬菌体具有优良的耐紫外线性、耐pH性能，现已存放在中 国典型培养物保藏中心进行保藏，保藏编号为CCTCC NO:M 2020252。
优选的，所述丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringae pv.Actinidiae phage PSA-P1)为烈性噬菌体，有一呈多面体立体对称的头部和可伸缩的尾部，头部直径为50～55nm，尾部的长度为15～20nm，尾部直径为6～10nm，属于Autographiviridae科噬菌体。
通过采用上述技术方案，从电镜中观察噬菌体的形貌，可知该噬菌体具有多面体立体对称的头部和可伸缩的尾部，有助于头部的核酸注入宿主菌中，还可有效识别宿主菌表面的特殊受体。
优选的，所述丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringae pv.Actinidiae phage PSA-P1)具有SEQ ID No.1所示的核苷酸序列。
优选的，所述丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringae pv.Actinidiae phage PSA-P1)在感染复数MOI＝0.000001条件下培养24h，效价达到7×10 10PFU/mL以上。
通过采用上述技术方案，感染复数(MOI)是噬菌体数量与细菌数量的比值，是研究噬菌体感染细菌与产出噬菌体子代量效关系的重要依据。本发明的丁香假单胞菌噬菌体只需添加少量，即可侵染丁香假单胞菌增殖增殖获得大量子代噬菌体。本发明为工业化生产噬菌体杀菌剂提供了优质噬菌体菌株来源。[0013]优选的，所述丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringae pv.Actinidiae phage PSA-P1)在pH＝3～12的条件下具有耐性，在96h内效价降低不超过4个数量级。
通过采用上述技术方案，可知在pH3或者pH12，即在酸性或者碱性条件下，本发明的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1具有优异的耐性，当防治环境在pH＝3～12之间时，本发明的丁香假单胞菌噬菌体均可以起到有效的防治作用。
优选的，所述丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringae pv.Actinidiae phage PSA-P1)经紫外辐射8h后，效价降低不超过1个数量级。
通过采用上述技术方案，当防治环境中紫外线较强时，采用本发明的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1具有良好的耐受性，效价降低较少， 能够对病原菌起到有效的防治作用。
第二方面，本发明提供一种丁香假单胞菌噬菌体的组合物，采用如下的技术方案：
一种丁香假单胞菌噬菌体的组合物，所述组合物中至少含有一株丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringae pv.Actinidiae phage PSA-P1)。
优选的，所述组合物包括化学性杀菌剂。
作为本发明的实施方案之一，将所述的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1和化学性杀菌剂作为组合物联合使用。作为示例性的说明，丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1与代森铵700倍液之间的比例关系可以由本领域技术人员结合本申请以及实际的应用领域以及本领域常识进行确定。
第三方面，本发明提供一种丁香假单胞菌噬菌体的试剂盒，采用如下的技术方案：
一种丁香假单胞菌噬菌体的试剂盒，所述试剂盒中带有所述丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringae pv.Actinidiae phage PSA-P1)或丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringae pv.Actinidiae phagePSA-P1的)组合物。
通过上述技术方案，本发明的丁香假单胞菌噬菌体应用于丁香假单胞菌的快速检测，包括但不限于以试纸、试纸盒等形式对丁香假单胞菌进行检测，或对临床样本中的目标致病菌进行筛选，有效确保检测的灵敏度。
第四方面，本发明提供一种丁香假单胞菌噬菌体的应用，采用如下的技术方案：
一种丁香假单胞菌噬菌体的应用，所述丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringae pv.Actinidiae phage PSA-P1)在10 1PFU/mL～10 8PFU/mL的效价范围内，对丁香假单胞菌的抑菌率达到54.1～94.9％。
第五方面，本发明提供一种丁香假单胞菌噬菌体组合物的应用，采用如下的技术方案：
一种丁香假单胞菌噬菌体组合物的应用，所述的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的组合物，用作为生物类消毒剂或生物类农药的有效成分，防治但不限于由丁香假单胞菌引起的细菌性疾病。
通过采用上述技术方案，丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1及其组合物可以用于治疗和预防由丁香假单胞菌、且不限于由丁香假单胞菌引起的细菌感染，用作防治由丁香假单胞菌、且不限于由丁香假单胞菌引起的病害的生物药剂。
综上所述，本发明具有以下有益效果：
1、本发明的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1是从自然界中分离的烈性噬菌体，对紫外线、pH具有较高的耐受性，适用在不同的防治环境中，能够对猕猴桃溃疡病起到较好的生物防治效果；
2、本发明的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1是从自然界中分离的烈性噬菌体，供试噬菌体不含毒力基因或不良基因，该噬菌体的DNA无法编码可能引起潜在的健康风险的蛋白，不存在携带溶源基因的可能；
3、本发明的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1具有高度亲和性及裂解能力，在培养24h内达到10 10PFU/mL以上的效价；丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1可以特异性地部分或完全灭活丁香假单胞菌，仅需少量初始噬菌体即可完成大量增殖，为工业化生产噬菌体杀菌剂提供优质噬菌体菌株来源；本领域技术人员可以根据本申请的记载及本领域常识将本申请所述的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1或其组合物制备成应用于检测、消毒及植物防护等方面的各种产品并加以工业应用；
4、本发明的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1为严格的烈性噬菌体，对宿主菌具有高度专一性和裂解性，并且具有较广的宿主范围，对45株丁香假单胞菌的裂解率高达91.1％；丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1可作为应用于环境消毒的各种产品的有效成分，例如包括但不限于以液体浸泡、喷洒、与含水性载体联合使用等形式对配水系统、灌溉设施、养殖业设施、公共及私人设施或其他环境表面进行消毒去污，可有效控制目标细菌的生长及活性；所述液体浸泡、喷洒形式包括但不限于洗涤剂、消毒剂、去污剂等；所述含水性载体包括但不限于磷酸盐缓冲液、TSB培养基、LB培养基、氯游离水等；
5、本发明的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1与非宿主性致病性细菌的互作，无法识别10株供试非宿主性致病性细菌中的任何一株，特异性良好；
6、本发明的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1稳定性良好，在 pH＝3～12的条件下具有耐性，在96h内效价降低不超过4个数量级；经紫外辐射8h后，效价降低不超过1个数量级；
7、本发明的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1，可用于制备组合物、试剂或试剂盒，应用于丁香假单胞菌的快速检测，包括但不限于以试纸、试剂盒等形式对目标样本中的丁香假单胞菌进行检测，或对临床样本中的目标致病菌进行筛选，有效确保检测的灵敏度；
8、丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1及其组合物对液体培养基中浓度为10 3PFU/mL的丁香假单胞菌具有良好的杀灭能力；当丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1浓度≥1×10 6PFU/mL时，其对丁香假单胞菌的杀灭率达91.8％以上，且对其他联用物质无拮抗作用；
9、本发明的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringae pv.Actinidiae phage PSA-P1)及其组合物可以由本领域技术人员根据本申请的记载和本领域常识制备成可应用于防治由丁香假单胞菌、且不限于由丁香假单胞菌引起的病害的生物药剂。
附图说明
图1是本发明的噬菌斑示意图；
图2是本发明的噬菌体的电镜结果示意图；
图3是溶原性试验中出现噬菌斑样品的结构示意图；
图4是溶原性试验中未出现噬菌斑样品的结构示意图。
具体实施方式
以下结合附图1～4和实施例对本发明作进一步详细说明。
以下实例中，所涉及菌株代号均为本公司的命名方式编号。
丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonassyringae pv.Actinidiae phage PSA-P1)，保藏编号为CCTCC NO：M 2020252，保藏单位为中国典型培养物保藏中心，保藏时间为2020年06月30日。
地毯草黄单胞菌噬菌体YHC5(Xanthomonas axonopodisphage YHC5)，保藏编号为CCTCCNO:M2018579，保藏单位为中国典型培养物保藏中心，保藏时间为2018年08月30日。
地毯草黄单胞菌柑橘致病变种(Xanthomonasaxonopodispv.citri)，保藏 编号为ACCC 03526，向保藏单位联系购买获得。
丁香假单胞菌黄瓜致病变种(Pseudomonassyringae pv.lachrymans)，保藏编号为ATCC7386，可通过向保藏单位联系购买获得。
丁香假单胞菌番茄致病变种(Pseudomonassyringae pv.tomato)，保藏编号为ATCC BAA-871D-5，可通过向保藏单位联系购买获得。
丁香假单胞菌烟草致病变种(Pseudomonassyringae pv.tabaci)，保藏编号为ATCC13453，可通过向保藏单位联系购买获得。
以下实例中，
TSB液体培养基的配方为：胰蛋白胨15g，大豆蛋白胨5g，氯化钠5g，蒸馏水1000mL；
TSA固体培养基的配方为：胰蛋白胨15g，大豆蛋白胨5g，氯化钠5g，琼脂15g，蒸馏水1000mL；
TSA平板：将TSA固体培养基灭菌后倾倒在无菌平板上，冷却凝固后制成TSA平板；
TSB半固体琼脂培养基配方为：胰蛋白胨15g，大豆蛋白胨5g，氯化钠5g，琼脂7g，蒸馏水1000mL；
SM液配方：氯化钠5.8g，硫酸镁2g，1mol/LTris-HCl 50mL，明胶0.25g，蒸馏水1000mL。
实施例1：丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的分离制备及纯化培养
本发明中丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的来源样品采集于江苏省南京市江宁区众彩农贸市场污水，经双层滤纸过滤后低速常温离心，再用0.22μm滤膜过滤上清。
噬菌体的分离：
(1)取10mL过滤后的上清液，加入10mL2倍TSB液体培养基中，同时加入1mL噬菌体宿主菌Psa-1对数期菌液，放置于28℃条件下过夜培养；
(2)取上述培养物，在8000rpm条件下离心10min，用0.22μm滤膜过滤上清，备用；
(3)取0.5mL噬菌体宿主菌Psa-1对数期菌液，加入5mL、40℃TSB半固体琼 脂培养基中混匀，倾倒于TSA平板上，制备成含有宿主菌的双层平板；
(4)取备用的上清液10μL，滴在已凝固的双层平板上，在无菌条件下风干后，放置于28℃过夜培养，形成噬菌体点滴斑。
噬菌体的纯化：
(1)用牙签挑取噬菌体点滴斑，移至1mLSM液中震荡1min；
(2)进行10倍梯度稀释，取10 2、10 4和10 6稀释液分别加入噬菌体宿主菌对数期菌液0.5mL，混合均匀；
(3)静置15min后，将上述混合液加入5mL、40℃TSB半固体琼脂培养基中，混匀后立刻倾倒于TSA平板上，摇匀平置5min，待其凝固，置于28℃温箱过夜培养后观察，获得含有单个噬菌斑的双层平板；
(4)挑起单个噬菌斑，移至1mL SM液中，按照上述方式纯化至少3次以上，最终在平板上形成形态大小一致的噬菌斑；
(5)用牙签挑取形态大小一致的单个噬菌斑，置于含有1mL对数期宿主菌菌液的50mLTSB液体培养基中，28℃条件下180rpm过夜摇培；
(6)取培养物在8000rpm条件下离心10min，用0.22μm滤膜过滤上清，得到纯化噬菌体溶液，即为丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringae pv.Actinidiae phage PSA-P1)。丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringae pv.Actinidiae phage PSA-P1)在丁香假单胞菌菌苔上产生单一的圆形噬菌斑，参照图1。丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonassyringae pv.Actinidiae phagePSA-P1)，保藏编号为CCTCC M:2020252。
实施例2：丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的电镜观察取实施例1制得的纯化噬菌体溶液作电镜观察：取20μL样本滴于铜网上，待其自然沉淀15min，用滤纸从侧面吸收多余液体，加1滴2％磷钨酸于铜网上，染色10min，用滤纸从侧面吸去染液，干燥后做电镜观察。
结果如图2所示，在电子显微镜下观察丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1形态发现，该噬菌体有一呈多面体立体对称的头部和较长的尾部，头部直径约为50～55nm；尾部的长度为15～20nm，尾部直径为6～10nm。丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1属于Autographiviridae科噬菌体。
实施例3：丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1颗粒制备及基因组的提取与测序
(1)取实施例1制得的纯化噬菌体溶液100mL，依次加入浓度为5mg/mL的DNaseI 20μL、RNaseA 20μL，37℃条件孵育60min后加入5.84g NaCl，待溶解后置于冰浴中1h；
(2)4℃条件下，11000rpm离心10min，将离心后的上清液转移至新的离心管中，加入固体PEG8000，使其终浓度为10％(w/v)，待PEG8000完全溶解后，冰浴1h；
(3)再在4℃条件下，11000rpm离心20min，加入1mLSM液重悬沉淀，即获得噬菌体颗粒浓缩液，4℃保存待用。
采用λ噬菌体基因组DNA试剂盒提取噬菌体核酸并进行测序。经过核苷酸测序，所述丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringae pv.Actinidiae phage PSA-P1)具有SEQ ID No.1所示的核苷酸序列。
将丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的序列在NCBI网站上比对可得该噬菌体属于Autographiviridae科噬菌体。
实施例4丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1效价的测定
用SM液做稀释液，将丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(由实施例1制得)的原液10倍梯度逐级稀释至l0 8倍。分别取l0 5、l0 6、l0 7及l0 8稀释度的噬菌体培养液l000μL与其宿主菌菌液300μL均匀混合，静置15min使其与细菌表面的受体充分结合。将上述混合液加入4mL冷却至50℃的半固体琼脂培养基中，混匀后立即铺于已凝固的固体琼脂平板上，待琼脂凝固后于28℃倒置培养6-8h。每个稀释度需做三个平行样，计数时取此稀释度的三个平行样的平均数。其中，噬菌体效价(PFU/mL)＝平均噬菌斑数×稀释倍数
从表1可以得出，丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1培养12h后具有10 10PFU/mL以上的效价。
表1培养12h后丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的效价
培养时间 4h 8h 12h
噬菌体PSA-P1效价(PFU/mL) 4.1x10 9 1.7x10 10 3.3x10 10
实施例5：丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的毒力基因或不良基因缺失检测试验
本实施例选取103种经鉴定源自病原细菌体内溶源性噬菌体的毒力基因如表2所示，通过测定丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的全基因组并对其进行生物信息学分析，以确定其是否含有下列毒力基因。结果显示，丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1不含有下列毒力基因或不良基因，所以无法编码可能引起潜在健康风险的蛋白，因此丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1不会影响人或动物体的健康。
表2病原细菌体内溶源性噬菌体的主要已知毒性基因
Figure PCTCN2020134391-appb-000001
Figure PCTCN2020134391-appb-000002
Figure PCTCN2020134391-appb-000003
Figure PCTCN2020134391-appb-000004
实施例6：毒理实验
实验小鼠20只，雌雄各半，适应性饲养三天后，随机分为两组(噬菌体组、对照组)，每组10只(雌雄各5只)，给予噬菌体组剂量为10 10PFU/kg的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1，对照组给予等量生理盐水，连续给药15d，将实验鼠断颈处死，检查内脏情况。
实验结果显示，此剂量的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1对小鼠日常行为没有影响。解剖检查内脏未见异常。丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1具有生物安全性，可作为农作物病害防治制剂。
实施例7：丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1对丁香假单胞菌最佳感染复数(MOI)的测定
挑取宿主菌丁香假单胞菌Psa-1单个菌落，接种到盛有3mLTSB液体培养基的试管中，在温度为28℃的摇床中，180rpm条件下过夜振荡培养，得到宿主菌悬液。将宿主菌悬液以1:100比例转接到10mLTSB液体培养基，在温度为28℃、转速为180rpm条件下振荡培养至对数前期。按照MOI分别为100、10、1、0.1、0.01、0.001和0.0001、0.00001、0.000001、0.0000001的比例分别加入噬菌体PSA-P1纯化液(由实施例1制得)和噬菌体宿主菌(MOI＝纯化噬菌体溶液效价/噬菌体宿主菌浓度)，加入TSB液体培养基使各管总体积相同。在28℃摇床中180rpm振荡培养24h。培养完毕后10000g离心10min并收集上清培养液，采用双层平板法测定各处理噬菌体效价。各点均作双份复管培养取平均值，以产生最高噬菌体效价的MOI为最佳感染复数。实验重复3次。
表3不同感染复数下丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的效价
MOI PSA-P1(PFU/mL) 宿主菌(cfu/mL) PSA-P1效价(PFU/mL)
100 10 7 10 5 1.2×10 8
10 10 7 10 6 3.3×10 8
1 10 7 10 7 4.6×10 8
0.1 10 7 10 8 1.4×10 9
0.01 10 7 10 9 5.8×10 9
0.001 10 7 10 10 7.2×10 9
0.0001 10 6 10 10 2.4×10 10
0.00001 10 5 10 10 4.5×10 10
0.00000 10 4 10 10 7.4×10 10
0.0000001 10 3 10 10 5.2×10 10
由表3可知，培养24h条件下，噬菌体PSA-P1效价达到最高7.4×10 10PFU/mL时，其MOI＝0.000001。说明仅需少量初始丁香假单胞菌噬菌体，即可完成大量增殖。噬菌体PSA-P1为工业化生产噬菌体杀菌剂提供了优质噬菌体菌株来源。
实施例8：丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的pH和温度稳定性的测定
8-1：不同pH条件下丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的稳定性取无菌EP管分别加入pH＝1～14的TSB液体培养基900μL，将上述EP管置于25℃的恒温水浴中，待温度平衡后加入l00μL纯化噬菌体溶液(实施例7制得)，使其初始效价为1×10 10PFU/mL，室温下静置。分别于反应1h、4h、8h、24h及96h时进行取样，将各处理样本进行适当稀释后采用双层平板法测定噬菌体效价。实验重复3次。
表4噬菌体PSA-P1在不同pH条件下的稳定性
Figure PCTCN2020134391-appb-000005
结果如表4所示，在pH＝5～10之间，噬菌体PSA-P1的效价均无显著变化，表明其在中性、微酸和微碱条件下有较好的稳定性。
在pH＝3的酸性条件下和pH＝12的碱性条件下，噬菌体PSA-P1的效价有一定程度的下降，但是与pH＝7条件下相比，效价下降约3个数量级，表明其在酸性和碱性条件下有较好的耐受性。在pH＝2的极酸性条件下和pH＝13的极碱性条件下，噬菌体PSA-P1的效价1个小时内均下降为0。
8-2：不同温度条件下丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的稳定性
将效价为1.0×10 7PFU/mL的噬菌体PSA-P1(实施例7制得)，分别置于4℃，25℃以及40℃条件下，定期取样检测其效价。
表5丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1在4℃条件下的稳定性
Figure PCTCN2020134391-appb-000006
表6丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1在25℃条件下的稳定性
Figure PCTCN2020134391-appb-000007
Figure PCTCN2020134391-appb-000008
表7丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1在40℃条件下的稳定性
Figure PCTCN2020134391-appb-000009
由表5～表7可知，4℃条件下，噬菌体PSA-P1具有较好的稳定性，存放3个月后，效价并无明显下降，且存放12个月后，效价下降依然未超过1个数量级；25℃条件下，噬菌体PSA-P1存放4周后效价无明显降低；40℃条件下，噬菌体PSA-P1在24小时内效价无显著下降，72小时后，其效价下降1个数量级。由此表明，噬菌体PSA-P1在不同温度条件下都具有较好的稳定性。
实施例9：丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1对紫外线的耐受能力测试
取10mL效价为1×10 8PFU/mL的PSA-P1噬菌体(由实施例7制得)平铺于90mm无菌培养皿中后将其放入超净工作台内，置于紫外灯(20w，20cm)下照射。分别于0min、20min、40min、1h、2h、3h、4h、5h、6h、7h、8h取样并将其置于暗处30min后采用双层平板法测定噬菌体效价。
表8丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1在紫外线照射下的稳定性
Figure PCTCN2020134391-appb-000010
结果如表8所示，紫外线照射8h时，丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的效价下降一个数量级，因此，本发明的噬菌体对紫外线的耐受性较强。
实施例10：丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1对不同来源地的丁香假单胞菌的裂解能力试验采用双层平板点滴法测定噬菌体的裂解谱。分别挑取自山东、四川、重庆、安徽、广东和河南等6省分离获得的45株丁香假单胞 菌的单菌落，将其接种于盛有3mL TSB液体培养基的试管中，28℃下180rpm过夜培养，制得各株细菌菌液。取500μL菌悬液分别与TSB半固体琼脂培养基混合铺于普通琼脂平板上，取5μL纯化噬菌体PSA-P1溶液(由实施例1制得)点滴于平板上，待自然风干后28℃过夜培养，观察结果。
表9丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1对不同来源地的丁香假单胞菌的裂解结果
Figure PCTCN2020134391-appb-000011
Figure PCTCN2020134391-appb-000012
注：“+++”完全透亮；“++”中等透亮；“+”轻微透亮；不裂解的为“-”。
结果如表9所示，噬菌体PSA-P1对不同来源地的丁香假单胞菌均具有较强的裂解能力，其裂解率可达91.1％。丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1具有较宽的裂解谱。
实施例11：丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1对非致病性有益细菌的裂解试验
挑取5株非致病性根瘤菌，5株非致病性枯草芽孢杆菌分别接种于盛有3mLTSB液体培养基的试管中，30℃下180rpm培养8h，制得各株细菌菌液。取300μL菌悬液分别与TSB半固体琼脂培养基混合铺于普通琼脂平板上。分别取5μL纯化噬菌体PSA-P1溶液(由实施例1制得)滴于平板上，待自然风干后30℃培养24h，观察结果。
表10丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1对非致病性有益细菌的裂解试验
Figure PCTCN2020134391-appb-000013
注：“+++”完全透亮；“++”中等透亮；“+”轻微透亮；不裂解的为“-”。
结果如表10所示，在本实施例中，噬菌体PSA-P1均无法识别上述10株非致病性细菌。说明供试噬菌体具有极强的宿主特异性，且对微生物群落无损伤作用。
实施例12：丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1溶原性鉴定将100μL(0PFU/mL，1.0×10 4PFU/mL，1.0×10 5PFU/mL，1.0×10 6PFU/mL，1.0×10 7PFU/mL)的噬菌体PSA-P1(实施例7制得)与100μL丁香假单胞菌Psa-1(1.0×10 8cfu/mL)分别混合后接种于含有10mLTSB液体培养基的50mL离心管中，28℃下振荡培养48h。将所获混浊培养液在TSA平板上梯度稀释涂布，置于28℃培养箱中培养48h。在TSA平板上挑取30～50个单菌落的中心部位，将其置于含有200μLTSB液体培养基的EP管中，于28℃下振荡培养24h；随后向EP管中加入终浓度为0.5μg/mL的丝裂霉素C，继续培养12h；将所获培养液用0.22μm滤膜过滤除菌后点滴在Psa-1双层平板上，置于28℃培养；同时将效价为1.0×10 7PFU/mL的噬菌体PSA-P1(实施例7制得)点滴在双层平板上作为阳性对照。24h后观察双层平板，如果出现噬菌斑，则证明噬菌体PSA-P1为溶原性噬菌体。
结果显示，对照平板均有噬菌斑出现(图3)，而所有试验平板均无噬菌斑出现(图4)，说明丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1不具有溶原性，为烈性噬菌体。
实施例13：浊度法检测丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1对丁香假单胞菌的抑菌率
挑取宿主菌Psa-1的单菌落接种于盛有3mLTSB的试管中，28℃下180r/min过夜培养至浑浊，制得宿主菌菌液；将效价为2×10 10PFU/mL的噬菌体PSA-P1(实施例7制得)以无菌水逐步梯度稀释至各处理组效价。如表11所示，取100μL Psa-1菌液与100μL各稀释处理的噬菌体PSA-P1分别加入各盛有10mLTSB液体培养基的50mL离心管中，同时以100μLPsa-1菌液与10mLTSB液体培养基混合后的处理作为阳性对照，28℃、120r/min培养24h，用浊度仪测各处理组浑浊度值。抑菌率＝(阳性处理浊度-处理组浊度)/阳性处理浊度×100％
表11丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1对丁香假单胞菌的抑菌效果
Figure PCTCN2020134391-appb-000014
Figure PCTCN2020134391-appb-000015
由表11可知，约10 1PFU/mL的噬菌体PSA-P1对Psa-1的抑菌率即可达到54.1％，这表明丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1在极低的剂量下即可有效抑菌杀菌。
实施例14：丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1在液体中的杀菌效果
培养丁香假单胞菌Psa-1至对数生长期，分装进不同试管中，用等体积的TSB液体培养基稀释菌液至丁香假单胞菌Psa-1的终浓度为1×10 3cfu/mL。向其中分别接入终浓度为1×10 2PFU/mL、1×10 3PFU/mL，1×10 4PFU/mL、1×10 5PFU/mL、1×10 6PFU/mL的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringaepv.ActinidiaephagePSA-P1)(实施例1制得)。同时设置对照组和空白组，对照组给予终浓度1x10 3cfu/mL的丁香假单胞菌Psa-1；空白组给予等量生理盐水。将各处理于28℃下150rpm振荡培养，4h后检测丁香假单胞菌的残留量。检测方法为：将各处理样本以无菌水进行稀释后，取100μL稀释液于TSA固体平板上涂布，28℃培养24h后统计平板上菌落数量。丁香假单胞菌数量＝TSA平板上菌落数量×稀释倍数×10。
表12不同浓度丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1在液体中的杀菌效果
Figure PCTCN2020134391-appb-000016
由表12可知，丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1在其终浓度为1×10 2PFU/mL时，即可良好地控制丁香假单胞菌Psa-1在液体培养基中的生长；当丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1终浓度≥1×10 4PFU/mL时，其对丁香假单胞菌杀灭率可达99％以上。
实施例15：丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的组合物的制备
1、将终浓度分别为1×10 2PFU/mL、1×10 3PFU/mL、1×10 4PFU/mL、1×10 5PFU/mL、1×10 6PFU/mL的纯化噬菌体PSA-P1溶液(实施例1制得)分别与终浓度为50％的代森铵700倍液(化学性杀菌剂)等体积均匀混合制成1:1的组合物1、组合物2、组合物3、组合物4和组合物5。
2、将终浓度分别为1×10 2PFU/mL、1×10 3PFU/mL、1×10 4PFU/mL、1×10 5PFU/mL、1×10 6PFU/mL的纯化噬菌体PSA-P1溶液(实施例1制得)分别与终浓度为1×10 6PFU/mL的地毯草黄单胞菌噬菌体液体等体积均匀混合制成1:1的组合物6、组合物7、组合物8、组合物9和组合物10。
3、将终浓度分别为1×10 2PFU/mL、1×10 3PFU/mL、1×10 4PFU/mL、1×10 5PFU/mL、1×10 6PFU/mL的纯化噬菌体PSA-P1溶液(实施例1制得)与终浓度为50％的代森铵700倍液、终浓度为1×10 6PFU/mL的地毯草黄单胞菌噬菌体液体等体积均匀混合制成1:1:1的组合物11、组合物12、组合物13、组合物14和组合物15。
实施例16：丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1组合物在液体中的杀菌效果
培养丁香假单胞菌Psa-1至对数生长期，分装进不同试管中，用等体积的TSB液体培养基稀释菌液至丁香假单胞菌Psa-1的终浓度为1×10 3cfu/mL，向其中分别接入实施例15制备的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的组合物，同时设置对照组和空白组，对照组给予终浓度1×10 3cfu/mL的丁香假单胞菌Psa-1；空白组给予等量生理盐水。将各处理于28℃下150rpm振荡培养，4h后检测丁香假单胞菌Psa-1的残留量，检测方法：将各处理样本以无菌水进行稀释后，取100μL稀释液于TSA固体平板上涂布，37℃培养24h后统计平板上菌落数量。丁香假单胞菌数量＝TSA平板上菌落数量×稀释倍数×10。
表13不同浓度丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1组合物在液体中的杀菌效果
Figure PCTCN2020134391-appb-000017
由表13可知，各浓度下的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1组合物对丁香假单胞菌均具有良好的杀灭作用。说明丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1可与其他物质联用防治细菌，且对其他物质无拮抗作用。
本实施例的组合不限于代森铵700倍液，还可以是多抗霉素，氟吗啉、烯酰吗啉、咪鲜胺、苯醚甲环唑、氟硅唑、腈菌唑、代森锰锌、甲基硫菌灵、多菌灵、百菌清、聚糖果乐等化学杀菌剂。本实施例还可以与化学消毒剂联用，起到防杀效果。
实施例17：丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1及其组合物对猕猴桃溃疡病的防治
猕猴桃树360棵，适应性培养1个月后，随机分为6组(噬菌体3组、组合物5、对照组和空白组)，每组60棵，噬菌体实验组分别给予剂量为1×10 4PFU/mL、1×10 5PFU/mL、1×10 6PFU/mL的供试噬菌体(实施例7制得)和1×10 5cfu/mL的丁香假单胞菌Psa-1；对照组给予1×10 5cfu/mL的丁香假单胞菌Psa-1；空白组给予等量生理盐水，采用茎干输液法接种1L，自接种起统计15d内猕猴桃溃疡病的发病率，发病率＝发病株数/总株数×100％。
表14丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1及其组合物对猕猴桃溃疡病的影响
时间 10 4PFU/mL 10 5PFU/mL 10 6PFU/mL 组合物3 对照组 空白组
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％
由表14可知，对照组的猕猴桃树在接种15d时，猕猴桃溃疡病的发病率达到100％。而噬菌体各实验组中，噬菌体PSA-P1浓度越高，猕猴桃树发病率越低；噬菌体PSA-P1浓度为10 6PFU/mL时，灌根15d后，猕猴桃树发病率保持在10％以内。表明丁香假单胞菌噬菌体及其组合物可以用作生物杀菌剂，有效防治猕猴桃溃疡病。
实施例18：丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1及其组合物的试剂盒的制备及使用
试剂盒中含有5～10mL的效价为1×10 7PFU/mL的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1液体或丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的组合物，1L TSB半固体培养基，1L TSA培养基。
试剂盒的使用方法为：取效价为1×10 7PFU/mL丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1液体或丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的组合物，采用双层平板点滴法来测定供试噬菌体的裂解谱。挑取待检测单菌落，将其接种于目标液体培养基中，于目标温度下结合待检测菌株生长特性进行振荡培养，制得待检测菌株菌液。取300μL待检测菌株菌悬液分别与5mL TSB半固体培养基混合铺于TSA平板上，取10μL丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1液体或丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的组合物点滴于平板上。待自然风干后根据待检测菌株生长特性于目标温度下进行培养，观察结果即可。
实施例19:
丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1及其组合物的试剂盒对柑橘溃疡病的应用
试剂盒1的主要组分为5～10mL的效价为3×10 8PFU/mL的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1液体。
试剂盒2主要组分为5～10mL的效价为3×10 8PFU/mL的地毯草黄单胞菌噬菌体YHC5液体。
试剂盒3主要组分为5～10mL终浓度为50％的代森铵700倍液。
试剂盒4主要组分为5～10mL的效价为3×10 8PFU/mL的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1液体、5～10mL的效价为3×10 8PFU/mL的地毯草黄单胞菌噬菌体YHC5液体、5～10mL终浓度为50％的代森铵700倍液。[0082]
试验过程：柑橘树120棵，适应性培养1个月后，随机分为6组(试剂盒1组、试剂盒2组、试剂盒3组、试剂盒4组、阳性对照组和阴性对照组)，每组20棵。采用茎干输液法分别将1L终浓度为1x10 3cfu/mL的地毯草黄单胞菌柑橘致病变种ACCC03526(Xanthomonasaxonopodispv.citri)对试剂盒1组、 试剂盒2组、试剂盒3组、试剂盒4组及阳性对照组共100棵柑橘树进行攻毒处理，阴性对照组20棵柑橘树以茎干输液法分别输入1L生理盐水。攻毒处理三天后，采用茎干输液法对攻毒处理的5组柑橘树分别接种试剂盒1组、试剂盒2组、试剂盒3组、试剂盒4组的千倍稀释液体各1L，阳性对照组与阴性对照组均给予等量生理盐水。自接种起15d统计内柑橘溃疡病的发病率，发病率＝发病株数/总株数×100％。
表15丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1及其组合物的试剂盒对柑橘溃疡病的影响
Figure PCTCN2020134391-appb-000018
如表15，试剂盒4组的柑橘发病率较试剂盒2组的更低，说明含有所述组合物的试剂盒4对柑橘溃疡病防治显著，有所述组合物的试剂盒4较仅含有地毯草黄单胞菌噬菌体YHC5的试剂盒2在控制柑橘发病率方面的效果更佳。
实施例20:
丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1及其组合物的试剂盒对黄瓜细菌性 角斑病的应用
试剂盒1、试剂盒2、试剂盒3、试剂盒4均采用实施例19的试剂盒。
试验过程：黄瓜藤120株，适应性培养1个月后，随机分为6组(试剂盒1组、试剂盒2组、试剂盒3组、试剂盒4组、阳性对照组和阴性对照组)，每组20株。采用灌根法分别将1L终浓度为1x10 3cfu/mL的丁香假单胞菌黄瓜致病变种ATCC7386(Pseudomonas syringae pv.lachrymans)对试剂盒1组、试剂盒2组、试剂盒3组、试剂盒4组及阳性对照组共100株黄瓜进行攻毒处理，阴性对照组20株黄瓜分别以灌根法施入1L生理盐水。攻毒三天后，采用灌根法分别接种试剂盒1组、试剂盒2组、试剂盒3组、试剂盒4组的千倍稀释液体各1L，阳性对照组与阴性对照组均给予等量生理盐水。自接种起统计15d内黄瓜细菌性角斑病的病率，发病率＝发病株数/总株数×100％。
表16丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1及其组合物的试剂盒对黄瓜细菌性角斑病的影响
Figure PCTCN2020134391-appb-000019
如表16，试剂盒4组的黄瓜发病率较试剂盒1组的更低，说明含有所述组合物的试剂盒4对黄瓜细菌性角斑病防治显著，有所述组合物的试剂盒4较仅含有丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的试剂盒1在控制黄瓜发病率方面的效果更佳。
实施例20:
丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1及其组合物的试剂盒对番茄细菌叶斑病的应用
试剂盒1、试剂盒2、试剂盒3、试剂盒4均采用实施例19的试剂盒。
试验过程：番茄藤120株，适应性培养1个月后，随机分为6组(试剂盒1组、试剂盒2组、试剂盒3组、试剂盒4组、阳性对照组和阴性对照组)，每组20株。采用灌根法分别将1L终浓度为1x10 3cfu/mL的丁香假单胞菌番茄致病变种ATCCBAA-871D-5(Pseudomonassyringaepv.tomato)对试剂盒1组、试剂盒2组、试剂盒3组、试剂盒4组及阳性对照组共100株番茄进行攻毒处理，阴性对照组20株番茄分别以灌根法施入1L生理盐水。攻毒三天后，采用灌根法分别接种试剂盒1组、试剂盒2组、试剂盒3组、试剂盒4组的千倍稀释液体各1L，阳性对照组与阴性对照组均给予等量生理盐水。自接种起统计15d内番茄细菌叶斑病的发病率，发病率＝发病株数/总株数×100％。
表17丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1及其组合物的试剂盒对番茄细菌叶斑病的影响
Figure PCTCN2020134391-appb-000020
Figure PCTCN2020134391-appb-000021
如表17，试剂盒4组的番茄发病率较试剂盒1组的更低，说明含有所述组合物的试剂盒4对番茄细菌叶斑病防治显著，有所述组合物的试剂盒4较仅含有丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的试剂盒1在控制番茄发病率方面的效果更佳。
实施例21:
丁香假单胞猕猴桃致病变种菌噬菌体PSA-P1及其组合物的试剂盒对菜豆晕疫病的应用
试剂盒1、试剂盒2、试剂盒3、试剂盒4均采用实施例19的试剂盒。
试验过程：菜豆120株，适应性培养1个月后，随机分为6组(试剂盒1组、试剂盒2组、试剂盒3组、试剂盒4组、阳性对照组和阴性对照组)，每组20株。采用灌根法分别将1L终浓度为1x10 3cfu/mL的丁香假单胞菌菜豆致病变种ATCC21781(Pseudomonassyringae pv.phaseolicola)对试剂盒1组、试剂盒2组、试剂盒3组、试剂盒4组及阳性对照组共100株菜豆进行攻毒处理，阴性对照组20株菜豆分别以灌根法施入1L生理盐水。攻毒三天后，采用灌根法分别接种试剂盒1组、试剂盒2组、试剂盒3组、试剂盒4组的千倍稀释液体各1L，阳性对照组与阴性对照组均给予等量生理盐水。自接种起统计15d内菜豆晕疫病的发病率，发病率＝发病株数/总株数×100％。
表18丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1及其组合物的试剂盒对菜豆晕疫病的影响
Figure PCTCN2020134391-appb-000022
Figure PCTCN2020134391-appb-000023
如表18，试剂盒4组的菜豆发病率较试剂盒1组的更低，说明含有所述组合物的试剂盒4对菜豆晕疫病防治显著，有所述组合物的试剂盒4较仅含有丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的试剂盒1在控制发病率方面的效果更佳。
实施例22:
丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1及其组合物的试剂盒对烟草野火病的应用
试剂盒1、试剂盒2、试剂盒3、试剂盒4均采用实施例19的试剂盒。
试验过程：烟草120株，适应性培养1个月后，随机分为6组(试剂盒1组、试剂盒2组、试剂盒3组、试剂盒4组、阳性对照组和阴性对照组)，每组20株。采用灌根法分别将1L终浓度为1x10 3cfu/mL的丁香假单胞菌烟草致病变种ATCC13453(Pseudomonas syringae pv.tabaci)对试剂盒1组、试剂盒2组、试剂盒3组、试剂盒4组及阳性对照组共100株烟草进行攻毒处理，阴性对照组20株烟草以灌根法分别施入1L生理盐水。攻毒三天后，采用灌根法分别接种试剂盒1组、试剂盒2组、试剂盒3组、试剂盒4组的千倍稀释液体各1L，阳性对照组与阴性对照组均给予等量生理盐水。自接种起统计15d内烟草野火病的发病率，发病率＝发病株数/总株数×100％。
表19丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1及其组合物的试剂盒 对烟草野火病的影响
Figure PCTCN2020134391-appb-000024
如表19，试剂盒4组的烟草发病率较试剂盒1组更低，说明含有所述组合物的试剂盒4对烟草野火病防治显著，有所述组合物的试剂盒4较仅含有丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的试剂盒1在控制发病率方面的效果更佳。
综上，本申请的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringae pv.Actinidiae phage PSA-P1)及其组合物安全性高，可用作为制备试剂盒以及生物类消毒剂或生物类农药的有效成分，防治但不限于由丁香假单胞菌引起的各类细菌性疾病。
通过实施例19-22，可以看出，单独使用丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1对攻毒实验所用的丁香假单胞菌具有杀灭效果，可对攻毒植株的病害起显著防治作用，但含有噬菌体PSA-P1的组合物对攻毒植株病害的防治效果更佳。
本具体实施例仅仅是对本申请的解释，其并不是对本申请的限制，本领 域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改，但只要在本申请的权利要求范围内都受到专利法的保护。

Claims (11)

  1. 一种丁香假单胞菌噬菌体，其特征在于，所述丁香假单胞菌噬菌体为丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringaepv.Actinidiaephage PSA-P1)，保藏编号为CCTCC NO：M 2020252。
  2. 根据权利要求1所述的一种丁香假单胞菌噬菌体，其特征在于：所述丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringaepv.Actinidiaephage PSA-P1)为烈性噬菌体，有一呈多面体立体对称的头部和可伸缩的尾部，头部直径为50～55nm，尾部的长度为15～20nm，尾部直径为6～10nm，属于Autographiviridae科噬菌体。
  3. 根据权利要求1所述的一种丁香假单胞菌噬菌体，其特征在于：所述丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringaepv.Actinidiaephage PSA-P1)具有SEQ ID No.1所示的核苷酸序列。
  4. 根据权利要求1所述的一种丁香假单胞菌噬菌体，其特征在于：所述丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringaepv.Actinidiaephage PSA-P1)在感染复数MOI＝0.000001条件下培养24h，效价达到7×10 10PFU/mL以上。
  5. 根据权利要求1所述的一种丁香假单胞菌噬菌体，其特征在于：所述丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringaepv.Actinidiaephage PSA-P1)在pH＝3～12的条件下具有耐性，在96h内效价降低不超过4个数量级。
  6. 根据权利要求1所述的一种丁香假单胞菌噬菌体，其特征在于：所述丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringaepv.Actinidiaephage PSA-P1)经紫外辐射8h后，效价降低不超过1个数量级。
  7. 含有权利要求1～6任意一项所述的一种丁香假单胞菌噬菌体的组合物，其特征在于：所述组合物中至少含有一株丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringaepv.Actinidiaephage PSA-P1)。
  8. 根据权利要求7所述的一种丁香假单胞菌噬菌体的组合物，其特征在于：所述组合物包括化学性杀菌剂。
  9. 含有权利要求7所述的一种丁香假单胞菌噬菌体的试剂盒，其特征在于：所述试剂盒中带有所述丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringaepv.Actinidiaephage PSA-P1)或丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringaepv.Actinidiaephage PSA-P1)的组合物。
  10. 一种丁香假单胞菌噬菌体的应用，其特征在于：所述丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1(Pseudomonas syringaepv.Actinidiaephage PSA-P1)在10 1PFU/mL～10 8 PFU/mL的效价范围内，对丁香假单胞菌的抑菌率达到54.1～94.9％。
  11. 一种丁香假单胞菌噬菌体组合物的应用，其特征在于：权利要求7～8任意一项所述的丁香假单胞菌猕猴桃致病变种噬菌体PSA-P1的组合物，用作为生物类消毒剂或生物类农药的有效成分，防治但不限于由丁香假单胞菌引起的细菌性疾病。
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